Transcriptome Profiling Revealed Basis for Growth Heterosis in Hybrid Tilapia (Oreochromis niloticus ♀ × O. aureus ♂)

Round 1

Reviewer 1 Report

Title: ‘Transcriptome Profiling Revealed Growth Heterosis in Hybrid Tilapia (Oreochromis niloticus × O. aureus)’ by Zhu et al.

Overall comments

The science is not a novel study, but this would be a valid addition to the scientific literature with investigations in hybrid tilapia. The research provides some transcriptomic data for further advanced studies on hybrid tilapia.

Abstract: Precise and reads well

Introduction is provided with good message along with relevant citations, however, it lacks the hypothesis of the research, hence authors are suggested to mention in the introductory section.

Materials and methods

Methodology is described very systematically; however there is no mention about RNA sample size used for trancriptomics and for qRT-PCR, and what gender the hybrid is? Is it a sterile fish? suggest to mention it in the methods.

Results

Line 171: ‘They were..................reference genome’ – it is a methodology, similarly there are some statements those should be brought into the methodology section.

Figure 2 and Figure 4. Legends (y-axis) are not readable

Lines 231-234: Rephrase the statements for better readership

Figure 5(a): axis legends are completely unreadable.. these distracts the readers.

Discussion

Overall, well written

Line 303: What is stronger stress? Does it mean stress tolerant? It is not clear...rephrase it.

Lines 378 -380 ‘This study showed that GSH-Px expression was significantly lower in hybrid tilapia compared with their parents. It was speculated that individuals with lower GSH-Px activity are susceptible to antioxidant protection [47]’: And line 386-388 ‘The pattern of altered defense-related gene expression observed in F1 indicates that the hybrid may have decreased capabilities of basal defense response [49]. Then how the hybrid tilapia is disease resistant. Justify your statements.

Conclusions

‘stronger disease resistance than their parents’ have you tested the hybrids through pathogenic challenge studies or is it just based on the transcriptomics?

Author Response

Please see the attachment.

Author Response File: Author Response.docx

Reviewer 2 Report

 The manuscript entitled “Transcriptome Profiling Revealed Growth Heterosis in Hybrid Tilapia (Oreochromis niloticus ♀ • O. aureus ♂) t” is an important study on the effect of Tilapia were first introduced into China in 1978 and became an economically important freshwater fish. Crossbreeding is a common conventional breeding method used for genetic improvement within tilapia species. The development of hybrid tilapia at the commercial level did not start until the 1980s in China, at which time it provided a great boost to the rapid development of the Chinese tilapia industry. According to the latest statistics from the China Fisheries Yearbook, China’s total tilapia production in 2020 was 1.655 million tons, ranking first in the world, for this reason this research is very interesting.

The introduction is relevant but must include new references. The discussion, in the light of results and knowledge, is relevant.

Your manuscript will not be accepted unless both the technical and grammatical revisions have been made successfully.

Based on these comments, I recommend a moderate revision of analytical aspects of this manuscript before final decision about its acceptance.

Moderate comment:

1. Introduction:

L32-77: Rewrite this part with new references it is a very old research (Gao, Y.; Zhang,  et al.; 2013 and Chen, Z. J. ; 2013)

See: The Biology of CancerSee: Recent study (Fazio F. et al., 2019) have shown a new analysis technique important in recognizing cells morphology through to Flow cytometry

Francesco Fazio, Concetta Saoca, Gregorio Costa, Alessandro Zumbo, Giuseppe Piccione, Vincenzo Parrino - Flow cytometry and automatic blood cell analysis in striped bass Morone saxatilis (Walbaum, 1792): A new hematological approach, Aquaculture 513 - 734398 (2019).

L42-77: Please insert this sentence: Some authors (Parrino et al., 2018 – 2021) have highlighted the effect of anthropogenic. 

Reference:

Parrino V. et al., (2018 and 2021) - Comparative study of haematology of two teleost fish (Mugil cephalus and Carassius auratus) from different environments and feeding habits. The European Zoological Journal, 85:1,194-200.

