PONE-D-21-36790Speculation on the possibility for introducing Anopheles stephensi as a species complex: secondary evidence based on odorant-binding protein 1 intron I sequencePLOS ONE

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( This study is supported by the National Research and Development Plan of China (No. 2016YFC1200500) and 111 project (B12003).

Prof Wu Zhongdao has received this funding.)

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Reviewer #1: Page 3: Authors claim "AnsteObp1 as a robust genetic marker for rapid and accurate

discrimination (taxonomic identification) of the An. stephensi species complex".

Page 11: " Finally, our investigations,

based on egg morphology and sequence analysis, endorse the use (independent) of the

AnsteObp1 intron I sequence as a new molecular tool for quick and reliable identification of all

the three biological forms of An. stephensi."

First: this claim had been proposed before on 2015, so this is not a "new" molecular tool.

Second: In this study only An. stephensi mysorensis lab strains were used. No wild anopheles and no type and intermediate forms were used.

Third: how would you use this marker to separate Type and Intermediate forms from Mysorensis?

The answer to this question is not clearly mentioned in the manuscript.

Phylogenetic trees in Fig.2-3 -4: Comparison between sequences is not clearly mentioned in the phylogenetic tree by name/region.

Reviewer #2: The manuscript by Jehangir Khan and coauthors tested the application of SNPs in the Anste Obp1 gene described in previous work for identification of biological form in the laboratory colony from China. The study is well done but it has quite a limited scope since it focuses only on one mosquito colony.

The title is not appropriate for this work as it does not introduce a new marker for species discrimination. I suggest changing the title to “Identification of a biological form in the Anopheles stephensi laboratory colony using the odorant-binding protein 1 intron I sequence.”

The authors should clearly state the limitations of their work.

First, the test was limited to a single laboratory colony. Second, the authors have not developed a rapid PCR-based test and identification of the biological form requires sequencing, which is labor- and time-consuming. Third, only a single (the last) SNP discriminate all three forms: “C” in mysorensis, “T” in intermediate, “G” in type. Other polymorphisms are shared between at least two forms.

The authors wrote that they counted for the number of ridges under stereomicroscope, however Figure 1 shows a scanning electron micrograph. Please, clarify and correct this, and also provide scale bars.

Please, correct the following:

Abstract:

An. stepehnsi species complex - change to - The An. stephensi species complex

vector competence (malaria) and ecology - change to - vector competence to the malaria parasite and

ecology.

To identify the species complex of our An. stephensi insectary colony - change to - To identify the members of the species complex in our An. stephensi insectary colony

Eggs were collected from individual mosquito - change to - Eggs were collected from individual mosquitoes

sequences in GenBank using MEGA vx. - change to - sequences in GenBank using MEGA 7.

An. stephensi sibling C - change to - An. stephensi sibling species C

Main text:

Despite efficient controlling strategies for malaria, An. stephensi is increasing in its geographic range (add reference)

chromosome karyotypes [30,31,32,33], cytogenetic properties [30,31].

Recently, AnsteObp1 is reported as a new marker for identification of the Asian main malaria vector, An. stephensi (add reference)

amino acids sequences of AnsteObp1among these members - change to - amino acids sequences of AnsteObp1 among these members

Wolbachia-based, Gene drive etc. - change to - Wolbachia-based, gene drive, etc.

Others demonstrated variations in the reproductive capacity within these biological forms of An. stephensi [34]. - change to - Others demonstrated variations in the reproductive capacity within An. stephensi [34].

Table 1: Size of ANSTEOBP1 is 900 bp, but it should be 845 bp.

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Reviewer #1: No

Reviewer #2: No

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Identification of a biological form in the Anopheles stephensi laboratory colony using the odorant-binding protein 1 intron I sequence

PONE-D-21-36790R1

Dear Dr. Khan,

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Kind regards,

Igor V. Sharakhov

Academic Editor

PLOS ONE

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