Rhynchophorus palmarum (Linnaeus, 1758) (Coleoptera: Curculionidae): Guarani-Kaiowá indigenous knowledge and pharmacological activities

Kellen Natalice Vilharva; Daniel Ferreira Leite; Helder Freitas dos Santos; Katia Ávila Antunes; Paola dos Santos da Rocha; Jaqueline Ferreira Campos; Claudiane Vilharroel Almeida; Maria Lígia Rodrigues Macedo; Denise Brentan Silva; Caio Fernando Ramalho de Oliveira; Edson Lucas dos Santos; Kely de Picoli Souza

doi:10.1371/journal.pone.0249919

Peer Review History

Original SubmissionNovember 20, 2020
31 Dec 2020 Decision Letter - Branislav T. Šiler, Editor PONE-D-20-36667 Rhynchophorus palmarum Linnaeus (Coleoptera: Curculionidae): Guarani-Kaiowá indigenous knowledge and pharmacological activities PLOS ONE Dear Dr. de Picoli Souza, 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. The authors are advised to carefully revise the statistical processing of the data, as described by Reviewer #1. The whole text should be meticulously checked for correct language usage and typograpgic errors. Reviewer #1 has required several additional clarifications, mostly related to the methodology used. Please submit your revised manuscript by Feb 12 2021 11:59PM. 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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 #2: Yes ******** 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: The manuscript (PONE-D-20-36667) entitled "Rhynchophorus palmarum Linnaeus (Coleoptera: Curculionidae): Guarani-Kaiowá indigenous knowledge and pharmacological activities" is about the therapeutic use of oils obtained from R. palmarum larvae. The authors determined the chemical characteristics of the obtained oil, and, for the first time, are described its antioxidant and healing properties. They paid a special attention to the Guarani-Kaiowá ethnic community and their knowledge of zootherapy. Submitted manuscript contains some valuable information, so, this interesting research deserves attention having in mind a possibility of vanishing of traditional knowledge and practices over time. Also, the presented results may be of some help in the future development of various pharmaceutical products. Unfortunately, I cannot recommend accepting the manuscript in its present form. Following are some comments and questions raised during the review of the text. Significant revision of English scientific writing is needed. ABSTRACT Abstract is concise and contains factual information. INTRODUCTION In the Introduction, the authors stated the objectives of their work that are supported by appropriate facts. MATERIALS AND METHODS The experiments are well designed and performed. But, some parts of Material and Methods section need a bit more details. Specifically, these issues are highlighted: Obtaining R. palmarum larvae oil In Supplementary material S1, the method of collecting larvae is already described in detail. Line 93-94 The authors mentioned that the larvae were collected in Takuara village. When this procedure was conducted? How many larvae were collected? Line 98 “...larvae were washed with distilled water, dried with paper towels, weighed, and frozen...“ What were the temperatures that larvae were kept on, until the beginning of the experimental procedure? Determination of the chemical composition of RPLO Line 124 “...followed by stirring and incubation of the mixture“ The time of incubation? Antioxidant activity of RPLO In formula for DPPH percentage, Abs sample is the absorbance of the sample and Abs control is the absorbance of control? Please, provide clarifications. Line 149-150 “The results of three experiments performed in triplicate were used to determine the antioxidant activity.“ How many samples were in each experiment? Antimicrobial activity of RPLO Line 176 “The experiments were carried out in triplicate. “ How many samples were in each of this triplicates? Cell culture, viability, and migration Please, provide clarifications for Abs treated cells and Abs control cells. Statistical analysis Parametric statistics should be used when the assumptions of the models (ANOVA) can be reasonably met. A test of homogeneity of variances (for example, Levene test for homogeneity of variances) must show that in all cases variances were not significantly heterogeneous, as well as a Kolmogorov-Smirnov test for normal distribution fitting. So, the authors should test data distribution by, for example, Kolmogorov-Smirnov test and point it out in Statistical analysis section. This is very important because normal distribution is prerequisite for using ANOVA test. RESULTS Antimicrobial activity of the RPLO Please, see the Table 3. Did you mean MIC and MBC, instead of CIM and MBC? Effect of RPLO on the viability of MRC-5 cells Maybe, it would be better to rephrase this section. E.g., at given concentrations, cell