Transcriptome Sequencing Analysis Reveals Dynamic Changes in Major Biological Functions during the Early Development of Clearhead Icefish, Protosalanx chinensis
, Yifan Zhou 1, Shiyue Guo 1 and Yang You 1,2,*Round 1
Reviewer 1 Report
Comments-fishes-1708583
Manuscript fishes-1708583 is a RNAseq study elucidating the dynamic changes of biological functions during early development of cleared icefish (Protosalanx chensis). A deep bioinformatics analysis was applied to describe the molecular mechanisms underlying the biological processes in the early development of cleared icefish. In my opinion, the ms is well-prepared and written, but the current form of ms is not suitable for publication yet. The quality of ms should be improved by addressing all the major and minor issues found in the text as listed below:
Abstract
- I would recommend the authors to follow the guideline from zebrafish in writing the gene names in the text. The authors can find the guideline on this link https://zfin.org. Apply this for the entire text.
Line 14 - a) “(Protosalanx chinensis)” can be omitted! It has been mentioned on the title!, note that the Latin name is only included at the first appearance of species mentioned in the text, and b) should be “we aimed to…..”
16 - “P. chinensis” can be omitted!
24 - it was found
Introduction
- see the previous comment regarding how to mention species in the text!
Line 46 - double check this statement! What does it mean? Is the information correct?
63 - should be “gene transcripts”
83 - Here, the authors mentioned that the present study aimed to gain a comprehensive understanding of the molecular characteristics and changes in nutritional requirements of fish at early development. Justification for nutritional requirements at early development of fish deserves to be explained. Why was it a concern in the present study by authors?
84-9 - this information can go to Materials and methods section!
Materials and methods
- see the previous comment regarding how to mention species and gene names in the text!
Lines 106 & 108 - “it” could be replaced with “the larvae”
115 - state the magnification applied for microscope observation!
122 - state the dye used for visualizing the RNA on the gel agarose!
124 - RNA was
125 - which was
134-5 - was it single-end or paired-end sequencing applied? State the length of reads resulted in!
148 - proposed or discarded!
172 - analyses
173-4 - I would recommend the authors to apply the Fisher exact test in defining the significantly enriched GO terms and KEGG pathways.
181 - module??
181-2 - How did the authors define the genes which have the highest correlation in each target module? State this!
187 - I suggest the authors to follow the guidelines from Zebrafish regarding to write the gene names in the text. The guideline can be found on the link https://zfin.org. Apply this for the entire text!
188 - How did the authors design the primers for qPCR analysis? Were they designed based on the transcriptome sequences obtained from this work or the sequences derived from public databases? The gene/transcript IDs (transcriptome sequences) or Accesion number of genes (sequences from databases) should be included in Table S5.
Results
- see the previous comment regarding how to mention species and gene names in the text!
Lines 201-2 - this sentence can be omitted! It has been stated in the previous section
208 - double check the BioProject Acc. No! It does not exit in NCBI!
212-4 - include the correlation value for both method, and state the statistical method used in M&M section
232-4 - this statement can be omitted! It is only repetition from the previous section!
250 - “…, and large…”
254 - “…was gradually..” and “…was enhanced…”
265 - are shown
280 - “.., and then …..”
370-1 - this sentence can be omitted!
373 - are shown
Discussion
- This section needs to be carefully read and rewritten as many grammatical errors are found
- see the previous comment regarding how to mention species and gene names in the text!
Lines 396-9 - I would be better to put this statement in introduction section. It is relevant with the previous comment on line 83!
400-1 - this sentence is only repetition and can be omitted!
404-5 - This sentence and Figure 9 should go to Results section!
413 - play
420, 426-8 - increased
441 - were
442 - showed
450, 459-60, 462 - were
466 - “(Figure 1)” can be omitted!
471 - was
474 - a) was, and b) avoid the use of “And” at the beginning of sentence!
476 - was
482 - showed
483 - “couldn’t be”, “was not very high”
487 - “were significantly”, “it was”
489 - were
491 - were also
497-501 - rephrase there two sentence and combine them as one
505 - What does the Chinese words mean?
509, 512 - were
520-3 - rewrite the sentence!
528 - the larva begins
529 - were
538 - “And” can be omitted!
Conclusions
- see the previous comment regarding how to mention species and gene names in the text!
551, 553 - was
553-4 - “The trend analysis and enrichment analysis indicated that” can be omitted!
557-9 - this statement is not a conclusion, It is just general information. The authors should specify this information based on the results of study.
Comments for author File: 
Comments.pdf
Author Response
Response to Reviewer 1 Comments
Thank you very much for your detailed comments and taking the time to read our manuscript. In response to the comments made, the specific changes have been made as follows:
Point 1: I would recommend the authors to follow the guideline from zebrafish in writing the gene names in the text. The authors can find the guideline on this link https://zfin.org. Apply this for the entire text.
Response 1: We thank the reviewer for pointing this out. We have corrected the relevant content in the manuscript.
