Effectiveness of a New Self-Marking Technique in Aedes aegypti under Laboratory Conditions

Round 1

Reviewer 1 Report

This is a very well-written paper and describes a very simple but useful technique. There are some extremely minor changes needed for the English, otherwise most everything looked to be ok. Have other Day-Glo colors been evaluated? Sometimes many small things in the environment will fluoresce green. They cannot be easily confused with a dust particle but they can interfere at times. Arc Yellow is a good choice to try. Very fine dust and bright color under the UV light. I agree that the small amount of dust on a male minimized the possibility of a transfer to females.

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Author Response

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Reviewer 2 Report

Title: Effectiveness of a new self-marking technique in Aedes aegypti under laboratory conditions

Authors: Gorgui Diouf, Momar Talla Seck, Assane Guèye Fall, Mireille Djimangali Bassène, Biram Biteye, Mame Thierno Bakhoum and Mamadou Ciss

Summary:

This paper addresses an important and yet somewhat neglected area of sterile insect technique (SIT). It builds on existing manuscripts in which sstandardized methods of marking for various Aedes. and Anopheles species have been developed and tested. Although results detailed within appear promising, I would advise that several more replicates need to be performed before more certainty can be applied to these results. I agree that self-marking negates some of the detrimental impacts imposed by chilling prior to marking, but it is finding a consistent and reliable method that has proved elusive thus far. I think the images of the self-marked males do not show a strong indication of marking. Perhaps, yes on day 1, but as time goes by, it would appear to me that the mark would be lost. I would be tempted to push even higher dust concentrations and think that this is a good preliminary paper but more work is needed before this can be touted as a viable self-marking method.

General Comments:

• Figure 1 – Check the English in the text boxes throughout. This figure may benefit from the addition of pictures at each step to aid visualization.
• Figure 2 – The axis titles and numbers are barely legible. This must be improved before publication can be considered.
• I would really like to see at least at image of the dusted pupal cups.

Specific Comments:

Introduction

L32-36: This sentence would benefit from being split into two more succinct ones.

L45: Remove “been”.

L61: Change “be safe for the environment” to “environmentally friendly” or the like.

Materials & Methods

L117-118: Change beginning of the sentence from “then” to something like “subsequently”, or “upon emergence, adults will be marked/ coated with fluorescent powder”.

L126-134: How many reps? The methodology implies that 100 individuals were scored (3 times) but it doesn’t say if this was from the same dusted pupal pot or were there several repetitions of each dust concentration. If the samples do all come from the same container, then this is not true replication as they would all then be from the same cohort. I'd like to see more rounds of each concentration tested to ensure it wasn't just a one-off result. If indeed the 3 reps are from 3 different containers, then state that clearer within the text.

L135: What was the reasoning behind only testing the highest dust concentration? Did you test the lower ones and find nothing? If so, it is important to state this either way.

Results

L176: Try to shorten this section title, it’s a little too wordy.

L179: “the all” should be “all the”.

L181: Add “present” to read “Fluorescent powder was present on…”

L190: You state dust transfer only occurred from marked males to unmarked females at the highest dust quantity. How was transfer scored and how many females were marked in the end? This is an important distinguishment to make.

Figure 2: Is this a consistent image across the males? what concentration is this from? Are the same parts of the body marked consistently? What day is this from? You need to be more descriptive here.

Table 1: What does the letter c denote? Normally lower-case letters are used to denote significant differences between columns and or rows, but only having one? Either add them all in or remove this one. Also, refer to it in the Figure legend which is currently absent. I also question the validity of the Mean column at the end.

Table 2: As above, no figure legend.

Discussion

L217: such “an” approach.

L223: This publication did not find chilling prior to dusting to be detrimental as the controls were also chilled and subject to the dust method, just not dusted. It is dust “amount” that is the most critical factor to subsequent adult quality.

Author Response

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Author Response File: Author Response.pdf

Reviewer 3 Report

The experiment is okay and looks like there are legitimate results but there are too many gigantic holes in the paper, which need fixing before it can be reviewed effectively.

Point 1: the introduction text looks like it's been lifted from somewhere else, talking about 4 different mosquito borne diseases AND insecticide resistance AND vector control strategies in relatively vague terms but then make very specific claims without accurate references. Authors either do not understand how to reference correctly or are writing general knowledge and throwing references in seemingly at random. Arboviruses, insecticde resistance, vector control, are not the subject of this paper. The authors can talk about these diseases as they exist but a detailed explanation of their outbreaks and impacts on human health are not needed for this study. It's really unnecessary here, the audience should be very aware of the status of mosquitoes and mosquito borne disease if they are reading this paper and really only a brief introduction along the lines of 'medically significant Aedes species are prevalent across the globe and threaten to spread arboviral diseases in changing climates (ref: https://journals.plos.org/plosone/article?id=10.1371/journal.pone.0210122).

Point 2: details of the fluorophore used completely absent from the manuscript. Some details, even if it's proprietary, are needed at least to know the wavelengths of activity (excitation / emission spectra) and accompanying safety documentation. If it's proprietary then the company needs to give it a specific name like 'compound 1' which the company needs to have referenced for future citations, otherwise it's impossible to use this research in any context.

Point 3: Figures have to be significantly improved, you can even see the MS word formatting in the figure 1 (bottom right). Text is illegible in many places.