viability was in what range? Compare to the control, which concentration of RPLO the most changes the viability of MRC-5 cells? In legend of Fig 4, please mention the number of samples. n= 9? Effect of RPLO on the migration rate of MRC-5 cells Line 299 "As shown in Fig 5A…" instead of "As shown in Fig 5…" Line 302-304 Please, rephrase the sentence. Maybe, something like the following: The cell migration rate was statistically increased by 60% in the wells treated with RPLO compared to the wells receiving the control treatment (Fig. 5B). Figure 5. Legend should be rewritten. It should be as clear and concise as possible. In addition, please mention the number of samples. Was it 9? Toxicity of RPLO in a C. elegans model In legend of Fig 6, please mention the number of samples. n= 10? DISCUSSION Please improve this section. The discussion should be clearly and concisely presented. Namely, the same things are repeated in several paragraphs – e.g. see lines 369-371 and 388-391; lines 402-408 and 418-425… REFERENCES Please, check this section once again. Reviewer #2: COMMENTS FOR AUTHORS Congratulations to a very interesting work. This big effort will certainly influence others to follow. It will be an important reference. The results and conclusions obtained in this study constitute a clear evidence of the need to conserve and promote the medicinal uses of insects (and animals in general) derived from a deep traditional knowledge. The paper should definitely be published, but it needs a minor revision. Below you will find a series of specific comments and suggestions, and I have just found some typographical errors, typos that I expose. Title Page 10 / line 1… Rhynchophorus palmarum (Linnaeus, 1758) (Coleoptera:... Abstract Page 11 / line 24… Brazil. This human community use the… Page 11 / line 25… snout beetle Rhynchophorus palmarum (Linnaeus, 1758) to... Page 11 / line 32… against Gram-positive and Gram-negative bacteria that… Introduction Page 12 / line 24… Traditional knowledge… the authors mention (they write) this concept numerous times. I think it is possible to use the abbreviation TK from this point on. Page 12 / line 24… species for their benefit (1). At this point it is possible to include more general and important references; for example: • Gibson, J. 2016. Community Resources: Intellectual Property, International Trade and Protection of Traditional Knowledge. Routledge, London. • Robinson, D.F., A. Abdel-Latif, and P. Roffe (eds.) 2017. Protecting Traditional Knowledge: The WIPO Intergovernmental Committee on Intellectual Property and Genetic Resources, Traditional Knowledge and Folklore. Routledge, London. • Nelson, M.K., and D. Shilling (eds.) 2018. Traditional Ecological Knowledge: Learning from Indigenous Practices for Environmental Sustainability. Cambridge University Press, Cambridge. Page 12 / line 57… the beetle Rhynchophorus palmarum (Linnaeus, 1758) (Coleoptera:... Page 12 / line 60… from the integument of R. palmarum larvae is… (not carcass). Page 12 / line 61… and skin infections (3). At this point it is possible to include more references; for example: • Cerda, H., R. Martínez, N. Briceno, L. Pizzoferrato, P. Manzi, M. Tommaseo Ponzetta, O. Marín, and M.G. Paoletti, 2001. Palm worm: (Rhynchophorus palmarum) traditional food in Amazonas, Venezuela-nutritional composition, small scale production and tourist palatability. Ecology of Food and Nutrition, 40: 13-32. • Cartay, R., V. Dimitrov, and M. Feldman, 2020. An insect bad for agriculture but good for human consumption: The case of Rhynchophorus palmarum: A social science perspective. In: Edible Insects, (Mikkola, H., ed.). IntechOpen, DOI: 10.5772/intechopen.87165. Available from: https://www.intechopen.com/books/edible-insects/an-insect-bad-for-agriculture-but-good-for-human-consumption-the-case-of-em-rhynchophorus-palmarum-e I even think that it is appropriate to bring reference number 16 to this point (Delgado et al. 2019). Materials and Methods Page 14 / line 115… at the bottom of Figure 3 appears, in parentheses, the word "arrow" in relation to image C. In this photograph I do not see the arrow. Page 15… references number 8 (line 120) and 9 (line 141) appear before number 7, which is on page 16 (line 154). Correct this. Page 15 / line 121… São Paulo Page 15 / line 140… described by Gupta and Gupta in 2011,… Page 16 / line 157… São Paulo Page 17 / line 178… São Paulo Results Page 22 / line 272… against Gram-positive and Gram-negative bacteria... Page 23 / Table 3… the abbreviations MIC and MBC are included in the title of the table… Why do the abbreviations “CIM” and “CBM” appear in the body of the table? Correct this. Discussion Page 26 / line 346… isolated from insects is… Page 26 / lines 347 and 348… change the order of the references: (3) (16), instead of (16) (3). Page 26 / line 