Point 2: Line 14 - a) “(Protosalanx chinensis)” can be omitted! It has been mentioned on the title! note that the Latin name is only included at the first appearance of species mentioned in the text, and b) should be “we aimed to…..”
Response 2: We have fixed the error.
Point 3: Line 16 - “P. chinensis” can be omitted!
Response 3: We have fixed the error.
Point 4: Line 24 - it was found
Response 4: We have fixed the error.
Point 5: Line 24 - it was found
Response 5: We have fixed the error.
Point 6: Line 46 - double check this statement! What does it mean? Is the information correct?
Response 6: Thank you for pointing this out, this part is correct, but it may be due to problems with our presentation, which makes it more difficult to understand. We have made the change. The new sentence reads as follows. [However, this species cannot be artificially cultivated, so that the annual replenishment in natural water can only rely on natural reproduction or artificial fertilized eggs, but the fertilization rate, hatching rate and survival rate of the larvae in the natural environment are all low, because it is rare winter spawning groups, and the effective accumulation temperature of hatching is about 5100 h°C, and the parental generation has no nurturing behavior, which makes the population of the clearhead icefish prone to large fluctuations due to environmental changes.]
Point 7: Line 63 - should be “gene transcripts”
Response 7: We have fixed the error.
Point 8: Line 83 - Here, the authors mentioned that the present study aimed to gain a comprehensive understanding of the molecular characteristics and changes in nutritional requirements of fish at early development. Justification for nutritional requirements at early development of fish deserves to be explained. Why was it a concern in the present study by authors?
Response 8: We thank the reviewer for pointing this out. We have made changes to this point. In order to make the logic smoother, we put it in lines 59-63, and the specific changes are as follows. [The lack of knowledge in this area seriously hinders people's understanding of the growth and development of the clearhead icefish, which in turn hinders the development of its aquaculture. Given its important economic value and important role in lake governance, it is an urgent need to understand its early development process in as much detail as possible.]
Point 9: Line 84-9 - this information can go to Materials and methods section!
Response 9: We have removed the information about this and only retained the following sections [which are as follows: YL (embryo stage), PM (1st day after hatching), KK (4th day after hatching), LC (7th day after hatching), and SL (10th day after hatching)]
Point 10: Lines 106 & 108 - “it” could be replaced with “the larvae”
Response 10: We have made changes to this point.
Point 11: Lines 115 - state the magnification applied for microscope observation!
Response 11: This is our mistake of not explicitly mentioning the magnification of the microscope in the manuscript, and we have made the following changes to this problem. [The collected samples were magnified 20-30x using the OLYMPUS SZX16 microscope to ensure that the samples were at the same level of development].
Point 12: Lines 122 - state the dye used for visualizing the RNA on the gel agarose!
Response 12: We have made the following changes to this point, as follows [Subsequently, the total RNA quality and integrity was measured using 1% agarose gel electrophoresis (voltage: 200V, time: 25 minutes), and Andy Safe™ Nucleic Acid Gel Stain was used for precast gel staining].
Point 13: Lines 124 - RNA was
Response 13: We have fixed the error.
Point 14: Lines 125 - which was
Response 14: We have fixed the error.
Point 15: Lines 134-5 - was it single-end or paired-end sequencing applied? State the length of reads resulted in!
Response 15: This is our mistake of not explicitly mentioning the magnification of the microscope in the manuscript, and we have made the following changes to this point. [Library quality was evaluated using the Agilent 2100 bioanalyzer system, and then, the libraries were loaded into the patterned nanoarrays and pair-end reads of 100 bp were read through on the BGISEQ-500 platform for subsequent data analysis.]
Point 16: Lines 148 - proposed or discarded!
Response 16: We have fixed the error. [all low-expression genes (FPKM value < 1) were discarded.]
Point 17: Lines 172 - analyses
Response 17: We have fixed the error.
Point 18: Lines 173-4 - I would recommend the authors to apply the Fisher exact test in defining the significantly enriched GO terms and KEGG pathways.
Response 18: We agree with the reviewer's proposal to use Fisher exact test to define significantly enriched GO terms and KEGG pathways, which can largely avoid the probability of false positives. However, due to the use of a corrected P value (Q value), some modules will not have significantly enriched KEGG paths, which will cause some interesting pathways to be excluded. And we noticed that there are many literature that also uses p-values to define the significantly enriched GO terms and KEGG pathways. So at this point we decided not to make changes.
Point 19: Lines 181 - module??
Response 19: yes, module, we have changed this point.
Point 20: Lines 181-2 - How did the authors define the genes which have the highest correlation in each target module? State this!
Response 20: It is our oversight not to describe this in detail, we have changed it as follows, [In order to identify the hub genes of each target module, the top 200 genes with the highest correlation in each target module according to the cor.geneModuleMembership were selected and used to make a protein–protein interaction (PPI) network. After that, the correlation between genes was calculated using the R package psych according to the expression levels of the genes at each stage, and then the packages stringr and dplyr were used to filter out the genes with low correlation (p>0.05, |R|<0.8) and output the result in a format accepted by Cytoscape (version 3.8.2) software. The correlation network was presented using Cytoscape (version 3.8.2) software, and the top 3-6 genes with the highest node degree were identified as hub genes]
Point 21: Lines 187 - I suggest the authors to follow the guidelines from Zebrafish regarding to write the gene names in the text. The guideline can be found on the link https://zfin.org. Apply this for the entire text!
