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Peer-Review Record

Black Soldier Fly Gut Microbiota Resists Invasion by Bacillus subtilis 168 and Pseudomonas putida KT2440

Appl. Microbiol. 2025, 5(3), 82; https://doi.org/10.3390/applmicrobiol5030082
by Joachim Carpentier, Grégoire Noël, Bo Li, Frédéric Francis and Rudy Caparros Megido *
Reviewer 1:
Reviewer 2: Anonymous
Reviewer 3:
Reviewer 4:
Appl. Microbiol. 2025, 5(3), 82; https://doi.org/10.3390/applmicrobiol5030082
Submission received: 30 June 2025 / Revised: 14 August 2025 / Accepted: 16 August 2025 / Published: 18 August 2025

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

The authors present a generally sound experiment to answer a reasonable question. I have made a few comments on the MS itself, but these are the biggest issues.

 

The statistics must be reported thoroughly. P-values alone are not suitable. For the non-microbiome analysis, the analysis used is not clear.

 

There needs to be text description prior to each figure and as such, figures cannot follow each other directly such as figure 3-5.

 

When reporting beta diversity, please perform a permanova to assess the significance of the differences between the groups. Then the p-value and stress values need to be quoted.

 

Line 289-295: The argument about the effect of B. subtilis on weight is not convincing enough.

 

Please use alternative terminology than “concerning xyz as this is used quite a few time and becomes repetitive”.

The discussion requires a limitation section.

Comments for author File: Comments.pdf

Author Response

Please see the attachment.

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

This paper describes the effect of feeding substrate spiked with Bacillus subtilis and Pseudomonas putida on the black soldier fly larvae and their microbiome. This topic is interesting, since bioconversation of agricultural waste to feed is an important step towards the circular economy and waste reduction. In addition, our knowledge on the impact of P. putida on BSF is very limited, so studies like this are important in discovering the benefits and disadvantages of microbes on the insect species produced for feeding or food purposes.

The text is well written, only a few minor typos were found. Self-citation is not relevant, only a small proportion of the cited papers were written by the authors of the present article. The same is true for the MDPI journals, only a few of the references belong to them. The level of similarity is low (see attached file).

My questions are as follows:
1. What was the origin of the bacterial strains? Do you know anything about their metabolic activities? Some B. subtilis and P. putida strains produce a wide range of antimicrobial proteins and a couple of metabolites, though, in these experiment there were no traces of such kind of activities.

2. You describe that "Larval development occurred over 11 days, with individuals from the CFB treatment being significantly lighter on days 7 and 11 compared to those reared on the CF and CFP diets (Figure 1)." (line 228-229) and "Between days 11 and 18, larvae evolved into prepupae. B. subtilis and P. putida produce prepupal weights identical to those of CF (Figure 1)." (line 232-233). It's in contrast with the findings of Yu et al. (2011; https://doi.org/10.1603/en10126) who found that the B. subtilis S15 strain (isolated from BSF larval gut) supplement resulted in a serious weight gain in the prepupal stage and the adult body length in the treated groups was significantly greater than in the control group.

3. Why P. putida was chosen as a model microbe? Its true, that there is a limited knowledge about its effect on BSF, but this species is neither serious pathogen nor really important in terms of feed safety. And, as it was mentioned above, there is no information about its origin or metabolic and antimicrobial activities, which might make it somewhat less interesting in terms of feed investigation.

Comments for author File: Comments.pdf

Author Response

Please see the attachment.

Author Response File: Author Response.pdf

Reviewer 3 Report

Comments and Suggestions for Authors

The paper is a significant contribution to the field of studies of functional roles of insect intestinal microbiomes. The case of black soldier flies is of special importance as this dipteran is exploited in a variety of biotechnological applications, from waste bioconversion to feed and food production. Moreover, these different directions of exploitation can be merged into a single pipeline, but this may pose threat to the consumers due to bacterial contamination. Will these products be safe? How the addition of bacteria influences the insects and what happens to the bacterial load? To answer this questions, detailed studies using microbiome analysis are required. In the manuscript under review, a substantial body of evidence has been accumulated and comprehensive explanations of possible reasons are given, describing the processes of bacteria-insect interactions. Meticulous microbiome tests are coupled with experimental introduction of two distinct bacterial species belonging to different taxonomic, ecological and functional groups. Due to this approach, differences could be found dependent upon biology of these bacteria representatives. It will be interesting to a broad audience, including specialists in entomology, microbiology, biotechnology, etc.