355… with obesity (20), diabetes (21),… enter spaces. Page 27 / line 362… functions (24-25),… enter a space. Page 27 / line 366… study by Souza et al. (30), the intake… placed here, this reference is better, at this point (not at the end of the sentence). Page 28 / line 401… in rats (47-48). … enter a space. Page 28 / line 408… to sunlight (51-52). … enter a space. Page 29 / line 414… extracted from the colubrid snake Spilotes pullatus (Linnaeus, 1758) (54),… Page 29 / line 415… extracted from Boa constrictor Linnaeus, 1758, which… Page 29 / line 419… for ingestion… Page 29 / lines 422 and 423… Studies examining the antioxidant activity of oils obtained from insects are scarce in the literature (XXXX);… At this point in the “Discussion” section, I believe that the inclusion of some references is very convenient. I provide you with some examples: • Ekpo, K.E., A.O. Onigbinde, and I.O. Asia, 2009. Pharmaceutical potentials of the oils of some popular insects consumed in southern Nigeria. African Journal of Pharmacy and Pharmacology, 3(2): 51-57. • Oonincx, D.G.A.B., S. van Broekhoven, A. van Huis, and J.J.A. van Loon, 2015. Feed conversion, survival and development, and composition of four insect species on diets composed of food by-products. PLoS One, 10(12): e0144601. • Sosa, D.A.T., and V. Fogliano, 2017. Potential of Insect-Derived Ingredients for Food Applications. In: Insect Physiology and Ecology (Shields, V.D.C., ed.), pp. 215-232. IntechOpen. http://dx.doi.org/10.5772/67318 • Marusich, E., H. Mohamed, Y. Afanasev, and S. Leonov, 2020. Fatty acids from Hermetia illucens larvae fat inhibit the proliferation and growth of actual phytopathogens. Microorganisms 8(9): 1423. Page 29 / line 431… conducted by Guidoni et al. (46), who… This is not reference number 42. Correct this. ****** 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: Yes:** José Antonio González [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: Revision_PONE-D-20-36667.doc https://doi.org/10.1371/journal.pone.0249919.r001
Revision 1
18 Feb 2021 Author Response Dear editor, We are very grateful for the comments raised during the review process. We would like to make it clear that all suggestions have been analyzed and the manuscript has been modified. The responses to each survey were entered as "> response". In the responses, the indicated lines correspond to the file "Revised Article with Changes Highlighted". The manuscript (PONE-D-20-36667) entitled "Rhynchophorus palmarum Linnaeus (Coleoptera: Curculionidae): Guarani-Kaiowá indigenous knowledge and pharmacological activities" is about the therapeutic use of oils obtained from R. palmarum larvae. The authors determined the chemical characteristics of the obtained oil, and, for the first time, are described its antioxidant and healing properties. They paid a special attention to the Guarani-Kaiowá ethnic community and their knowledge of zootherapy. Submitted manuscript contains some valuable information, so, this interesting research deserves attention having in mind a possibility of vanishing of traditional knowledge and practices over time. Also, the presented results may be of some help in the future development of various pharmaceutical products. Unfortunately, I cannot recommend accepting the manuscript in its present form. Following are some comments and questions raised during the review of the text. Significant revision of English scientific writing is needed. ABSTRACT Abstract is concise and contains factual information. INTRODUCTION In the Introduction, the authors stated the objectives of their work that are supported by appropriate facts. MATERIALS AND METHODS The experiments are well designed and performed. But, some parts of Material and Methods section need a bit more details. Specifically, these issues are highlighted: Obtaining R. palmarum larvae oil In Supplementary material S1, the method of collecting larvae is already described in detail. Line 93-94 The authors mentioned that the larvae were collected in Takuara village. When this procedure was conducted? How many larvae were collected? Line 98 “...larvae were washed with distilled water, dried with paper towels, weighed, and frozen...“ What were the temperatures that larvae were kept on, until the beginning of the experimental procedure? > In this version of the manuscript we added further information regard the process of oil obtainment, informing the number of larvae, temperature of storage and further details requested (line 98-99). Determination of the chemical composition of RPLO Line 124 “...followed by stirring and incubation of the mixture“ The time of incubation? > We inserted in this version of the manuscript the time of incubation, of 15 min (line 125). Antioxidant activity of RPLO In formula for DPPH percentage, Abs sample is the absorbance of the sample and Abs control is the absorbance of control? Please, provide clarifications. > We appreciate this observation and now, we included more information about the formula (line 148). Line 149-150 “The results of three experiments performed in triplicate were used to determine the antioxidant activity.