microRNA profile of Hermetia illucens (black soldier fly) and its implications on mass rearing

The growing demands on protein producers and the dwindling available resources have made Hermetia illucens (the black soldier fly, BSF) an economically important species. Insights into the genome of this insect will better allow for robust breeding protocols, and more efficient production to be used as a replacement of animal feed protein. The use of microRNA as a method to understand how gene regulation allows insect species to adapt to changes in their environment, has been established in multiple species. The baseline and life stage expression levels established in this study, allow for insight into the development and sex-linked microRNA regulation in BSF. To accomplish this, microRNA was extracted and sequenced from 15 different libraries with each life stage in triplicate. Of the total 192 microRNAs found, 168 were orthologous to known arthropod microRNAs and 24 microRNAs were unique to BSF. Twenty-six of the 168 microRNAs conserved across arthropods had a statistically significant (p < 0.05) differential expression between Egg to Larval stages. The development from larva to pupa was characterized by 16 statistically significant differentially expressed microRNA. Seven and 9 microRNA were detected as statistically significant between pupa to adult female and pupa to adult male, respectively. All life stages had a nearly equal split between up and down regulated microRNAs. Ten of the unique 24 miRNA were detected exclusively in one life stage. The egg life stage expressed five microRNA (hil-miR-m, hil-miR-p, hil-miR-r, hil-miR-s, and hil-miR-u) not seen in any other life stages. The female adult and pupa life stages expressed one miRNA each hil-miR-h and hil-miR-ac respectively. Both male and female adult life stages expressed hil-miR-a, hil-miR-b, and hil-miR-y. There were no unique microRNAs found only in the larva stage. Twenty-two microRNAs with 56 experimentally validated target genes in the closely related Drosophila melanogaster were identified. Thus, the microRNA found display the unique evolution of BSF, along with the life stages and potential genes to target for robust mass rearing. Understanding of the microRNA expression in BSF will further their use in the crucial search for alternative and sustainable protein sources.

Thank you so much for your review and comments. We have gone through and made the changes and edits you have suggested.
1. Please find the attached word file with some track changes.
We have gone through and corrected each of the track changes found in the attached document.
We have gone through and made sure they are all italicized.
Reviewer #2: In this paper authors analyzed the profile of microRNAs in all the developmental stages of Hermetia illucens. The aim was to relate them to possible functions in order to understand, in future works, the specific gene regulation to improve mass rearing techniques. I have some doubts that must be addressed, especially regarding the comparison among different life stages, it is not clear, and it seems that in many points sentences seem to be contradictory.
We have made addressed each of the issues raised below and tried to clarify our analyses between life stages. Fixed Lines 61-68: "The black soldier fly (Hermetia illucens; BSF) is a species found worldwide and can be used as a sustainable animal and fish feed (6-9). Depending on the organic substrate the black soldier fly larvae (BSFL) are fed and the extraction process , they can contain a range of 40-60% protein, up to 47% lipid , and 3-8% chitin in prepupae (10-15). This makes them not only a good protein source, but shows potential for a variety of derived products, and has minimal environmental impacts. Due to these facts, and that they are not a nuisance species, or mechanical disease vector (16, 17), they are an ideal candidate for large scale rearing for food sustainability and waste reduction (18, 19)." We have added in references suggested, and a range for the composition of prepupae that have been found on different substrates.
3. RESULTS: In the sentence "A total of 44 miRNAs (23% of the total microRNAs) showed differential expression between at least two life stage with an LFC" write "stages" Corrected Line 128: "A total of 44 miRNAs (23% of the total microRNAs) showed differential expression between at least two stages with an LFC of ±2."

DISCUSSION:
What does it mean "Focusing on arthropod species only"? Which species? I don't understand as the authors said that they performed comparisons with arthropod species.
We changed the sentence at lines 172-173 as follows: "Focusing our miRNA discovery pipeline on known arthropod species miRNA only, allowed for a stronger comparative analysis of potential microRNA function in BSF." "Only three conserved microRNAs were found to be specific to a single life stage: hil-miR-6-2-5p (Egg), hil-miR-3884-3p (Larva), and hil-miR-12418-3p (Pupa)." In the adult stages there are exclusively common microRNAs? Moreover, as the authors identify stage-specific microRNAs, I suggest to perform also a comparison among common microRNAs in all the analysed stages and not exclusively Egg to Larva, Larva to Pupa, Pupa to Female, and Pupa to Male.
We have changed that sentence to be accurate to the conserved miRNA. An error had occurred. Line 189-190: "Seventy-four conserved microRNAs were found to be specific to a single life stage, while only 27 were found across all life stages (S1 Table)." We are not sure about the second statement on performing a comparison among common miRNAs in all the "analysed stages and not exclusively…" As this is a baseline study showing profile of these novel miRNAs, we did not think a comparison of the miRNAs without a high LFC that were found commonly across all life stages would be significant for publication. We included them and their expression values in the supplemental tables, S3 & S4.
We removed "higher" from the text for clarity at line 191. The adult stages had specific unique miRNAs, but the female adult had one unique miRNA not found in the male adult. We could combine them so it just states "adult" but felt this was more specific.
"However, these 10 unique microRNAs provide insight into the egg, pupa, and adult life stages of the BSF and can be used to compare the developmental regulatory differences of microRNAs to those of other insect species (26)" and what about the larval stage?
"In all three cases the females had expression whereas the males had none." What does this sentence mean?
Line 201: We removed this sentence.
"miRNA target prediction" why did the authors choose to explore in detail only 3 microRNAs?
The three explored were selected for their direct implications to mass rearing which we thought had the most impact on the thesis of the paper. We added the following at line 220: "the target genes discussed below were selected for having the greatest potential impacts on the mass rearing of BSF". This sentence needs a reference "The microRNA has been seen to increase in expression over the third larval instar, with highest expression levels in the pupa stage, in D. melanogaster." Line 236: Added reference 44 at the end the sentence.
"Once validated, the microRNA can be harnessed in breeding protocols to improve mass rearing of BSF." please provide the readers concrete ideas of using these microRNA to improve BSF mass rearing.
Lines 249-252: Once validated, the microRNA can be harnessed in breeding protocols to improve mass rearing of BSF by understanding how they adapt to different environments (28, 46, 54) and is being used in the grape and olive industries (55, 56)." Why did the authors choose to perform specifically these comparisons? "Five different paired life stages were compared: Egg to Larva, Larva to Pupa, Pupa to Female, and Pupa to Male." Line 282: We have changed the wording in the methods section by removing the word "paired" to show that we did not only compare Egg to Larva, Larva to Pupa, Pupa to Female, and Pupa to Male. In fact, we did all versus all comparison. The Log fold change of all these comparisons for the Unique miRNA (added as supplementary Table 3 and file labelled S3 Table), and the evolutionary conserved miRNA (added as supplementary Table 4 and file labelled S4 Table).

METHODS
We have subsequently edited Figure 2 so that it only shows the statistically significant fold changes for the unique miRNAs.
6. FIGURE: "Finally, 21 microRNA were identified with an LFC of ±10 (11% of total miRNA). These included 18 miRNAs conserved across arthropods and 3 miRNAs unique to BSF (Figure 1; represented. Please fix this, moreover, looking at the image, I supposed a comparison among all the samples. As I wrote before, it is not clear in the main text.