PONE-D-24-00912Assessing complex member composition and insecticide resistance status of Anopheles gambiae s.l. in three coastal health districts of Côte d'Ivoire.PLOS ONE

Dear Dr. Ives,

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Reviewers' comments:

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Comments to the Author

1. 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 #2: Partly

Reviewer #3: Yes

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2. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

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3. Have the authors made all data underlying the findings in their manuscript fully available?

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Reviewer #1: No

Reviewer #2: Yes

Reviewer #3: Yes

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Reviewer #1: No

Reviewer #2: Yes

Reviewer #3: Yes

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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: Nice paper on Anopheles gambiae s.l. resistance level to pyrethroid insecticides and mechanisms involved in three coastal health districts of Côte d'Ivoire.

However, the level of English use lowered the quality of the manuscript. I would suggest the authors to really take time and rephrase most the sentences to an acceptable level.

Reviewer #2: To select the appropriate vector control products for us in a particular region of a country, it is important to determine which vector species are present and to establish the susceptibility of each species to the insecticides formulated into the products available.

The manuscript states that 'Dead and surviving mosquitoes’ exposure in WHO tube bioassays and CDC bottle tests were separately stored individually in Eppendorf tubes in silica gel and kept at −20 °C for further identification of An. gambiae s.l. complex members and kdr and Ace-1 mutations'. This would have facilitated the determination of the susceptibility of each molecular form of An. gambiae s.l. to each insecticide tested. However, the susceptibility data are presented simply as An gambiae s.l.. Assuming that the molecular form of each dead and alive mosquito in a susceptibility test has been determined as stated, it would be much more valuable to present the susceptibility data for each molecular form in each village.

There are numerous instances of the incorrect reference being identified in the text.

Reviewer #3: This manuscript describes the results of assessing complex member composition and insecticide resistance status of Anopheles gambiae s.l. in three coastal health districts of Côte d’Ivoire. The paper appears to be methodologically sound, and the analyses are well described in the manuscript. The strength of the paper is the fact that the authors are able to compare between three health districts,

Comments 1: Line 1 and 2. It’s will be important to reformulate the title of the manuscript

I’m suggested: Assessing of species composition and insecticide resistance of Anopheles gambiae complex member in three coastal health districts of Côte d’Ivoire

The aim of the study was to investigate the complex members and the insecticide resistance in the Anopheles gambiae s.l. populations in coastal Côte d'Ivoire.

a. Abstract:

You can choice to write in the manuscript between “An. gambiae s.L.” and “An. gambiae complex”. It's a totology when you write An. gambiae s.L. complex

Comment 1: Line 33: Author should put the 4.7% of hybrid species for what species?

Comment 2: Author should put the abbreviation (An. gambiae s.L) throughout the abstract subsections as the species name “Anopheles gambiae sensu lato” was already mentioned at the beginning of the abstract in line 41.

Comment 3: Abstract and throughout the manuscript, common Latin terms such as “s.s” and “s.L” are not italicized.

Comment 4: The authors are not mentioned in the abstract and the methodology if this is a cross-sectional survey, a longitudinal survey…etc. Add in the abstract and the methodology (Study site)

b. Introduction:

Ccomment 1: This is a study of the An. gambiae complex, except that I have only seen two species of the complex mentioned in the manuscript. The authors needs to define the An. gambiae complex and to cite the nine member of An. gambiae s. L. These articles will help you to cite the different species of the An. gambiae complex member, those which contribute to transmission and those which do not contribute. Loughlin SO (2020) https://doi.org/10.1080/20477724.2020.1722434. Abel et al. (2024) https://doi.org/10.1186/s13071-023-06102-7

Comment 2: The authors are not mentioned the Entomological Inoculation Rate (EIR) of An. gambiae s.L in coastal Côte d'Ivoire (in the studied sites).

c. Methodology:

Comment 1: Line 126 and 135. The larval method collected is not mentioned for what? What do you collected in the water wells, natural breeding sites, gardens field …etc larval only or not?