“ How many samples were in each experiment? > This was another important observation raised by referee. We included more details about this topic, informing that three independent assays. In each assay, we used triplicates for each oil concentration (n=3). Thus, each assay was carried out with n= 3. The sum of three independent assays resulted in a total samples of n= 9 (line 152-153). Antimicrobial activity of RPLO Line 176 “The experiments were carried out in triplicate. “ How many samples were in each of this triplicates? > In each assay, we used triplicates for each oil concentration (n=3). Thus, each assay was carried out with n= 3. The sum of three independent assays resulted in a total samples of n= 9 (3 assays using a 3 samples) (line 180). Cell culture, viability, and migration Please, provide clarifications for Abs treated cells and Abs control cells. > We appreciate this observation and now, we included more information about the formula (line 206). Statistical analysis Parametric statistics should be used when the assumptions of the models (ANOVA) can be reasonably met. A test of homogeneity of variances (for example, Levene test for homogeneity of variances) must show that in all cases variances were not significantly heterogeneous, as well as a Kolmogorov-Smirnov test for normal distribution fitting. So, the authors should test data distribution by, for example, Kolmogorov-Smirnov test and point it out in Statistical analysis section. This is very important because normal distribution is prerequisite for using ANOVA test. > We thank the referee regards this so important observation. In this version of the manuscript, we carried out further statistics analyses, such as suggested. So, we included the test of homogeneity of variances (Levene) and for normal distribution (KS test). Since non-significant values were obtained, we continued using ANOVA (line 242-244). RESULTS Antimicrobial activity of the RPLO Please, see the Table 3. Did you mean MIC and MBC, instead of CIM and MBC? > These mistakes were corrected. Effect of RPLO on the viability of MRC-5 cells Maybe, it would be better to rephrase this section. E.g., at given concentrations, cell viability was in what range? Compare to the control, which concentration of RPLO the most changes the viability of MRC-5 cells? > This section was rewritten to improve the clarity (line 294-298). In legend of Fig 4, please mention the number of samples. n= 9? > The mention regards the n=9 was included (line 302). Effect of RPLO on the migration rate of MRC-5 cells Line 299 "As shown in Fig 5A…" instead of "As shown in Fig 5…" Line 302-304 Please, rephrase the sentence. Maybe, something like the following: The cell migration rate was statistically increased by 60% in the wells treated with RPLO compared to the wells receiving the control treatment (Fig. 5B). > Based in observation of referee, we modified the section, improving the clarity of suggested sentences (line 306-310). Figure 5. Legend should be rewritten. It should be as clear and concise as possible. In addition, please mention the number of samples. Was it 9? > Actually, the legend contained excessive sentences. This mistake was corrected and the number of samples was included in this version (line 314-322). Toxicity of RPLO in a C. elegans model In legend of Fig 6, please mention the number of samples. n= 10? > This was a good observation. This assay contains a n= 90, formed by triplicates containing 10 worms/well (n= 30). Once the assay was carried out three times independently, the number of 90 was obtained (line 332). DISCUSSION Please improve this section. The discussion should be clearly and concisely presented. Namely, the same things are repeated in several paragraphs – e.g. see lines 369-371 and 388-391; lines 402-408 and 418-425… > We included modifications in discussion topic in order to make more conscious. However, in some points different approaches were necessary. So, whenever possible we improved the section. REFERENCES Please, check this section once again. > We made corrections in references section. Attachments Attachment Submitted filename: Response to reviewers.doc https://doi.org/10.1371/journal.pone.0249919.r002
