PONE-D-22-33514Our good neighbors: Understanding ecosystem services provided by insectivorous bats in RwandaPLOS ONE

Dear Dr. Walker,

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Reviewers' comments:

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Reviewer #1: Partly

Reviewer #2: Partly

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

Reviewer #1: Yes

Reviewer #2: Yes

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Reviewer #1: Yes

Reviewer #2: Yes

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Reviewer #1: Yes

Reviewer #2: 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: The authors investigated the diet of several insectivorous bat species in Rwanda, the presence of pest species and the potential role of molossid bats as pest suppressors. I really enjoyed the manuscript and I believe it would make a great contribution to the bat conservation research. However, I do think that some aspects must be improved/clarified before publication (technical replicates, inclusion of contaminated samples in the analyses). Please find my major and minor comments below.

L64 : Please, precise what GDP stands for.

Table 1: It’s a tiny detail but it seems that the table is cropped a little bit on the right side

L151: typo, space is missing between “1” and “and”

L148: I understand that you’re are limited in page number, but maybe provided a little bit more information here (in my understanding you did a two step PCR but that’s something you can precise here for example, as well as amplicon size for each primer pair etc)

L152: Following which PCR conditions? The same than for ANML? (amplicon size etc)

153: What about the technical replicates? If you used technical replicates, please indicate how many and how the data were processed accordingly. If not, please justify why as it is highly highly recommended in metabarcoding study to overcome PCR and sequencing stochasticity.

L160: For each run, please indicate the volume of library that was loaded & %PhiX. Please also indicate whether one run was done for ANML and one for SFF or whether they were mixed. Also, please indicate how many raw reads you obtained for each run in the results.

L166: Any filtering using the NTC or technical replicates? If not, please justify your choice.

L173: I don’t understand, why did you exclude ASV of bat species? It would have been interesting to compare the ANML & SFF IDs, especially as you got mixed bat ID within samples.

L176: I’m sorry I don’t understand how the mock communities informed to set 4000 reads as a threshold, could you elaborate a little bit more on that please?

L183: Why using a different classifier approach?

L186: Please precise the tool (function/package) that you used for the calculation.

L192: Any check on geographic ranges (e.g. species not occurring in Rwanda or Africa)?

L196 : I wonder whether a quick reminder of the digestion time would be useful as you are measuring before/after (here or even in the Discussion).

L200/201 : Is the « : » missing? (like 1830 should be 18 :30 ?)

L224 : Why only 19, did the four remaining samples not amplify (SFF marker) or did they have less than 4000 reads?

L226: Same question, why 21/23 (although I counted 22 samples in your Suppl. table - maybe double check the numbers and percentages)?

L230: Please add that there are six samples showing a mix of different bat species (RWCAD00554, 655, 960, 15154, 14153, 1263). I strongly recommend discarding them when doing analysis per bat species, even when the number of reads of the second bat specie is ~low (<100) because it is impossible to know if some prey belong to one or another bat species.

L234: Be consistent with sample ID (RWCA00251, RWCAD00655 etc). RWCAD00655 identification mismatch could be due to contamination with the other bat species. Also, I would not mention this sample here as you indicated a few lines above that you have discarded it.

L241: Why giving more details on Molossids and not on the other families?

L250: Why these 18 individuals? Is the previous paragraph based on these 18 individuals? If so, please clarify.

L258: I would suggest reminding the number of samples per species when describing their diet, ex. “Nycteris spp. (n=1) was found to consume (…) “ . Something to keep in mind when discussing the results in the Discussion.

L264 : typo, Blattodea

L276: typo, “%” is missing for Odonata

L347-350 : results section?

L388: I wonder whether you could add Order/family of the pest species to the Table 3. I do think it would be very helpful to make the link with the text that describes per family and order.

Global comment on Discussion: In my opinion some parts of the Discussion are too repetitive with the Results. Your data are very interesting, but sometimes it feels like you could go a little further (e.g. L380 to L387: what could explain the differences between your study and the other studies?). Also, sample size is (very) limited for some species, it would be relevant to mention it in the Discussion.

Literature: Check reference format. Sometimes info is missing (ex #10 volume/pages) and species name should be in italic.

Fig2: Please add in the caption what the grey shaded area represents.

Fig3: Why only 17 samples are shown?

Fig4: Please, precise the unit of the Y axis (grammes). Why is only the genus shown? This analysis is by species, isn’t it?

Table 3: It would be great to have another column showing in which area (North, East, West, South) they were detected in your study to make the link with the Discussion.

Supplementary tables: Please, add a caption above both tables. Table Final results bats: Please check and homogeneize bat species name: Hipposideros ruber or Hyposiderus ruber; Rhinolophus not Rhinolofus etc. Why is Visual ID at the genus level in 2019?

“Ambiguous_taxa”? Please clarify (should be the lowest common shared assignment i.e. Chrysomya?) [sample D014153]

Reviewer #2: This is a valuable manuscript that investigates the diet of bat species in Rwanda and demonstrates, through molecular diet analysis and insect sampling in the field, that these bats consume several agricultural pests. The authors are to be commended for addressing a timely topic in a region where it is understudied compared to other areas. However, there is room for improvement, as outlined below.

1. Scientific articles should frame hypotheses and predictions rather than research questions. I urge the authors to reframe the objectives of their study by presenting well-thought-out hypotheses and measurable predictions. While the research questions are clear, reframing them in this way should not require significant effort.

2. In the introduction, better coverage of European studies would increase the international appeal of the manuscript. Recent articles from France, Italy, Portugal, and the UK cover the topic and should be included.

3. In many sections of the manuscript, the authors imply that bats "control" pests, but the term control implies a demographic effect on the prey or visible reduced damage to the cultivation. I recommend using more neutral wording such as suppression or consumption unless studies providing strong evidence of control are mentioned.

4. The authors' method of estimating the amount of prey consumed by measuring the weight of bats leaving and returning to the roost after a hunting night is not reliable. To make this approach more dependable, the authors should have used a repeated-measures analysis, capturing the same bat twice in the two temporal phases to determine the weight difference as the amount of food eaten. Since this was not done, I suggest dropping this section entirely. The manuscript already contains a wealth of valuable and interesting information. Also, please refrain from discussing non-significant results like they were significant. P>0.05 means there is no difference.

Minor comments:

1) In line 68, the authors state that the risk of bats carrying and transmitting SARS-CoV2 is "low to non-existent”, but in fact it is null – please correct

2) Line 114: clarification is needed on whether the nets were kept open for three hours.

3) Table 2: Hipposideros is misspelt.

4) Wordiness can be reduced for better readability, e.g., "Chaerephon pumilus was found to prey on all insect orders” would become “ Chaerephon pumilus preyed on all insect orders

5) Line 306: clarification is needed on how many bats were used to make this estimate (but please see my major point on this part of the study).

6) In the discussion, the authors should avoid presenting results and instead focus on discussing them. Percentages of prey occurrence should not be repeated here, they are already shown in the results.

7) In line 413, it should be “Noctuid”

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Reviewer #1: No

Reviewer #2: No

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Our good neighbors: Understanding ecosystem services provided by insectivorous bats in Rwanda

PONE-D-22-33514R1

Dear Dr. Walker,

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,

Camille Lebarbenchon

Academic Editor

PLOS ONE