PONE-D-19-33599

Leishmania  detection and blood feeding preferences of phlebotomine sand fly species common in the Mediterranean area

PLOS ONE

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Reviewers' comments:

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Comments to the Author

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Reviewer #1: Yes

Reviewer #2: No

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

Reviewer #1: Yes

Reviewer #2: 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: Yes

Reviewer #2: Yes

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4. Is the manuscript presented in an intelligible fashion and written in standard English?

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Reviewer #1: Yes

Reviewer #2: No

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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: General comments:

Clearly written and comprehensible article bringing interesting information.

Title: “…common in the Mediterranean area” – the title is exaggerated – it is definitely not an analysis of sand flies common in the Mediterranean – only in the western part and still not all common species, rather only selected ones, which were common on localities (in Sicily) where the authors made their catches (over only two seasons). PLS change “…common in Sicily”. Introduction should, therefore, also focus primarily on Sicily and, where appropriate, on southern Italy. Most parts describing the faunistic situation should be move from Intro to Discussion.

It is really interesting that it was not possible to detect/identify any reptilian blood - it would like to comment on it with at least one sentence.

It would be appropriate to include a paragraph where the authors comment on the existence of different haplotypes. There were always two haplotypes for both Leishmania species (L.t. and L.d.), as well as for unnamed trypanosome species - at least they would like to mention or comment this situation. This is particularly interesting in the case of L.donovani / L.infantum - is it really possible to assume that both species were captured/detected?

Specific comments:

26. etc. n = XXX

35. etc. (27/82) – not clear, need explanation

36. blood rat

45. why only warm-blooded ?

68. etc. X% or X %

136/137: for more info about the used traps add “see Fig. 1”

160. individually ? = 1866 DNA extractions?

246. You should add the information that obtained sequences (of L.t. and L.d.) were not 100% identical – both species form two “haplotypes”

255. … the same for two left sequences (a monophyletic cluster with another L.t. strain)

259. add information about occurrence in: unfed / blood fed / gravid

264. add Tarentola annularis from Senegal

269. two (2)

270. T. varani / T. sp.

Tab 2. n (instead no); n = XY; It would be beneficial to add info about Leishmania/Trypanosoma infection into this table.

316 (express it also in percentage … 96 % - congratulation; and 64 %)

320/Tab 3. rooster ? / hen-chicken-fowl

Fig. 4. Unify column color/pattern for presented animals (use the same pattern for the same animal); y axis – better “No. blood fed females”

Tabs 3. and 4. Move to Supplementary material ???

453-4. It is/was not clear that Trypanosoma DNA was detected only in blood fed S.m. females??? This info must be add 327/8 and PLS add also info which blood sources were detected.

Reviewer #2: The manuscript describes detection and identification of trypanosomatids and bloodmeals in Sicilian sand flies. Despite the results are interesting, their interpretation is not correct and some parts of the manuscript (mainly Abstract and Introduction) need to be rewritten.

General or major comments

1. Some sand fly species studied are NOT „commonly present in the Mediterranean area“ or “common in the Mediterranean area”. Please change the info on lines 22, 125-126, 345-346. Title is also misleading, more specific one would be useful (e.g. “Identification of trypanosomatids and bloodmeals of sand flies in Sicily”).

2. It is necessary to mention (on lines 32-33) that Trypanosoma DNA found in S. minuta clusters with reptile Trypanosomatids. Therefore, finding of Trypanosoma DNA in S. minuta should not be interpreted as a support for S. minuta role in transmission of human pathogens. There is no single evidence that Trypanosoma DNA found originate from species infecting mammals. The sentence on lines 42-43 needs to be changed.

3. Surprisingly, authors did not find a single reptile blood in S. minuta which contradicts frequent finding of reptile trypanosomes. Again, this should be mentioned in the Abstract.

4. Introduction is too long. Text on lines 104-108 and 116-119 could be deleted as most of information is repeated again in Discussion.

Table 3 should include also the numbers of females with nonidentified bloodmeals: P. perniciosus (56?) and S. minuta (12?).

Table 4 should be moved into Supplement.

Minor comments

Single finding of Leishmania DNA in P. perniciosus should not be interpreted as confirmation of the vector role of this sand fly species (line 39) or highlighting its role in Mediterranean area (lines 455-456). It may agree with well-known role of this vector with circulation of L. infantum in western Mediterranean. Please, change sentences accordingly.

The second sentence of Introduction should be changed. It is not correct in two aspects: 1. There is a single bacterial pathogen transmitted by sand flies (not “plethora” as written by the authors) and number of sand fly-borne viruses is much lower than in mosquitoes. 2. Leishmania of subgenus Mundinia are very likely transmitted by biting midges, not by sand flies.

Lines 60-61: Text should be changed to clarify that both information is valid only for western Europe.

Line 65: clarify what do you mean by “in free territories”

Lines 73-80: Part about imported cases should be reduced or changed. The reference by Fotakis et al (2019) does not concern the Italy. Moreover, this study (published in a good journal) suffers of serious mistakes: only sand fly heads were used for Leishmania DNA and despite of this nonsense extremely high percentage of sand flies were claimed as positive! Such results cannot be trusted and should not be cited. If authors decide to mention/discuss the potential spread L. tropica in Italy, then there are two recent papers about this topic published in Int. J. Parasitol, showing high susceptibility of P. perniciosus and P. tobbi to L. tropica.

Lines 363-364: P. sergenti is NOT “the sole vector able to transmit L. tropica”. The vectorial role of P. arabicus is well described in many papers since 2003. Moreover, there are two papers on other sand flies as potential L. tropica vectors published this year, see above.

Lines 383-385: the information about potential of L. tarentolae to infect humans is misleading and should be deleted (together with both references).

Finding of reptile Trypanosoma in S. minuta is not unexpected (as said on line 404).

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Reviewer #1: No

Reviewer #2: No

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Identification of trypanosomatids and blood feeding preferences of phlebotomine sand fly species common in Sicily, Southern Italy

PONE-D-19-33599R1

Dear Dr. Brianti,

We are pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it complies with all outstanding technical requirements.

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Vyacheslav Yurchenko

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

Authors have adequately addressed all the reviewers' concerns and the manuscript can now be accepted for publication.

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 #1: 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 #1: Yes

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

Reviewer #1: N/A

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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 #1: Yes

**********

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 #1: 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 #1: I am completely satisfied. The authors have adequately addressed all my comments and I feel that this manuscript is now acceptable for publication.

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Reviewer #1: No