Differential expression of gut protein genes and population density of Arsenophonus contributes to sex-biased transmission of Bemisia tabaci vectored Cotton leaf curl virus

Ikbalpreet Singh; Ramandeep Kaur; Ashok Kumar; Satnam Singh; Abhishek Sharma

doi:10.1371/journal.pone.0259374

Peer Review History

Original SubmissionMay 5, 2021
22 Jun 2021 Decision Letter - Rajarshi Gaur, Editor PONE-D-21-14850 Is Cotton Leaf Curl Virus transmission by Bemisia tabaci (Gennadius) (Hemiptera: Aleyrodidae) sex-biased? PLOS ONE Dear Dr. Singh, Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process. Please submit your revised manuscript by Aug 06 2021 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file. 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The conclusions must be drawn appropriately based on the data presented. Reviewer #1: Partly Reviewer #2: Partly ******** 2. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: Yes Reviewer #2: Yes ****** 3. Have the authors made all data underlying the findings in their manuscript fully available? The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified. Reviewer #1: Yes Reviewer #2: Yes ****** 4. Is the manuscript presented in an intelligible fashion and written in standard English? PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here. Reviewer #1: Yes Reviewer #2: Yes ****** 5. Review Comments to the Author Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters) Reviewer #1: Review Comments to the Authors: This study by Singh et al focuses on the sex-biased transmission efficiency of Cotton Leaf Curl Virus (CLCuV) by Bemisia tabaci. The gender dependent differential variation was validated by using quantitative real-time PCR through the relative expression of CLCuV coat protein. The female whiteflies acquired and transmitted higher viral load compared to males. The authors identified that the higher density of Arsenophonus and differential expression of Cyclophilin, Knottin, Hsp40, Hsp70 of the midgut genes may possibly contribute to higher vector competency to females compared to males. This study advances our understanding to key players involved in sex biased CLCuV transmission. Overall, the study is novel and well conceptualized, however there are some points which need to be addressed before consideration to publish. Major comments: 1. The CLCuV coat protein real-time PCR primers used for this study should be tested on non-infected whitefly as well as non-infected plant to ensure there is not any non-specific binding. 2. Line 264-266 explained Fig 3c and indicating expression levels of CLCuV coat protein, but the figure legends are indicating these are Arsenophonus levels. Please update the figure legends. 3. As reported, there is differential level of expression of the genes in male and female so the efficiency of knock down (KD) using same amounts dsRNA for both male as well as female could be different and that may affect the observations. Indeed, the transmission efficiency of KD female is reduced more compared to KD males which is in consistent with the study. Ideally, it is important to normalize the gene expression in knockdown male as well as females. In short, authors should provide data for the expression levels of Arsenophonus in tetracycline fed/control male as well as female to validate the knock down for Arsenophonus. Also, it would have been interesting to have the comparative expression levels of GroEL during acquisition or transmission to strengthen and complement the data. 4. The authors should provide data for the expression levels of Cyclophilin in dsRNA fed/control male and female to validate the authenticity of knockdowns. Similar data should be provided for Knottin, Hsp40, Hsp70 as proof of concept. 5. Fig.6c data plot should be in comparison to male versus GFP fed control. Similarly, for fig.6d data plot should be comparison to female versus GFP fed control. Please update the labels on data plots. 6. It was quite evident that the expression levels of Arsenophonus, Knottin, Hsp40, Hsp70 in viruliferous flies are higher in comparison to non- viruliferous flies irrespective of gender. But the expression levels of Cyclophilin are lower in viruliferous flies in comparison to non-viruliferous flies. The authors should address that in the manuscript. Minor comments: 1. The male and female should be represented with different color for better representation of the graphs. 2. The authors should label Y-axis for all the data plots for consistency throughout the manuscript. 3. The statistics analysis is missing for the graphs Fig.1 c, Fig.3 d, Fig.4 a, c, d, Fig.5 a, b, c, Fig.6 d, Fig.7 c. 4. Primers in Table 1 should be cross validated as the forward and reverse primers for HSP40 cannot be identical. 5. Please provide the details (accession number or Uniprot ID) in methods section for the CLCuV coat protein sequence used for designing the primers for real-time PCR. Also, adding details of the source of Cotton Leaf Curl Rajasthan Virus would be better. 