Reviewer 1: The manuscript "Identification and application of piwi-interacting RNAs from seminal plasma exosome in Cynoglossus semilaevis" (GICS-D-19-01116) identified piRNAs from male and sex reversed "pseudo-male" C. semilaevis seminal plasma exosomes. Their results concluded six piRNAs that expressed significantly higher in males than in pseudo-males which could be potential bio-markers for C. semilaevis sex typing. This study presents an interesting data set on germ line piRNAs in a teleost fish. Even that the finding of piRNAs as sex biomarker is interesting, this is not the first sex markers and DNA sex markers for this species have been widely used in previous studies. Thus, one suggestion that the statement of "first male specific biomarkers in C. semilaevis" should not be made. The manuscript is simply descriptive and the writing still needs extensive proof reading to get rid of grammatical issues, as well as more detailed explanation in almost every section. Altogether, I think this manuscript at this stage is not suitable for publication in BMC Genomics, only if the authors manage to clarify the writing of their manuscript.

Major comments:
1. Please add correct line numbers in the revised manuscript. It is difficult to track the information in the manuscript.

The line numbers have been added 

2. The manuscript is simply descriptive with only sequencing results listed. A lot of information are missing. For example, what is the major object of this study, how did the authors design the experiment to fulfill the object, et al. Moreover, the introduction is poorly organized without a clear logic line. Here is only my personal suggestion for organizing the introduction, like what is piRNAs - how do they function in germlines - what is the difference of piRNA in germline cells and exosomes, and why it is important to study piRNAs in exosomes - introduce the tongue sole sex reversal system and the development of sex markers in tongue sole - the importance of studying piRNAs in tong sole germ lines and their potential as sex markers.

We have improved the introduction of the manuscript by focusing on the functional interpretation of piRNAs and target genes. In addition, the text has been revised for grammar and spelling errors, and redundant text has been deleted

3. The writing has not been revised seriously before submission and contains a lot of grammatical errors (see details below in the minor comments) and would benefit from proof reading by a native English speaker.
 Have corrected these error and a native English speaking editor has improved the writing.

4. The piRNA play important roles in germline, why this study pay the major attention on seminal exosomes but not directly on the germline cells. Please explain more detail in the introduction why it is necessary to study the seminal exosomes rather than germline cells piRNAs. If the aim of the study is to identify sex markers, in my opinion, the order of best materials is DNA > mRNA > small RNA > exosomes small RNA, which exosomes small RNA is the least efficient and stable material for this purpose. It is not enough to just simply mention that small RNAs in exosomes have been reported as biomarkers in other studies, then it must be applied in C. semilaevis piRNAs. Please clarify the major advantage of exosome piRNAs.

Seminal exosomes act as intergenerational transmission media of genetic information; therefore, it is more direct to study the intergenerational inheritance of pseudomales than to study the germline cells. Exosome communication is also a widespread cell communication and signal transmission pathway, as demonstrated by many previous studies. Thus, we chose to study seminal exosomes and not the germline cells. In the improved introduction, we focused on our main point concerning differentially expressed piRNAs and their regulated function. The potential application of piRNAs in exosomes as sex markers may be not the most efficient method; however, it seems to work and could have many potential uses, especially if the selected piRNAs play a regulatory role.  

5. In the abstract, page2 line 34, "these are first male specific biomarkers in C. semilaevis" which is not true. There are already successfully applied sex-specific DNA biomarkers for male and pseudo-male of C. semilaevis, which should be more accurate that the small RNA-based markers identified in this study which could be highly affected by unstable gene expression profiles with qRT-PCR, especially with small RNAs whose expression level are normally much lower than mRNAs. The sex-specific markers in C. semilaevis should be implemented more in detail in the introduction. Moreover, it should be mentioned how did the authors distinguish male and pseudo-male in your study before your experiment in the methods section, otherwise it is still contradictory that you conclude to identify first male biomarkers in C. semilaevis but you already distinguished them before your experiment using what makers?

We appreciate the advice of the reviewer and have deleted the inappropriate statement. In addition, we have added information from previous studies on sex-specific DNA biomarkers of C. semilaevis and emphasized that our markers are only a supplement and enrichment of previous markers. We also supplied the markers which we used in the materials and methods section to identify the sex of experimental fish before we developed our piRNA markers.

6. For experimental design, the authors sequenced two pools of male (30) and pseudo-male (30) small RNA without replication, which can not be applied with statistical significance analysis. It is better to perform the sequencing with replicates, for example, three independent male pools (10 individuals for each pool) and three independent pseudo-male pools (10 individuals for each pool).

That is a very good point. We did not set up a repeat in the sequencing section  to ensure sufficient small RNA samples for sequencing. Therefore, we conducted qRT-PCR to verify our selected piRNAs expression levels. Fortunately, the validation results are basically consistent with the sequencing results.

Minor Comments:
1. Page 3 line 48, "play a rolefunction via the formation of forming …" should be revised.

This has been revised

2. Page 3 line 51, "piRNAs PiRNAs are…" is redundant.

The redundant word has been deleted

3. Page 4 line 11-14, 20-26, need references.

References have been added

4. "Signature piRNA" has been used throughout the manuscript, what is the specific meaning of "signature" here?

It means markers with significant differential expression as verified by qRT PCR

5. Page6 line 28, "novel piRNAs are between 21 and 38 bp", what is the criteria to distinguish miRNA from piRNAs about 21 bp?