19 Feb 2021 Decision Letter - Branislav T. Šiler, Editor PONE-D-20-36667R1 Rhynchophorus palmarum Linnaeus (Coleoptera: Curculionidae): Guarani-Kaiowá indigenous knowledge and pharmacological activities PLOS ONE Dear Dr. de Picoli Souza, 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. ============================== The authors have revised the manuscript according to the Reviewer #1' report only. The revision based on the reports of both Reviewers are needed. In addition, Response to reviewers should be better structured and authors' reposes have to be provided to each point raised by the Reviewers, instead of being provided for some of them. Cover letter needs to be updated. ============================== Please submit your revised manuscript by Apr 05 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: http://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols We look forward to receiving your revised manuscript. Kind regards, Branislav T. Šiler, Ph.D. Academic Editor PLOS ONE [Note: HTML markup is below. Please do not edit.] [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. https://doi.org/10.1371/journal.pone.0249919.r003
Revision 2
9 Mar 2021 Author Response Dear editor, We are very grateful for the comments raised during the review process. We would like to make it clear that all suggestions have been analyzed and the manuscript has been modified. The responses to each survey were entered as ‘> response’. Reviewer #1: COMMENTS FOR AUTHORS The manuscript (PONE-D-20-36667) entitled "Rhynchophorus palmarum Linnaeus (Coleoptera: Curculionidae): Guarani-Kaiowá indigenous knowledge and pharmacological activities" is about the therapeutic use of oils obtained from R. palmarum larvae. The authors determined the chemical characteristics of the obtained oil, and, for the first time, are described its antioxidant and healing properties. They paid a special attention to the Guarani-Kaiowá ethnic community and their knowledge of zootherapy. Submitted manuscript contains some valuable information, so, this interesting research deserves attention having in mind a possibility of vanishing of traditional knowledge and practices over time. Also, the presented results may be of some help in the future development of various pharmaceutical products. Unfortunately, I cannot recommend accepting the manuscript in its present form. Following are some comments and questions raised during the review of the text. Significant revision of English scientific writing is needed. ABSTRACT Abstract is concise and contains factual information. INTRODUCTION In the Introduction, the authors stated the objectives of their work that are supported by appropriate facts. MATERIALS AND METHODS The experiments are well designed and performed. But, some parts of Material and Methods section need a bit more details. Specifically, these issues are highlighted: Obtaining R. palmarum larvae oil In Supplementary material S1, the method of collecting larvae is already described in detail. Line 93-94 The authors mentioned that the larvae were collected in Takuara village. When this procedure was conducted? How many larvae were collected? Line 98 “...larvae were washed with distilled water, dried with paper towels, weighed, and frozen...“ What were the temperatures that larvae were kept on, until the beginning of the experimental procedure? > In this version of the manuscript we added further information regard the process of oil obtainment, informing the number of larvae, temperature of storage and further details requested. Determination of the chemical composition of RPLO Line 124 “...followed by stirring and incubation of the mixture“ The time of incubation? > We inserted in this version of the manuscript the time of incubation (15 min). Antioxidant activity of RPLO In formula for DPPH percentage, Abs sample is the absorbance of the sample and Abs control is the absorbance of control? Please, provide clarifications. > We appreciate this observation and now, we included more information about the formula. Line 149-150 “The results of three experiments performed in triplicate were used to determine the antioxidant activity.“ How many samples were in each experiment? > This was another important observation raised by referee. We included more details about this topic, informing that three independent assays. In each assay, we used triplicates for each oil concentration (n=3). Thus, each assay was carried out with n= 3. The sum of three independent assays resulted in a total samples of n= 9. Antimicrobial activity of RPLO Line 176 “The experiments were carried out in triplicate. “ How many samples were in each of this triplicates? > In each assay, we used triplicates for each oil concentration (n=3). Thus, each assay was carried out with n= 3. The sum of three independent assays resulted in a total samples of n= 9 (3 assays using a 3 samples) Cell culture, viability, and migration Please, provide clarifications for Abs treated cells and Abs control cells. > We appreciate this observation and now, we included more information about the formula. Statistical analysis Parametric statistics should be