6. It is also recommended to provide additional details in methods sections for the software or tools used to analyze real time data, statistical and image analysis. 7. Line 220 please provide the full form for DOI. Reviewer #2: Whitefly, Bemisia tabaci (Gennadius) is an important pest of cotton as vector of Cotton Leaf Curl Virus (CLCuV) and the paper showed B. tabaci AsiaII-1 haplotype showing higher virus transmission efficiency of females than males. The transmission is related to the density of endosymbiont Arsenophonus, but the expression of some midgut proteins genes i.e. Cyclophilin, Knottin, Hsp40, Hsp70 may be possibly associated with the vector competency of both sex. All the results might not give us explicit conclusions that females treated by knocked-down or antibiotics could directly show higher competency to transmit the virus. My suggestion is that authors just focus on the role of one of endosymbionts and its midgut protein on the sex-biased transmission of whitefly. However, the title “Is Cotton Leaf Curl Virus transmission by Bemisia tabaci (Gennadius) (Hemiptera: Aleyrodidae) sex-biased?” does not fully explain and cover all main ideas of the manuscript, i.e. the relation the sex-biased of transmission with endosymbionts and their proteins. ****** 6. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files. If you choose “no”, your identity will remain anonymous but your review may still be made public. Do you want your identity to be public for this peer review?** For information about this choice, including consent withdrawal, please see our Privacy Policy. Reviewer #1: No Reviewer #2: No [NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files.] While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email PLOS at figures@plos.org. Please note that Supporting Information files do not need this step. Attachments Attachment Submitted filename: Peer Review Report PLOS.docx https://doi.org/10.1371/journal.pone.0259374.r001
Revision 1
28 Jul 2021 Author Response Editor’s comments: 1 Please ensure that your manuscript meets PLOS ONE's style requirements, including those for file naming Manuscript has been modified according to Plos one style as suggested. 2 We note that you have referenced (ie. Bewick et al. [5]) which has currently not yet been accepted for publication. Please remove this from your References and amend this to state in the body of your manuscript: (ie “Bewick et al. [Unpublished]”) as detailed online in our guide for authors Reference has been deleted as per suggestion 3. We note that Figure 2 and Supplementary Figures 1, 2 and 3 in your submission contain copyrighted images. All PLOS content is published under the Creative Commons Attribution License (CC BY 4.0), which means that the manuscript, images, and Supporting Information files will be freely available online, and any third party is permitted to access, download, copy, distribute, and use these materials in any way, even commercially, with proper attribution. We require you to either (1) present written permission from the copyright holder to publish these figures specifically under the CC BY 4.0 license, or (2) remove the figures from your submission: Figure 2 and Supplementary Figures 1, 2 and 3 have been clicked by us in our lab/ greenhouse setup. So we do not need to seek any permissions for using these figures in our manuscript. However, S1 Fig. contains pictures of cages purchased from Bugdrom Store (Taiwan). The permission from the manufacturer has been sought to use the picture of cages in manuscript (permission under the CC BY 4.0 license has been attached) Reviewer 1: 1. The CLCuV coat protein real-time PCR primers used for this study should be tested on non-infected whitefly as well as non-infected plant to ensure there is not any non-specific binding. The CLCuV coat protein primers has been validated on non-viruliferous and viruliferous whiteflies and plants. Details added in Fig 1. 2. Line 264-266 explained Fig 3c and indicating expression levels of CLCuV coat protein, but the figure legends are indicating these are Arsenophonus levels. Please update the figure legends. Done 3As reported, there is differential level of expression of the genes in male and female so the efficiency of knock down (KD) using same amounts dsRNA for both male as well as female could be different and that may affect the observations. Indeed, the transmission efficiency of KD female is reduced more compared to KD males which are in consistent with the study. Ideally, it is important to normalize the gene expression in knockdown male as well as females. In short, authors should provide data for the expression levels of Arsenophonus in tetracycline fed/control male as well as female to validate the knock down for Arsenophonus. Also, it would have been interesting to have the comparative expression levels of GroEL during acquisition or transmission to strengthen and complement the data. We thanks reviewer for this comment. The knocked down after tetracycline feeding in female and male relative to control female and male whiteflies has been validated and added in main text along with figure and statistical data. As per suggestion comparative expression levels of GroEL during acquisition or transmission has been conducted and incorporated in Fig 4. 