Parrino V., De Marco G., Minutoli R., Lo Paro G., Giannetto A., Cappello T., De Plano L. M., Cecchini S., & Fazio F. - Effects of pesticides on Chelon labrosus (Risso, 1827) evaluated by enzymatic activities along the north eastern Sicilian coastlines (Italy). The European Zoological Journal, vol. 88:1, p. 540–548; - ISSN:https://doi.org/10.1080/24750263.2021.1905090; (2021).

Recent study (Acar Ü., et al., 2018) showed the role of supplementation on growth performance, some hematological:

Acar Ü. et al., (2018) - Effects of Different Levels of Pomegranate Seed Oil on Some Blood Parameters and Disease Resistance Against Yersinia ruckeri in Rainbow Trout. Frontiers in Physiology, vol. 9, p. 1-7.

2. Materials and Methods:

L79-152: All sampled of fishs were exposed to imidacloprid concentrations by adding water were clinically healthy? Please described the absence of lesion.

L157-160: Reactions were performed under the 157 following conditions: 94 °C for 4 min, followed by 40 cycles of 94 °C for 15 s, 60 °C for 20 158 s, and 72 °C for 40 s. β-actin was used as an internal control in qRT-PCR. The relative 159 expression levels of the target genes were calculated using the 2-ΔΔCt approach ...? Are there protocols on this or does the hydrolysis reaction respond better? Explain this concept better ...

Statistical:

 

The statistical analysis used is appropriate.

3. Results:

The authors should be improve this part, please better the results shown in figures.

 

4. Discussion: 

L299-391: This part is very long, reduce it and discuss only the obtained results. 

Reference missing (please insert some references).

In this study, comparative transcriptome analysis was 307 performed on livers of hybrid tilapia (NA) and their parents (N and A). In total, 2319 DEGs 308 were identified. Trend co-expression analysis showed that a large proportion of genes (> 309 67.1%) were non-additive in hybrid tilapia from heterozygous crossing. Excluding the 310 influence of DEGs between N and A, 666 up-regulated and 802 down-regulated DEGs 311 were identified between parents and hybrid tilapia… Please explain this concept better ...

5. Conclusions:

It is OK

Author Response

Please see the attachment.

Author Response File: Author Response.docx

Reviewer 3 Report

This is an interesting study and the manuscript was well prepared. However, I have a very strong concern about the bioinformatics protocol used, that may have generated many artifacts.

 

I find this differential expression bioinformatics protocol very problematic. It is not clear how close or distant is the genome of Oreochromis aureus from the reference niloticus. For any gene that has diverged a bit more in the aureus lineage, its sequence reads are not going to align well to the reference niloticus genome. Therefore, an artifactual difference will emerge. Thus, this analysis only makes sense for genes that are very highly conserved between the two parental lineages. Only for this set of conserved genes one can draw safe conclusions.

My strong suggestion is to do a comparative study between the two genomes and generate a “Consensus” reference genome that contains only the highly conserved genes between the two parental lines. Then, re-run the entire protocol on this “Consensus” reference genome. Only this makes sense.

 

An additional control would be to measure the FPKMs of aureus reads twice, by using both the aureus and the niloticus genomes as reference and see for how many genes they see difference in estimated FPKMs.

 

How did the authors ensure that the conditions were the same for the hybrid and the two parental lineages? Gene expression is very dynamic and changes according to different conditions/stimuli.

 

Why did the authors decide to study gene expression in liver, for understanding heterosis?

 

Line 12-13: Please rephrase. Its not clear if the hybrid has superior features.

 

Line 51-5 : hybrid Chinese soft-shelled turtle (Pelodiscus sinensis), with which other species? Same for the next two hybrids.

 

Line 65: compared to

 

Line 85-86: Is this difference in body-mass statistically significant? Did the authors measure enough samples from each group and what statistic did they perform?

 

Line 88-89: Better state here as well that 3 fish were used for RNA-seq from each group.

 

Line 110: Please state here the read length.

 

Tables 2 and 3 should have information for each biological replicate.

 

Line 180: differentially expressed

 

Line 420: The bioproject codes should also be mentioned in the materials and methods.

Author Response

Please see the attachment.

Author Response File: Author Response.docx

Round 2

Reviewer 3 Report

The authors have addressed my minor concerns, but they have not addressed my most major concern, therefore, it is impossible for me at this stage to judge if their analysis is sound or just an artifact.

Author Response

Please see the attachment

Author Response File: Author Response.docx