Comment 2: For rearing adult, author need to mention the technics used of rearing adult mosquito and the water type

Comment 3: Line 136: WHO tube bioassay assesses the degree of susceptibility for what?

Comment 4, Line 137 to 139: The author mentioned that the diagnostic dose tests of pyrethroids (alphacypermethrin, deltamethrin, and permethrin) were conducted in 2018, 2019, and 2020. The following tests were conducted exclusively in 2020 on the same population of An. gambiae complex (Fo, F1, F2…)?

Comment 5: The bottle analysis method is a monitoring tool for detecting and characterizing changes in insecticide susceptibility in vector populations?

Comment 6: Mosquito sample processing for DNA extraction was not mentioned clearly, does the author used whole mosquito for extraction or it was dissected to head and thorax and abdomen?

Identification of target site mutation (L202)

Comment 7: L202: For molecular Identification of target site mutation, author need to mention the primers used and PCR probes sequences and brief about the PCR methods.

Comment 8: L205-205: The author said that the krd (Kdr-East and Kdr-West) mutation was investigated using real-time PCR (TaqMan assay). To my knowledge, the probes (Kdr-East and West) attach to the FAM, I need to know how do you differentiate it? Did you do two separate the mix-PCR (WT, sensimix, water and Kdr-E FAM; WT, sensimix, water and Kdr-W FAM) or in the same mix did you put the Kdr-E and Kdr-W?

Data analysis

Comment 9: Please explain how this statistical analysis plan answer the stated question,: role of individual members of An. gambaie compex to malaria transmission?

Comment 10: Were there any considerations for possible non-blood-fed females from exophagic, zoophagic and endophilic mosquitoes? Any chance that some mosquitoes feed?

d. Results:

Species composition of the Anopheles gambiae complex

Comment 1, L236: For molecular identification of Anopheles gambiae complex member species, author mentioned the SINE200 or SINE-PCR using (Line 187 to 188). To my knowledge, SINE-PCR makes it possible to identify three species of the An. gambiae complex, namely: An. gambiae ss, An. arabesnsis and An. coluzzii. This is what I don't see in this manuscripts, in these results, the author identify two species and the hybride, were these others species in the complex member?

Comment 2: L237: the author said that 672 An. gambiae s.l. was selected from the three studied sites for species identification, however only 671 samples were used in An. gambaie s.l species molecular identification (there is 1 sample undetectable), because when I do the calculations of 593+51+27=671, where is the remaining 1 mosquito?

Comment 3: Figure 2 is not adopted for these results, remove that. It is better to represent the distribution of An. gambiae s.l. species on a table

Comment 4: L244, Remove the “of members” and write in the Table XX: Distribution of Anopheles gambiae s.l. in the three studied sites

Insecticide susceptibility in An. gambiae s.l

Comment 5: How many Anopheles have been testing with each insecticide, years and the different studied sites? The number of each An. gambiae s.L. alive and death (e.g: 2% (20/672))?

Intensity of resistance section

Comment 6, L273-273: The author said that: these results confirmed the strong resistance of An. gambiae s.L., for what? An. coluzzii or hybrid…etc.

Resistance mutation (L352)

Comment 7: Was the Kdr reading validated in relation to the value of the curve (CT), at the N point or by the value of FAM? What are the CT values (≤25 was positive in Kdr-E or W; 26-30 was RS-E or W) that you consider positive for the Kdr-E and the Kdr-W (RR)? For RS and SS?

- Wreting mistakes were recognized in line, L393 “Cô te d'Ivoire” and in, L394 “Bouaké ”.

Discussion

Comment 1: The authors should provide information, however, moderate resistance intensity was recorded with a 10X concentration of alphacypermethrin in the Aboisso populations, and additional investigations are required for better understanding. In this study, pre-exposure to PBO showed significantly increased pyrethroid-induced mortality in all An. gambiae s.l. populations. However, full susceptibility was not recovered using PBO, except with the strain of Jacqueville with deltamethrin. Is there any reports document to describe this resistance in Côte d’Ivoire?