4. The authors should provide data for the expression levels of Cyclophilin in dsRNA fed/control male and female to validate the authenticity of knockdowns. Similar data should be provided for Knottin, Hsp40, Hsp70 as proof of concept The relative expression Cyclophilin, Knottin, Hsp40 and Hsp70 in dsRNA fed and control male and female has been validated using RT-PCR and statistical analysis was performed by Student t-Test. The data has been added in main text body and Fig 5, 6, 7 and 8 5. Fig.6c data plot should be in comparison to male versus GFP fed control. Similarly, for fig.6d data plot should be comparison to female versus GFP fed control. Please update the labels on data plots. Fig. 6c and 6d has been rephrased to updated to panel Fig. 7C and labels have been updated as per suggestions. Minor Comments 1. The male and female should be represented with different color for better representation of the graphs. Male and female has been represented by different colours in all the figures 2. The authors should label Y-axis for all the data plots for consistency throughout the manuscript. Done 3. The statistics analysis is missing for the graphs Fig.1 c, Fig.3 d, Fig.4 a, c, d, Fig.5 a, b, c, Fig.6 d, Fig.7 c. The data have been analysed by Student’s T test and this has been mentioned in the text 4. Primers in Table 1 should be cross validated as the forward and reverse primers for HSP40 cannot be identical. This has been changed as suggested. 5. Please provide the details (accession number or Uniprot ID) in methods section for the CLCuV coat protein sequence used for designing the primers for real-time PCR. Also, adding details of the source of Cotton Leaf Curl Rajasthan Virus would be better. Accessions have been mentioned in the text. Inadvertently we have written the virus name as Cotton Leaf Curl Rajasthan Virus in place of Cotton Leaf Curl Multan-Rajasthan Virus and this is the correct classification as per ICTV Code. The virus strain has been continuously maintained on cotton plants and is crosschecked time to time for its purity. 6. It is also recommended to provide additional details in methods sections for the software or tools used to analyze real time data, statistical and image analysis. Details have been added in materials methods. 7. Line 220 please provide the full form for DOI. The DOI has been replaced by DPI (Days Post Inoculation) Reviewer 2 1. Whitefly, Bemisia tabaci (Gennadius) is an important pest of cotton as vector of Cotton Leaf Curl Virus (CLCuV) and the paper showed B. tabaci AsiaII-1 haplotype showing higher virus transmission efficiency of females than males. The transmission is related to the density of endosymbiont Arsenophonus, but the expression of some midgut proteins genes i.e. Cyclophilin, Knottin, Hsp40, Hsp70 may be possibly associated with the vector competency of both sex. All the results might not give us explicit conclusions that females treated by knocked-down or antibiotics could directly show higher competency to transmit the virus. My suggestion is that authors just focus on the role of one of endosymbionts and its midgut protein on the sex-biased transmission of whitefly. However, the title “Is Cotton Leaf Curl Virus transmission by Bemisia tabaci (Gennadius) (Hemiptera: Aleyrodidae) sex-biased?” does not fully explain and cover all main ideas of the manuscript, i.e. the relation the sex-biased of transmission with endosymbionts and their proteins. This is a good suggestion and keeping in view the reviewers comments we have revised the title of manuscript title highlighting Arsenophonus and gut proteins. Attachments Attachment Submitted filename: Response to Reviewers.docx https://doi.org/10.1371/journal.pone.0259374.r002
4 Oct 2021 Decision Letter - Rajarshi Gaur, Editor PONE-D-21-14850R1Differential expression of gut protein genes and population density of Arsenophonus contributes to sex-biased transmission of Bemisia tabaci vectored Cotton leaf curl virusPLOS ONE Dear Dr. Singh, Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process. Please submit your revised manuscript by Nov 18 2021 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file. Please include the following items when submitting your revised manuscript: A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). You should upload this letter as a separate file labeled 'Response to Reviewers'. A marked-up copy of your manuscript that highlights changes made to the original version. You should upload this as a separate file labeled 'Revised Manuscript with Track Changes'. An unmarked version of your revised paper without tracked changes. You should upload this as a separate file labeled 'Manuscript'. If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter. Guidelines for resubmitting your figure files are available below the reviewer comments at the end of this letter. If applicable, we recommend that you deposit your laboratory protocols in protocols.io to enhance the reproducibility of your results. Protocols.io assigns your protocol its own identifier (DOI) so that it can be cited independently in the future. For instructions see: https://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols. Additionally, PLOS ONE offers an option for publishing peer-reviewed Lab Protocol articles, which describe protocols hosted on protocols.io. Read more information on sharing protocols at https://plos.org/protocols?utm_medium=editorial-email&utm_source=authorletters&utm_campaign=protocols. We look forward to receiving your revised manuscript. Kind regards, Rajarshi Gaur Academic Editor PLOS ONE Journal Requirements: Please review your reference list to ensure that it is complete and correct. If you have cited papers that have been retracted, please include the rationale for doing so in the manuscript text, or remove these references and replace them with relevant current references. Any changes to the reference list should be mentioned in the rebuttal letter that accompanies your revised manuscript. If you need to cite a retracted article, indicate the article’s retracted status in the References list and also include a citation and full reference for the retraction notice. Reviewers' comments: Reviewer's Responses to Questions Comments to the Author 1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation. Reviewer #1: (No Response) Reviewer #3: All comments have been addressed ******** 2. Is the manuscript technically sound, and do the data support the conclusions? The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented. Reviewer #1: Yes Reviewer #3: Yes ****** 3. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: Yes Reviewer #3: N/A ****** 4. Have the authors made all data underlying the findings in their manuscript fully available? The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified. Reviewer #1: Yes Reviewer #3: Yes ****** 5. Is the manuscript presented in an intelligible fashion and written in standard English? PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here. Reviewer #1: Yes Reviewer #3: Yes ****** 6. Review Comments to the Author Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters) Reviewer #1: This study by Singh et al, provides substantial evidence of correlation between sex based differential gene expression and transmission efficiency of Cotton Leaf Curl Virus (CLCuV) by Bemisia tabaci. The study was conducted using B. tabaci Asia II-1 halotype and the virus transmission efficiency of females was reported significantly higher over males. The authors have addressed majority of the comments from both the reviewers. However, I have mentioned few important concerns which should be addressed before consideration for publishing. Therefore, I would recommend acceptance with minor revision. 1- The color codes for the data plots are not consistent for example female are orange and controls are white in Fig-1 but in Fig 3B and 3C both male and female are blue, and controls became orange. Please try to maintain consistent colors throughout the manuscript. 2- Please provide the accession number if available for the coat protein sequence of the CLCuMuV-Ra which was used to design the real time primers for this study. 3- The real time PCR reaction conditions mentioned in lines 119-121 are different from lines 150-152, but both were used for the detection of coat protein either from whiteflies or from infected plants. Please justify. 4- Lines 229 and 230 please correct nl to microliters. 5- Fig1 E the fluid loss assay data is not clear as blue dots are not easy to visualize. 6- Lines 279-280 belong to Fig 3 legends to my understanding. Please correct accordingly. 7- The relative levels of Arsenophonus in non-viruliferous whiteflies are significantly higher than viruliferous whiteflies (Fig 3A) which is not corelating with the relative levels of GroEL. The relative amounts of GroEL are lower in non-viruliferous whiteflies in comparison with viruliferous whiteflies (Fig 4A) Please cross-check if there is labelling error in data plots of Fig3A or justify. 8- Line 343 the expression levels of cyclophilin in non-viruliferous males and females are not at par in Fig 5A. As Fig 5A data plots clearly indicate that females have higher levels of cyclophilin in viruliferous as well as non-viruliferous whiteflies. Please modify accordingly. 