Anopheles maculipennis Complex in The Netherlands: First Record of Anopheles daciae (Diptera: Culicidae)

Round 1

Reviewer 1 Report

This is a nice and interesting study to study on the distribution of the 24 members of this complex in the Netherlands, and reports on the presence of Anopheles daciae. The authors analyzed 541 specimens of 26 An. maculipennis s.l. from 161 locations covering the entire territory. The methods were adequate and the text were smooth. I recomanded to accept this manuscript.

Author Response

Dear reviewer, thanks for the review of our manuscript and for your kind words.

Reviewer 2 Report

The article provides relevant information on the occurrence of species of the Anopheles maculipennis complex in the Netherlands and also records, for the first time, solid evidence of the occurrence of the species Anopheles daciae in that country, through an ITS2 analysis. Although it is a descriptive study, the sample size is remarkable and the association of the different species with the different Land Cover Class is very interesting and the set of results justifies the publication

However, some improvements could be added:

Material and Methods

- the addition of a map depicting  the sampled locations would greatly enrich the reading and the detection of eventual patterns of species ocorrence and Land Cover Classesresults;

-the methodology used in the characterization of the habitat could be briefly explained.

 

Results:

I failed in an attempt to access the sequences deposited in Genebank, with the codes provided.

Although there is a legend for figure 3, the figure does not appear neither in the text nor in the supplementary material.

 

Discussion

The data presented in Figure 1 and Table 1 are little explored in the Discussion.

L.252 the term "randomly selected" should be replaced because  selected and  randomly are antagonistic therms

Author Response

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Author Response File: Author Response.docx

Reviewer 3 Report

This study aimed at investigating the distribution of the members of the Anopheles maculipennis complex in the Netherlands using ribosomal internal transcribed spacer 2 (ITS2) sequencing. First, the study presents the first report of An. daciae in the Netherlands. Second, the authors showed that An. messeae is the most frequent species in the country during summers and occurred in sympatry with An. daciae in winters. Third, by using land cover use data, the authors also showed that the occurrence of An. messeae was significantly higher in artificial habitats.

In my opinion, the authors collected an impressive number of specimens from most Dutch regions to improve our understanding of the distribution of the An. maculipennis species throughout the Netherlands, an interesting topic that could be of interest to the readership of Diversity. The scientific context, objective, methods, results, and discussions are clear. However, some points need to be addressed that are outlined below.

First, as the manuscript does not currently provide any conclusions, I recommend adding one paragraph which will highlight the main conclusions of the study. For example, this study contributes to a better estimation of the risk of mosquito-borne disease in the country. Second, as the Introduction section of the manuscript is very succinct, providing a more detailed description of the scientific context will significantly improve the quality and impact of the study. Third, some improvements are required regarding the organization of the manuscript.

ABSTRACT

l25: I suggest quickly explaining/referring that Anopheles daciae is part of the species of the An. maculipennis complex, e.g. “…the Netherlands, including Anopheles daciae that has recently been found in countries bordering the Netherlands”.

l35: I abstract does not currently bring any discussion/conclusions. I recommend adding at least one sentence at the end of the abstract which will provide the main conclusions.

l36-37: As “Anopheles maculipennis” is already part of the study title, I advise removing it from the keyword list.

INTRODUCTION

l43-47: These two sentences seem contradictory, i.e. “Four members of the Anopheles maculipennis complex (= s.l.) are reported as present” VS. “This latter species is not mentioned as present in the Netherlands”. To avoid confusion, I suggest rephrasing.

l72-75: As An. daciae is highlighted in the present study, I recommend providing more information about what is currently known about this species in the Introduction. For example, is this species capable of carrying pathogens of medical importance such as malaria? Is its ecological niche different than the other species of the complex (e.g. overwintering sites…)? Similarly, I would also consider providing more information about the other species of the complex that are present in The Netherlands (e.g. their niches, such as brackish waters, artificial habitats…; some of this information is currently described in the Discussion section, e.g. l256-262…).

MATERIALS AND METHODS

l82-83: I suggest adding a short sentence describing how specimens were identified as part of the Anopheles maculipennis complex. For example, “…morphologically identified belonging to the Anopheles maculipennis s.l. (REF)”. This information is provided in another paragraph (l96-98), but some re-organization is required to clarify the methodology, i.e. 1) sampling, 2) morphological identification, 3) number of Anopheles maculipennis specimens.

l111, 129, 130, 135, 141 …: Here, and everywhere else, I recommend providing the version of each software or program (e.g. Genious Prime v.x, ArcGIS v.x…).

l121: As An. funestus is a complex of species, I advise using the full species name of the ITS2 sequences which was used as an outgroup for the phylogenetic analysis (e.g. An. funestus sensu stricto). This comment should also be applied to figure 1 (i.e. tip label and/or caption).

l122-125: As new results should not be described in the Methods, I suggest removing the end of that sentence, i.e. “and in the present survey”.

l130-131: I would consider indicating that Spider is a package of R, e.g. “…the R v.x package Spider v3.6.2 [25,26]’. The version of R which was used should also be provided.

RESULTS

l144-145 and l181-183: In my opinion, Figure 2, i.e. the map of The Netherlands showing the distribution of the different species, should also show the land cover to illustrate one of the main results of the study.

l145-148: I would consider removing this information which is already indicated within the Data Availability Statement.

l154, 176, and 180: Replace “Figure 2” with “Figure 1”.

Figure 1: To avoid confusion, I suggest clarifying the first sentence of the caption (i.e. list all the species that belong to the Anopheles maculipennis complex). Moreover, I recommend adding a symbol on the tip labels which indicates the species which are present in The Netherlands (e.g. “Anopheles messeae*”; caption: “*species reported in The Netherlands”).

DISCUSSIONS

l221-223: I would consider adding a perspective about using whole-genome data to explore these questions with more confidence (i.e. find extended molecular evidence of species divergence within the complex). Some examples can be taken from other vector complexes, e.g. these approaches were/are already used for the main malaria vectors in Africa, i.e. the An. gambiae complex and the An. funestus complex.

l238-242: These sentences, which are interesting results, should be moved to the Results section.

l271: I suggest finishing the manuscript with a small paragraph summarizing the main conclusions of the study.

Author Response

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