used when the assumptions of the models (ANOVA) can be reasonably met. A test of homogeneity of variances (for example, Levene test for homogeneity of variances) must show that in all cases variances were not significantly heterogeneous, as well as a Kolmogorov-Smirnov test for normal distribution fitting. So, the authors should test data distribution by, for example, Kolmogorov-Smirnov test and point it out in Statistical analysis section. This is very important because normal distribution is prerequisite for using ANOVA test. > We thank the referee regards this so important observation. In this version of the manuscript, we carried out further statistics analyses, such as suggested. So, we included the test of homogeneity of variances (Levene) and for normal distribution (KS test). Since non-significant values were obtained, we continued using ANOVA. RESULTS Antimicrobial activity of the RPLO Please, see the Table 3. Did you mean MIC and MBC, instead of CIM and MBC? > These mistakes were corrected. Effect of RPLO on the viability of MRC-5 cells Maybe, it would be better to rephrase this section. E.g., at given concentrations, cell viability was in what range? Compare to the control, which concentration of RPLO the most changes the viability of MRC-5 cells? > This section was rewritten to improve the clarity. In legend of Fig 4, please mention the number of samples. n= 9? > The mention regards the n=9 was included. Effect of RPLO on the migration rate of MRC-5 cells Line 299 "As shown in Fig 5A…" instead of "As shown in Fig 5…" Line 302-304 Please, rephrase the sentence. Maybe, something like the following: The cell migration rate was statistically increased by 60% in the wells treated with RPLO compared to the wells receiving the control treatment (Fig. 5B). > Based in observation of referee, we modified the section, improving the clarity of suggested sentences. Figure 5. Legend should be rewritten. It should be as clear and concise as possible. In addition, please mention the number of samples. Was it 9? > Actually, the legend contained excessive sentences. This mistake was corrected, and the number of samples was included in this version. Toxicity of RPLO in a C. elegans model In legend of Fig 6, please mention the number of samples. n= 10? > This was a good comment. This assay contains a n= 90, formed by triplicates containing 10 worms/well (n= 30). Once the assay was carried out three times independently, the number of 90 was obtained. DISCUSSION Please improve this section. The discussion should be clearly and concisely presented. Namely, the same things are repeated in several paragraphs – e.g. see lines 369-371 and 388-391; lines 402-408 and 418-425… > We included modifications in discussion topic in order to make more conscious. However, in some points different approaches were necessary. So, whenever possible we improved the section. REFERENCES Please, check this section once again. > We made corrections and addition of further references in this version. Reviewer #2: COMMENTS FOR AUTHORS Congratulations to a very interesting work. This big effort will certainly influence others to follow. It will be an important reference. The results and conclusions obtained in this study constitute a clear evidence of the need to conserve and promote the medicinal uses of insects (and animals in general) derived from a deep traditional knowledge. The paper should definitely be published, but it needs a minor revision. Below you will find a series of specific comments and suggestions, and I have just found some typographical errors, typos that I expose. Title Page 10 / line 1… Rhynchophorus palmarum (Linnaeus, 1758) (Coleoptera:... > We included in this version the correct taxonomic mention to C. Linnaeus, 1758. Abstract Page 11 / line 24… Brazil. This human community use the… Page 11 / line 25… snout beetle Rhynchophorus palmarum (Linnaeus, 1758) to... Page 11 / line 32… against Gram-positive and Gram-negative bacteria that… > Regards the three mentions, we would like to keep the first one (line 24), since the first author of the manuscript is an indigenous researcher woman. For this reason, the expression ‘my people’ was considered pertinent. The changes in lines 25 and 32 were carried out. Introduction Page 12 / line 24… Traditional knowledge… the authors mention (they write) this concept numerous times. I think it is possible to use the abbreviation TK from this point on. > We appreciate the important observation. Along the manuscript we used the abbreviation TK replacing the expression Traditional knowledge. Page 12 / line 24… species for their benefit (1). At this point it is possible to include more general and important references; for example: • Gibson, J. 2016. Community Resources: Intellectual Property, International Trade and Protection of Traditional