Here are some example edits that need to be made line numbers given on the left:

16 - remove 'consistently' it doesn't fit in the sentence and it also the 'consistency' of the method will be visible in the results.

17 - is this actually a fluorescent compound? I.e. is it a compound which absorbs an electromagnetic photon within a specific wavelength range and releases another lower energy photon?

17 - please specify what the powder is exactly, far too vague. What is the chemical specifically. If you need to get this information from the company then do so, there's no way you can publish a paper using an unknown substance, or without clearly marking it as a safe compound with some specific link to the company which can provide MSDS for the material and comment on it's potential for being used on animals which are then released into the wild. If there's toxicological impacts of the dye, this may not be considered suitable outside of laboratory conditions and must be clearly specified here.

20 - how were marked mosquitoes assessed, manual inspection, imaging? If this powder is fluorescent was a fluorescent detection system used?

32 - References used are poor and do not refer to the claims made. The following sentence makes huge number of claims with no references: 'In the last decades, major outbreaks of DF [1-4], yellow fever (YF) [5,6], Chikungunya [7-10] and Zika [11,12] have affected the populations of many countries causing deaths and other disabilities such as lasting sensory-motor damage, impaired cognitive and language development, as well as neurodevelopmental disorders of children affected by the Zika epidemic'

• Specific references needed PER DISEASE for each claim made unless a reference provided specifically encapsulates the totality of the statement
• References list random countries and many references such as reference 4 refer to insecticide resistance and are only secondarily related to dengue. Good references exist in the ref 4 but are sub-ref: https://apps.who.int/iris/bitstream/handle/10665/241453/WER8445_469-469.PDF
• No references for each impact of each disease. But this sentence isn't relevant to the study anyway.
• 'countries' is vague and not supported by references. Why not just list Senegal specific outbreaks?

42: no references again but a major claim about tradition use of vector control, also no definition of vector given, but mosquito-borne disease is, so the terminology jumps around.

43: 'abusively' opinionated language doesn't fit in a scientific paper. Also the references here show the spread of insecticide resistance in geographically distinct areas, it's such a huge area of research there have been many reviews on the subject, specific studies do this claim no justice (see: https://scholar.google.co.uk/scholar?q=mosquito+insecticide+resistance+review&hl=en&as_sdt=0&as_vis=1&oi=scholart)

47: ref 17 about deep sequencing, specific resistance refs or reviews for each drug needed. Again I will say that this study is just about mosquito surveillance which is NOT the same as mosquito control or monitoring for drug resistance, so these details are really not suited for this manuscript.

49: references not relevant.

Author Response

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Reviewer 4 Report

The paper brings an interesting idea and a new way to mark mosquitoes, specially for MRR experiments.

The introduction section needs a better organization between MRR and SIT programs, since they have very distinct objectives.

The paper also lacks a comparison of a standard method side-by-side with their innovative way to demonstrate the benefits it has. And finally, it is only in the end that the authors state the need to test the method under field or semi-field conditions, although the manuscript says that is ready to go.

Comments for author File: Comments.pdf

Author Response

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Round 2

Reviewer 2 Report

Dear authors. I'd firstly like to thank you for taking all of my comments and suggestions on board and for making all of the noted suggested changes. I am pleased after reading through the new manuscript with the overall layout and the improvements to all of the Figures. The manuscript flows much better now and I feel that in its current form, it is both publication worthy and ready. Good work and I look forward to seeing more innovative research which boosts the mosquito SIT field further towards operational level. Well done!

Author Response

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Reviewer 3 Report

Same image in figure 1 and figure 3. Combine figures, no need for them to be separated. 

The following data needs to be shown in figures in the paper or in tables
"oncerning the persistence of the florescent powder on mosquitoes, observation at 228
time of death showed that the powder persisted in 100% of male mosquitoes (n=630) 229
throughout the duration of the experiment (n= 20 days; Fig. 3)." Figure 3 is just a single picture already presented before in the manuscript, ideally show in a table or figure the number marked at the start and the number at set timepoints throughout if data exists. Otherwise it cannot be commented on as it's an opinion and not a scientific finding without actual measurements and data analysis. Also n=20 days doesnt make any sense. N is the number of individuals not the number of days, line 230.

Various places where the font / typeface changes in the manuscript and there's blank pages.

Author Response

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Reviewer 4 Report

Thank to all authors to revise and prepare an updated version, addressing or clarifying some of my comments.

My last comment is more related to the introduction, that has the epidemiological path to later connect with the SIT and the use of MRR. I would remove or reduce to one or two sentences and try to keep more about the objective and challenges of marking mosquitoes and why it is needed.

Overall, the manuscript brings a very interesting and useful piece of information, that from my perspective, still have room for improvement to become a more solid and robust publication.

Author Response

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Round 3

Reviewer 3 Report

Thanks for the rebuttal, I would still recommend combining the figure 1 and figure 3 because the issue is it's the same data presented twice. Each figure should be an independent experiment in a scientific paper, this is the same experiment and the same exact data, so it can be presented as 3 images with the third being at 20 days, then you can still mention it later but it just leads readers to confusion when they see the same image twice. On first read it forces you to go back and then you lose confidence in the science. For the interest in making sure readership has confidence in the data and the quality of presentation I would recommend combining the figures, it doesn't make a huge difference but it would just be a bit cleaner.

Author Response

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