9- Fig 7B the control females have higher levels of Hsp40 than control males while Fig 7A clearly states females express lesser amounts of Hsp40. Similarly, Fig 8B the control females have slightly higher levels of Hsp70 than control males which is contradictory to data in 8A. Please correct the controls accordingly to support the data for 7A and 8A. 10- Fig 5C, 6C, 7C, 8C the control females have lower virus transmission rate than control males while Fig 1 clearly indicates female have higher transmission rate than males it is not consistent with the hypothesis throughout the manuscript. Please add corrections accordingly or provide justification. Reviewer #3: The MS entitled "Differential expression of gut protein genes and population density of Arsenophonus contributes to sex-biased transmission of Bemisia tabaci vectored Cotton leaf curl virus" is in present form is suitable for the publication ****** 7. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files. If you choose “no”, your identity will remain anonymous but your review may still be made public. Do you want your identity to be public for this peer review?** For information about this choice, including consent withdrawal, please see our Privacy Policy. Reviewer #1: No Reviewer #3: No [NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files.] While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email PLOS at figures@plos.org. Please note that Supporting Information files do not need this step. Attachments Attachment Submitted filename: review comments_.docx https://doi.org/10.1371/journal.pone.0259374.r003
Revision 2
16 Oct 2021 Author Response We are grateful to the reviewers for spending their time for the critical and valuable comments in the manuscript. We extend our heartfelt thanks to Reviewer 1 for critical analysis of the figures which has helped us to correct the inadvertent cut and copy mistakes in the Fig 3A and 7 B. We have tried to address each and every query raised by the reviewer. The point to point rebuttal has been given below: Reviewer comment:The color codes for the data plots are not consistent for example female are orange and controls are white in Fig-1 but in Fig 3B and 3C both male and female are blue, and controls became orange. Please try to maintain consistent colors throughout the manuscript Authors response: Colour codes of the figures have been modified as suggested. Reviewer Comment: Please provide the accession number if available for the coat protein sequence of the CLCuMuV-Ra which was used to design the real time primers for this study. Authors response: The accession no of CLCuMuV-Ra virus is provided in line no 98.As per suggestion this is also incorporated in lines 131and 132 Reviewer Comment: The real time PCR reaction conditions mentioned in lines 119-121 are different from lines 150-152, but both were used for the detection of coat protein either from whiteflies or from infected plants. Please justify. Authors Response: The conditions have been made uniform. Apologies for this error, inadvertently the protocol for three step amplification, which is also commonly used in our lab was written at 119-121. Reviewer Comment: Lines 229 and 230 please correct nl to microliters Authors Response: The total diet consumption in male and female (n=50) whiteflies was 2.4 and 3.4 µl after subtracting the diet loss from control (Fig 1E) and the diet consumption was converted in nl/ adult. So the nl is the correct representation. The same has been explained in text section. Reviewer Comment: Fig1 E the fluid loss assay data is not clear as blue dots are not easy to visualize Authors Response: The images have been converted into tiff format and are clearer. Thanks to reviewer for this comments as we have missed to put the methodology for this experiment and the same has been incorporated in the manuscript under “Feeding and Fluid loss assay” Reviewer Comment: Lines 279-280 belong to Fig 3 legends to my understanding. Please correct accordingly Authors Response: The subtitle of the next paragraph was merged with Fig. 2 legends. It has been corrected by spacing. Reviewer comment: The relative levels of Arsenophonus in non-viruliferous whiteflies are significantly higher than viruliferous whiteflies (Fig 3A) which is not corelating with the relative levels of GroEL. The relative amounts of GroEL are lower in non-viruliferous whiteflies in comparison with viruliferous whiteflies (Fig 4A) Please cross-check if there is labelling error in data plots of Fig3A or justify. Authors Response: We appreciate the reviewer’s critical analysis for Fig 3 A and & 7 B as both figures were wrongly pasted from excel. In Fig 3A the cq-values were mistakenly plotted which shows huge variability across the y-axis of plots. The graphs for Fig 3A has been replotted with correct values and incorporated in the Manuscript. Reviewer comment: Line 343 the expression levels of cyclophilin in non-viruliferous males and females are not at par in Fig 5A. As Fig 5A data plots clearly indicate that females have higher levels of cyclophilin in viruliferous as well as non-viruliferous whiteflies. Please modify accordingly. Authors Response: Here we mean to say that the expression of cyclophilin in non-viruliferous males and females are statistically at par with each other as we these values are not significantly different as per Student’s t test. We have reformatted the line by addition ‘statistically at par with each other’ Reviewer Comment: Fig 7B the control females have higher levels of Hsp 40 than control males while Fig 7A clearly states females express lesser amounts of Hsp40 Authors Response: As mentioned earlier, the inadvertent mistake in plotting Fig. 7B, the data has been crosses checked and the correct plot has been incorporated in the manuscript. The percent knockdown 80.7 was correctly mentioned in the text earlier. Reviewer Comment: Similarly, Fig 8B the control females have slightly higher levels of Hsp70 than control males which is contradictory to data in 8A. Please correct the controls accordingly to support the data for 7A and 8A. Fig 5C, 6C, 7C, 8C the control females have lower virus transmission rate than control males while Fig 1 clearly indicates female have higher transmission rate than males it is not consistent with the hypothesis throughout the manuscript. Please add corrections accordingly or provide justification. Authors Response: We agree to the reviewer perspective but each graph of panel is as a result of one experimentation under one set of conditions. It is not possible to compare the y-axis of the every plot in each panel of the figures or even across the figures. The reason is that every graph is a result of different biological sample under different set of experimental conditions which gives variation in housekeeping gene and PCR efficiency in spite of all efforts to keep the things same throughout the experimentation process. This can also be visualized from the variation in Standard deviation across all the figures. The results were interpreted on the basis of target gene in male and female relative to its reference control normalized with housekeeping gene at that point of time or that very single qPCR reaction so the two figures in each panel represent a relative expression in two different set of qPCR reactions. Moreover, for Fig 8B slighter variation is clearly indicated by the ±0.01 value of SD bar in female control as compared to male controls.Thus we again emphasis that these graphs are generated on the basis of multiple experiments and replicates, the comparison of Y-axis values across graphs, which show slight experimental variations in Fig 5C, 6C, 7C, 8C is not possible. https://doi.org/10.1371/journal.pone.0259374.r004
19 Oct 2021 Decision Letter - Rajarshi Gaur, Editor Differential expression of gut protein genes and population density of Arsenophonus contributes to sex-biased transmission of Bemisia tabaci vectored Cotton leaf curl virus PONE-D-21-14850R2 Dear Dr. Singh, We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements. Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication. An invoice for payment will follow shortly after the formal acceptance. To ensure an efficient process, please log into Editorial Manager at http://www.editorialmanager.com/pone/, click the 'Update My Information' link at the top of the page, and double check that your user information is up-to-date. If you have any billing related questions, please contact our Author Billing department directly at authorbilling@plos.org. If your institution or institutions have a press office, please notify them about your upcoming paper to help maximize its impact. If they’ll be preparing press materials, please inform our press team as soon as possible -- no later than 48 hours after receiving the formal acceptance. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact onepress@plos.org. Kind regards, Rajarshi Gaur Academic Editor PLOS ONE https://doi.org/10.1371/journal.pone.0259374.r005
Formally Accepted
26 Oct 2021 Acceptance Letter - Rajarshi Gaur, Editor PONE-D-21-14850R2 Differential expression of gut protein genes and population density ofArsenophonus contributes to sex-biased transmission of Bemisia tabacivectored Cotton leaf curl virus Dear Dr. Singh: I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department. If your institution or institutions have a press office, please let them know about your upcoming paper now to help maximize its impact. If they'll be preparing press materials, please inform our press team within the next 48 hours. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information please contact onepress@plos.org. If we can help with anything else, please email us at plosone@plos.org. Thank you for submitting your work to PLOS ONE and supporting open access. Kind regards, PLOS ONE Editorial Office Staff on behalf of Professor Rajarshi Gaur Academic Editor PLOS ONE https://doi.org/10.1371/journal.pone.0259374.r006

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