Knowledge. Routledge, London. • Robinson, D.F., A. Abdel-Latif, and P. Roffe (eds.) 2017. Protecting Traditional Knowledge: The WIPO Intergovernmental Committee on Intellectual Property and Genetic Resources, Traditional Knowledge and Folklore. Routledge, London. • Nelson, M.K., and D. Shilling (eds.) 2018. Traditional Ecological Knowledge: Learning from Indigenous Practices for Environmental Sustainability. Cambridge University Press, Cambridge. > In this version of the manuscript these important references were included to improve the sources of information regards Traditional Knowledge (TK). Page 12 / line 57… the beetle Rhynchophorus palmarum (Linnaeus, 1758) (Coleoptera:... Page 12 / line 60… from the integument of R. palmarum larvae is… (not carcass). Page 12 / line 61… and skin infections (3). At this point it is possible to include more references; for example: • Cerda, H., R. Martínez, N. Briceno, L. Pizzoferrato, P. Manzi, M. Tommaseo Ponzetta, O. Marín, and M.G. Paoletti, 2001. Palm worm: (Rhynchophorus palmarum) traditional food in Amazonas, Venezuela-nutritional composition, small scale production and tourist palatability. Ecology of Food and Nutrition, 40: 13-32. • Cartay, R., V. Dimitrov, and M. Feldman, 2020. An insect bad for agriculture but good for human consumption: The case of Rhynchophorus palmarum: A social science perspective. In: Edible Insects, (Mikkola, H., ed.). IntechOpen, DOI: 10.5772/intechopen.87165. Available from: https://www.intechopen.com/books/edible-insects/an-insect-bad-for-agriculture-but-good-for-human-consumption-the-case-of-em-rhynchophorus-palmarum-e I even think that it is appropriate to bring reference number 16 to this point (Delgado et al. 2019). > We also appreciate the suggestions in order to improve the final quality of introduction section, including additional references suggested. Materials and Methods Page 14 / line 115… at the bottom of Figure 3 appears, in parentheses, the word "arrow" in relation to image C. In this photograph I do not see the arrow. Page 15… references number 8 (line 120) and 9 (line 141) appear before number 7, which is on page 16 (line 154). Correct this. Page 15 / line 121… São Paulo Page 15 / line 140… described by Gupta and Gupta in 2011,… Page 16 / line 157… São Paulo Page 17 / line 178… São Paulo > In this version, the Figure 3 mentioned before is Figure 2, since the map image was removed. The legend was corrected, as well the further points raised in lines 121, 140, 157, and 178. Results Page 22 / line 272… against Gram-positive and Gram-negative bacteria... Page 23 / Table 3… the abbreviations MIC and MBC are included in the title of the table… Why do the abbreviations “CIM” and “CBM” appear in the body of the table? Correct this. > The changes suggested by reviewer were accepted and the corrections in abbreviations CIM and CBM by MIC and MBC. Discussion Page 26 / line 346… isolated from insects is… Page 26 / lines 347 and 348… change the order of the references: (3) (16), instead of (16) (3). Page 26 / line 355… with obesity (20), diabetes (21),… enter spaces. Page 27 / line 362… functions (24-25),… enter a space. Page 27 / line 366… study by Souza et al. (30), the intake… placed here, this reference is better, at this point (not at the end of the sentence). Page 28 / line 401… in rats (47-48). … enter a space. Page 28 / line 408… to sunlight (51-52). … enter a space. Page 29 / line 414… extracted from the colubrid snake Spilotes pullatus (Linnaeus, 1758) (54),… Page 29 / line 415… extracted from Boa constrictor Linnaeus, 1758, which… Page 29 / line 419… for ingestion… > All these observations are pertinent, for this reason the mentioned lines were changed according referee’s suggestions. Page 29 / lines 422 and 423… Studies examining the antioxidant activity of oils obtained from insects are scarce in the literature (XXXX);… At this point in the “Discussion” section, I believe that the inclusion of some references is very convenient. I provide you with some examples: • Ekpo, K.E., A.O. Onigbinde, and I.O. Asia, 2009. Pharmaceutical potentials of the oils of some popular insects consumed in southern Nigeria. African Journal of Pharmacy and Pharmacology, 3(2): 51-57. • Oonincx, D.G.A.B., S. van Broekhoven, A. van Huis, and J.J.A. van Loon, 2015. Feed conversion, survival and development, and composition of four insect species on diets composed of food by-products. PLoS One, 10(12): e0144601. • Sosa, D.A.T., and V. Fogliano, 2017. Potential of Insect-Derived Ingredients for Food Applications. In: Insect Physiology and Ecology (Shields, V.D.C., ed.), pp. 215-232. IntechOpen. http://dx.doi.org/10.5772/67318 • Marusich, E., H. Mohamed, Y. Afanasev, and S. Leonov, 2020. Fatty acids from Hermetia illucens larvae fat inhibit the proliferation and growth of actual phytopathogens. Microorganisms 8(9): 1423. Page 29 / line 431… conducted by Guidoni et al. (46), who… This is not reference number 42. Correct this. > In this version we fixed this mistake. https://doi.org/10.1371/journal.pone.0249919.r004