The references are sufficient, the methodology is adequate, the results are comprehensive and conclusions are convincing. The paper is well-structured and relatively clearly written. However, from the stylistic point of view, many terms and expressions are utilized repeatedly. Synonyms are strongly recommended.

Other comments are listed below.

L29: insect taxonomy is welcome

L32-33: it's either "at the end of the larval stage" or "in 11-14 days". If you want to highlight both points, split and rephrase

L35: the house fly is the single species, multiple form is odd here

L50: protozoa = protists

L55-70: consider insects not only as food (for human consumption) but also feed (in fishery, poultry etc)

L71: bacterial taxonomy is appropriate here

L72: aerobic = anoxybiont

L90: change to past tense for uniformity

L144: change to past tense for uniformity

L231: what are the “mean larvae”?

L244 and possibly elsewhere: use italics for genus and species epithets in Latin

L310-311: at first mentioning, the term "antimicrobial peptides" requires abbreviation which is used further

L316: which caterpillars are implied? How this relies to the study?

L319: microflora = microbiota

L360-361: what does it mean “abundance level were expected”? What exactly was expected?

L379: what does it mean “Lactobacillus spp. were correlated”?

L395-399: matrix which disrupts; cells which provides – please rephrase and break the sentence down for clarity

L418: Lactobacillus is a genus, while Lactobacillus spp. is a set of species within this genus

Comments on the Quality of English Language

L216: odd semicolon at the end of subheading

L258: as being - one of these two words is odd

L320: structure varies with feeding different diets = structure varies depending upon diet

L355: English word “abundance” doesn’t require plural form, even when applied to multiple objects

L356-357: punctuation should be checked

L361: level … were – a possible mixture of singular and plural forms

L385: range = ranged

L393: the term “bacteria” is plural so the use of indefinite article is inappropriate

L396: singular subject vs plural predicate

L413: the semicolon is odd

Author Response

Please see the attachment.

Author Response File: Author Response.pdf

Reviewer 4 Report

Comments and Suggestions for Authors

This paper tested the effects of adding pathogenic Pseudomonas and Bacillus to BSF substrate. it found the insect's gut microbiota did not change with the presence of these pathogens in the diet, which is a noteworthy find. The Pseduomonas was excluded from the gut [destroyed?] while the Bacillus was briefly carried in the gut.

The title implies that gut microbiota stability boosts resistance to bacterial invasion. One could just as easily say resistance to bacterial invasion boosts gut microbiota stability. You are ultimately saying "A causes A," as a stable microbiome implies resistance to invasion. Determining the direction of causality is impossible from your data, so I strongly suggest changing the title to something descriptive and specific: "Black Soldier Fly Gut Microbiota Resists Invasion by Bacillus subtilis and Pseudomonas putida"

Double-check your line spacing in the paper, as it is not consistent.

Introduction
38-49 delete. Focus this paper on BSF gut microbes.
49-61 Rephrase to only cite papers about the BSF microbiome, and go into more detail. For example, in line 86 you mention a "core microbiome," but you haven't mentioned it before. A few sentences about what the core microbiome typically comprises would be useful in the introduction.

Materials & Methods
-Where did the bacteria come from? What strain?

Results
-239 "By day 7, all modalities differ significantly." Maybe replace "By" with "On," as "by" implies they remained significantly different from then onwards. On Day 11, CF and CFP look identical, so I don't think they differ significantly from each other, and on D18 they are all identical.
-Figure 2 is quite striking! You explain it well in the discussion.
-Table 3 is excellent. A clear representation of what a core microbiome looks like. 

Discussion
298-300 It is not clear who the subject of this sentence is: who is isolating, promoting, and managing strains? Rewrite it in the active voice.

Author Response

Please see the attachment.

Author Response File: Author Response.pdf

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

Not all of the issues have been addressed although most comments have been attended to. 

Author Response

Comment 1 : Not all of the issues have been addressed although most comments have been attended to. 

Response 1 : it is now done.

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