PiRNAs are mainly 26–31 nt , while microRNAs (miRNAs) are 21–24 nt. However, these are not absolute ranges. In the present study, we distinguished miRNAs from piRNAs of about 21 bp. The sequence alignment with the known database was considered, especially in the prediction of novel piRNAs, in which a small number of 21-nt piRNAs were predicted to align with piRNAs in the database 

6. Page7 line 1-16, why the authors first used TPM>150 and FC>100 as criteria and then TPM >400 and FC>200. The authors only predicted target genes of the 44 piRNAs from the second criteria. Nothing was mentioned why there should be two different criteria and what has been done with the 87 piRNAs from the first criteria. It seems redundant to report all these result in this section? Remove or explain why to use two criteria, and replace "condition" with "criteria" in these sentences.

We appreciated the opinion of the reviewer. We adopted a step-by-step filtering strategy to select the significant differential piRNAs. 87 piRNAs were identified among all differential piRNAs from the first screen with TPM>150 and FC>100 as criteria. After a preliminary screen that identified too many significant piRNAs for further analysis, we adopted stringent criteria of TPM > 400 and FC >200, which identified 44 piRNAs from the first screen 87 piRNAs for subsequent analysis. We believe this more stringent screening allowed us to find more significant piRNAs.

7. Page7, line 36-47, 37 of the 44 piRNAs' target genes are related to sex differentiation et all, whereas at page 8, line 1-9, "most of the target genes are involved in plasma membrane, integral component of membranc, …….", but not to sex differentiation. What is the exact enriched function of the target genes? Please revise and clarify the results of target gene enrichment analysis.

Each piRNA corresponds to multiple target genes and sex-related target genes are a tiny fraction of all the target genes of the 44 piRNAs. In fact, employing the target genes of 44 piRNA for KEGG pathway enrichment, the top two enriched functions were lipid-carbohydrate metabolism and signal transduction pathway, as the primary and secondary enrichment of KEGG is generalized. However, this did not affect the selection of sex-related genes from all target genes and their corresponding piRNAs. We identified the sex-related target genes and their corresponding piRNAs through KEGG pathway enrichment, and reduced the effective piRNAs from 44 to 37. The details can be seen in Supplementary Fig 4: Screening process for the differential expression of piwi interacting RNAs (piRNAs) between two donor groups

8. Page 10, line 9 "Comparing Compared with …, piRNAs has have less conservative conserved sequences….", line 15 "There were are ……". Obvious mistakes, please revise these sentences.

These sentences have been revised

9 Page 10, line 45, reference 27 should be 28; line 51, reference 28 should be 27.
 
This has been corrected

10. In the methods, please explain in detail how you distinguished male and pseudo-male specimens.

This explanation has been added to the methods

11. Page 15, line 47, the fold change criteria for differentially expressed piRNAs are missing.

We have added the missing fold change criteria. 

12. Page 16, line 39, "in three duplicates" should be "in triplicate", and line 42 "quantification of miRNA" should be "quantification of piRNA".

We have revised the text as requested.

Reviewer 2: This manuscript identified seminal plasma exosomes from male and sex reversed ''pseudo-males'' of half-smooth tongue sole and compared their difference of piRNAs. Then，the authors confirmed six sex-related signature piRNAs as male specific biomarkers in C. semilaevis. Their results may provide an effective tool to explore sex typing and a clue for future research on the generative mechanism of sex reversed ''pseudo-males'' in C. semilaevis. However, the content of the article deserved a stronger improvement of the writing.

For example

1) Throughout MS, Tables and Figures, "male (ZZ♂) and pseudo-male (ZW♂)" should be corrected by "male (ZZ♂) and pseudo-male (ZW♂)" in order to discriminate ZW♀.

The text has been revised as suggested.

2) P2, L42, "may rovide guidance" is a mistake spell.

This mistake has been corrected

3) The last paragraph in P3 and the first paragraph in P4 should be merged.

These paragraphs have been merged

4) P5, L12, "with mean sizes of 108.2 nm and 116.3 nm inmales and pseudo-males, respectively", should provide standard deviation.

We have added the standard deviations

5) Section "Target prediction for candidate signature piRNAs and GO enrichment and KEGG pathway enrichment analysis." It's better to firstly describe the results about " GO term categories generated from the 6231 genes targeted by the 44 signature piRNAs", then present the results about "the 37 candidate signature piRNAs related to sex".

We have changed the order

6) Section "Verification by qRT-PCR", did the authors choose "piRNAs with a non-zero expression in the ZZ♂ group, but much higher expression in the ZW group" to verify? And did they identify signature piRNAs of pseudo-males? 

Yes, we also chose the piRNAs with a non-zero expression in the ZZ♂ group, but much higher expression in the ZW group from sequencing; however, the qRT-PCR results did not match well to the sequencing results.

7) Figure 6, why all six sex-related signature piRNAs had the highest expression in individual ZZ♂5 and the lowest expression in individual ZZ♂3? Were the samples at different gonad development stage？
We appreciate the reviewer's opinion. That is a very careful observation.
Individuals ZZ 5 and ZZ 10 had higher expression in all six signatures, while ZZ 3 and ZZ 6 had lower expression. All the samples were obtained at the same stage (random sampling from different ponds in the same factory were carried out). We speculated that the environment or individual differences of culture ponds may result in the relatively consistent expression of six piRNA and this may reflect to a certain extent that the piRNAs we selected are representative.