Comment 2: L449-450. The author said that the pyrrole chlorfenapyr and neonicotinoid clothianidin have new modes of action and are good candidates for malaria vector control in areas comprised of mutation and/or metabolic resistance to pyrethroids. Can you say that you can advise the authorities of your country to change the molecule or the insecticide (pyrethroids) to impregnate the mosquito nets by pyrrole chlorfenapyr and neonicotinoid clothianidin in relation to this study? Explain

Comment 3: Please, author should revise his discussion accordingly from Line 407 to 460

Conclusions

Comment 1: Lines 463-463: Check whether the revised sentence: The present study showed that An. gambiae s.l. was mainly composed of An. coluzzii, followed by An. gambiae s.s and hybrids in costal Côte d’Ivoire

I suggested: The present study showed that An. gambiae s.l. was predominate by An. coluzzii, followed by An. gambiae s.s and hybrids in the three studied sites of costal Côte d’Ivoire

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Reviewer #1: No

Reviewer #2: No

Reviewer #3: Yes: Jacques Dollon NTABI MBAMA

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PONE-D-24-00912R1Assessing of species composition and insecticide resistance of Anopheles gambiae complex members in three coastal health districts of Côte d’IvoirePLOS ONE

Dear Dr. Ives,

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.

There is just one final minor comment from Reviewer #3 that needs to be addressed. Please make the requested change, or provide a justification if you believe it is unnecessary. Once this is done, I will be pleased to accept your manuscript for publication.

Please submit your revised manuscript by Oct 31 2024 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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• 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,

Luca Nelli, PhD

Academic Editor

PLOS ONE

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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.

Additional Editor Comments:

Can you please adress this final comment from reviewer #3?

Add in abstract and methodology

Commnt 1: For the An. 32 gambiae s.l. populations were primarily composed of Anopheles coluzzii (88.24%, n = 312), followed by Anopheles gambiae sensu stricto (7.56%) and the crossbreed (4.17%), I am suggested that: In the total of 672 An. gambiae S.L. analysed, 27 (4.17%) mosquitoes were not discriminated against; thus, they are part of other species of An. gambiae complex

Comment 2: If the hybrids identified here do not belong to the An. gambiae complex and are not distinct species, why do you identify those in An. gambiae complex if you used PCR? I think your work is based on the member An. gambiae? Replace the hybrids with: other species of An. gambiae complex

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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 #2: All comments have been addressed

Reviewer #3: All comments have been addressed

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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 #2: Yes

Reviewer #3: Yes

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3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #2: Yes

Reviewer #3: Yes

**********

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 #2: Yes

Reviewer #3: Yes

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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 #2: 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 #2: The authors have addressed well the comments and suggestions made on the original manuscript. They have provided a solid link between the members of the Anopheles gambiae s.l. species complex at the study sites and their insecticide resistance profiles, both in terms o insecticide susceptibility and the mechanisms of resistance they possess.

Reviewer #3: Add in abstract and methodology

Commnt 1: For the An. 32 gambiae s.l. populations were primarily composed of Anopheles coluzzii (88.24%, n = 312), followed by Anopheles gambiae sensu stricto (7.56%) and the crossbreed (4.17%), I am suggested that: In the total of 672 An. gambiae S.L. analysed, 27 (4.17%) mosquitoes were not discriminated against; thus, they are part of other species of An. gambiae complex

Comment 2: If the hybrids identified here do not belong to the An. gambiae complex and are not distinct species, why do you identify those in An. gambiae complex if you used PCR? I think your work is based on the member An. gambiae? Replace the hybrids with: other species of An. gambiae complex

Because this complex are nine species

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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 #2: No

Reviewer #3: Yes: Jacques Dollon NTABI MBAMA, PhD

Postdoc follow

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Assessing of species composition and insecticide resistance of Anopheles gambiae complex members in three coastal health districts of Côte d’Ivoire

PONE-D-24-00912R2

Dear Dr. Ives,

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.

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Kind regards,

Luca Nelli, PhD

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

Reviewers' comments: