RNA-Seq Analysis on the Microbiota Associated with the White Shrimp (Litopenaeus vannamei) in Different Stages of Development

Round 1

Reviewer 1 Report

A brief summary

The manuscript had made the analysis of the microbiome with Litopenaeus vannamei in the different development stages. And the functional and metabolic analysis indicated that the joint metabolism between microbiome and L. vannamei may be present. But it needs more experiments to approve that.

General concept comments

(1) The manuscript needs line number to be easily modified.

(2) The abstract should be more concise. And in this section, the “and” in the “Proteobacteria, Firmicutes and Actinobacteria” should not be italic.

(3) The introduction should provide some known description about the microbiome in the L. vannamei or other shrimps.

(4) In the Materials and Methods section, this sentence “The ribosomal RNA depletion of each sample was performed for eukaryotes with the Low Input RiboMinus Eukaryote System V2 (AMBION) and prokaryotic ribosomal RNA depletion was performed with the Ribominus Transcriptome Isolation Kit, Bacteria (INVITROGEN) according to the manufacturer’s instructions.” was unclear. I guess the ribosomal RNA depleted samples was the eukaryotic and prokaryotic ribosomal RNA depletion samples.

(5) In the Result section, Figure 1 did not show all the phyla and Class found in the L. vannamei. The color was also not clear for us to identify each phyla or class. This problem also exists in the figure 2-4.

(6) In the discussion section, the description about the immune response was not necessary. This section can be streamlined.

Author Response

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Author Response File: Author Response.pdf

Reviewer 2 Report

In my review of “RNA-Seq analysis on the microbiota associated with the White shrimp (Litopenaeus vannamei) in different stages of development” the authors obviously have done a great deal of work and put effort into their research. Unfortunately, the manuscript doesn’t convey all that they anticipated it would suggest and it misses their own objective. Overall, it feels unfinished and a bit disjointed. It is intriguing data, however the overarching goal is never fully achieved.

At the start, the authors state that “potential use of these types of studies lies in the functional characterization of the microbiome with a given nutritional management under culture conditions.” And reiterate this point in the abstract. Hence, I found it perplexing when reading the Materials & Methods that the nutritional information was vague at best, listing crude protein, crude fat, pigments, vitamins, etc. without any comprehensive information. How might nutritional management be significant if readers never know what the feeding formula was? Perhaps the authors could improve this by including a table with the omitted components for each portion mentioned in the M&M section so that we have that detail. Otherwise, the point is missed. Moreover, the conclusion mentions in section 4.1 as “In the present study, the carbohydrates were supplied in the feeding formula…” with nothing more about what was in the feeding formula or why it was mentioned as a significant part of the study at the outset as there was zero follow through? If there was indeed follow through, please include that data.

I found it brilliant that the authors mention various factors that may impact the environment in the first paragraph of the Introduction. Again, as with the previous comment, there is no follow through in the Conclusion. It would be beneficial to have the authors speculate on how probiotics/microbiome might affect one or more of the three conditions mentioned. It feels unfinished to offer this information and never complete the thought process.

The authors use the pronoun “his” to refer to the microbiome several times in the manuscript. Anthropomorphizing microbes using specific gendered pronouns seems peculiar and out of place in a professional science manuscript. Perhaps the authors should consider a neutral term that does not allocate gender to a microbe.

In the Materials & Methods section, 2.2, could the authors explain what was used to attain nuclease-free tubes for the organisms?  Additionally, and this is most likely a typographical error, but I don’t think 750 liters reagent was used, perhaps the authors meant ul?

M&M section 2.2, Larvae paragraph, could you provide a reference for the sodium acetate precipitation please?

M&M section 2.2, Juvenile & Adult paragraph, the authors mention the reference to a “Chomczinsky and Sacchi protocol with modifications” but do not include those modifications. Please insert the changes made to that protocol so that readers know.

In the results section, several figures report the percentages of phyla, class, order, and family. It would be great if the authors could indicate where there are significant differences. Such as, in the Larvae bacterial order where specific orders were 41% but 29% in Juvenile and 25% in Adults.

All the figures in the Results section are genuinely too small to thoroughly examine. Please enlarge them so that readers may discern the details. Also, please consider the colours of the bars. Specifically, when endeavoring to read the text and examine the chart, the similar colors of different categories became quite confusing (e.g., yellow in a is for poorly char and in b for information storage). Perhaps use the same colour per category with added headings on the chart for sections a and b indicating Prokaryotic and Eukaryotic.

The authors may want to consider moving figures 5, 6, and 7 to the supplementary section and enlarging them to a single page each. As they are now, it is impossible to discern anything from the small lines and text.

The Discussion has a repetitive formula for each paragraph beginning with reiterating what was observed in their study, what other studies have found and repeating that this study agreed with those studies. It would be more informative if the authors speculated as to why they observed their results, and what else might be contributing factors. Going back to that Introduction first paragraph (i.e., farm effluents, disease, antibiotic resistance) of thoughts would be a great step as well as considering seasonal changes, microbial loop dynamics, unexpected sources such as oil spills, invasive species, etc. These all may and do have influences, what do the authors think about their results as associated to their initial thoughts and more?

The brief Conclusion paragraph caused further confusion. The authors again mention the culture conditions and then also include management conditions denoting presence of insecticidal compounds and antibiotics. Looking back over the manuscript, the M&M mentions nothing about the analysis of the culture conditions of the ponds where the shrimp were selected. There is no chemical analysis of the environment nor is there any indication that it is known that there was a specific insecticide or antibiotic used. In the Discussion, page 13, second paragraph starting “In advanced stages of…” the authors mention finding genetic evidence in low representation from the module, however, this doesn’t explain what or how these microbiomes are interacting with their hosts or environments. It would be extremely helpful if the authors could include further data regarding exactly what chemicals were present, how the shrimp reacted and anything else that would clarify this concluding remark.

Lastly, with the enormous amount of information that the authors did collect, I’m surprised that they finished with merely one paragraph of vague deductions. It would be a much sounder manuscript if the authors essentially took the first portion of the paper and unified that with the data, added in the missing data and made some insightful propositions.

Author Response

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Author Response File: Author Response.pdf

Round 2

Reviewer 1 Report

All my concerned in the last reviewing have been addressed well. However, the English writing should be proofed carefully again. Please finish it at this round.

Author Response

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Author Response File: Author Response.docx

Reviewer 2 Report

Overall, the authors have done an excellent job improving and revising their manuscript. I only have a few items; on line 84, is “his” a typo? And it feels as though the conclusion needs one more piece of content describing what possible future research/work the authors propose.

Author Response

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Author Response File: Author Response.docx