18 Mar 2021 Decision Letter - Branislav T. Šiler, Editor PONE-D-20-36667R2 Rhynchophorus palmarum (Linnaeus, 1758) (Coleoptera: Curculionidae): Guarani-Kaiowá indigenous knowledge and pharmacological activities PLOS ONE Dear Dr. de Picoli Souza, 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. ============================== Some additional work on statistical processing is needed as stated in the Reviewer's #2 report (provided in the attachment). Figure captions also need the authors' attention. Please put references in the text into square brackets. ============================== Please submit your revised manuscript by May 02 2021 11:59PM. 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Attachments Attachment Submitted filename: PONE-D-20-36667R2_R2.doc https://doi.org/10.1371/journal.pone.0249919.r005
Revision 3
25 Mar 2021 Author Response The manuscript PONE-D-20-36667R2 entitled “Rhynchophorus palmarum (Linnaeus, 1758) (Coleoptera: Curculionidae): Guarani Kaiowá indigenous knowledge and pharmacological activities” was reviewed and the authors have addressed all the issues that reviewers had raised. I suggest that the manuscript should be accepted after an additional minor revision. We would like to thank Dr. José Antonio González for taking care of the smallest details that will certainly enrich our manuscript. Below are the responses to the surveys, which are marked in blue. Minor comment: The Material and Methods section has been greatly improved. But, Statistical analysis description could be a bit more precise, something like this: The data were expressed as mean ± standard error of the mean (SEM). Prior to the statistical analysis, Levene´s test of homogeneity of variances was used to assume that variances are equal across groups or samples and Kolmogorov-Smirnov test was used to assess the normality of the data. The mean values of the cell migration rate were analyzed by One-way analysis of variance (ANOVA) followed by Dunnett's post-test. In in vivo assays, a t-test was used to determine differences between groups. All analyses were performed using GraphPad Prism 5 software. The results were considered to be statistically significant at p ≤ 0.05. > We appreciate the suggestion, in this version the manuscript the section Statistical analysis was rewritten observing the improvement of clarity. Also, Figure legends would be rewritten, e.g. Fig 3. Viability of MRC-5 cells incubated with different concentrations of RPLO for 24 h. The graph shows the means ± SEM of three independent experiments performed in triplicate (n= 9). The observed changes were not statistically significant (t-test). Fig 4. The effect of RPLO on the migration rate of MRC-5 cells. (A) Representative image from assay. The wound areas were determined at 0 h and after 24 h of incubation. Control cells were incubated with culture media. Cells incubated with 0.5% of oil were named RPLO. Scale bar is 1 mm. (B) The graph shows the means ± SEM of three independent experiments performed in triplicate (n= 9). An asterisk () indicate statistically significant differences between mean values (one-way ANOVA, Dunnett's post-test, p <0.05). Fig 5. Acute toxicity of RPLO in the C. elegans experimental model in vivo. Viability was recorded at (A) 24 h and (B) 48 h of treatment. The graph shows the means ± SEM of three independent experiments performed in triplicate (n= 90). The observed changes were not statistically significant (t-test). > The same care regards the previous appointment was took in consideration, in order to become the Figure legends clear. Attachments Attachment Submitted filename: Response to Reviewers.doc* https://doi.org/10.1371/journal.pone.0249919.r006
29 Mar 2021 Decision Letter - Branislav T. Šiler, Editor Rhynchophorus palmarum (Linnaeus, 1758) (Coleoptera: Curculionidae): Guarani-Kaiowá indigenous knowledge and pharmacological activities PONE-D-20-36667R3 Dear Dr. de Picoli Souza, 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, Branislav T. Šiler, Ph.D. Academic Editor PLOS ONE Additional Editor Comments (optional): Reviewers' comments: https://doi.org/10.1371/journal.pone.0249919.r007
Formally Accepted
6 Apr 2021 Acceptance Letter - Branislav T. Šiler, Editor PONE-D-20-36667R3 Rhynchophorus palmarum (Linnaeus, 1758) (Coleoptera: Curculionidae): Guarani-Kaiowá indigenous knowledge and pharmacological activities Dear Dr. de Picoli Souza: 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 Dr. Branislav T. Šiler Academic Editor PLOS ONE https://doi.org/10.1371/journal.pone.0249919.r008

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