Response 21: We thank the reviewer for pointing this out. We have revised.
Point 22: Lines 188 - How did the authors design the primers for qPCR analysis? Were they designed based on the transcriptome sequences obtained from this work or the sequences derived from public databases? The gene/transcript IDs (transcriptome sequences) or Accesion number of genes (sequences from databases) should be included in Table S5.
Response 22: We apologize for this mistake and have added the information to the manuscript. [In order to verify the reliability of the transcriptome data, six genes were randomly selected for qRT-PCR (prss (LS_GLEAN_10003514), apob-48 (LS_GLEAN_10003911), cd36 (LS_GLEAN_10003179), apoa-1 (LS_GLEAN_10004674), muc2 (LS_GLEAN_10008706), slc6a19 (LS_GLEAN_10011640)) (Table S5). Primer Premier5.0 was used to design primers for selected genes, and then synthesized by Yixin Biotechnology (Shanghai) Co., Ltd]
Point 23: Lines Lines 201-2 - this sentence can be omitted! It has been stated in the previous section
Response 23: We have removed this part.
Point 24: Lines 208 - double check the BioProject Acc. No! It does not exit in NCBI!
Response 24: This was our mistake, started thinking “NO.PRJNA687639” was “number PRJNA687639”. We have change this to [The original data were stored in NCBI under BioProject accession: PRJNA687639]
Point 25: Lines 212-4 - include the correlation value for both method, and state the statistical method used in M&M section
Response 25: We have supplemented this in M&M section and made modifications to this part. [Origin software was used for qRT-PCR and transcriptome trend analysis. In correlation analysis, the relative expression qRT-PCR and FPKM values were corrected using log-transformation to avoid arithmetic error and large negative values in transformation, a pseudo-count of 1was added to each value before log-transformation [20]. Use SPSS software to perform spearman correlation analysis, and the scatter plots were visualized using Origin software] [In addition, Real-time fluorescence quantitative (qRT-PCR) analysis showed that the expression trends of the six genes were basically consistent with the transcriptome analysis results, showing a trend of gradual increase in expression level over time (Figure S1). The significant positive correlations (rs = 0.624, P < 0.01) were observed between the RNA-seq and qRT-PCR data (lg-transformed) (Figure S2), which indicating that the transcriptome data was reliable]
Point 26: Lines 232-4 - this statement can be omitted! It is only repetition from the previous section!
Response 26: We have removed this part.
Point 27: Lines 250 - “…, and large…”
Response 27: We have fixed the error.
Point 28: Lines 254 - “…was gradually..” and “…was enhanced…”
Response 28: We have fixed the error.
Point 29: Lines 265 - are shown
Response 29: We have fixed the error.
Point 30: Lines 280 - “.., and then …..”
Response 30: We have fixed the error.
Point 31: Lines 370-1 - this sentence can be omitted!
Response 31: We have removed this part.
Point 32: Lines 373 - are shown
Response 32: We have fixed the error.
Point 33: Lines 396-9 - I would be better to put this statement in introduction section. It is relevant with the previous comment on line 83!
Response 33: We made appropriate changes in the paper based on your comments. We deleted this sentence in this part, and made changes to the foreword accordingly, see response 8 for details.
Point 34: Lines 400-1 - this sentence is only repetition and can be omitted!
Response 34: We have removed this sentence.
Point 35: Lines 404-5 - This sentence and Figure 9 should go to Results section!
Response 35: Thank you very much for your suggestion. Since we want to have a general summary of the whole result, we put it in the first part of the discussion. So, from this point of view, we want to keep this part without changing it.
Point 36: Lines 413 - play
Response 36: We have fixed the error.
Point 37: Lines 420, 426-8 – increased
Response 37: We have fixed the error.
Point 38: Lines 441 - were
Response 38: We have fixed the error.
Point 39: Lines 442 - showed
Response 39: We have fixed the error.
Point 40: Lines 450, 459-60, 462 - were
Response 40: We have fixed the error.
Point 41: Lines 466 - “(Figure 1)” can be omitted!
Response 41: We have removed this.
Point 42: Lines 471 - was
Response 42: We have fixed the error.
Point 43: Lines 474 - a) was, and b) avoid the use of “And” at the beginning of sentence!
Response 43: We have made changed in this.
Point 44: Lines 476 – was
Response 44: We have fixed the error.
Point 45: Lines 482 - showed
Response 45: We have fixed the error.
Point 46: Lines 483 - “couldn’t be”, “was not very high”
Response 46: We have fixed the error.
Point 47: Lines 487 - “were significantly”, “it was”
Response 47: We have fixed the error.
Point 48: Lines 489 - were
Response 48: We have fixed the error.
Point 49: Lines 491 - were also
Response 49: We have fixed the error.
Point 50: Lines 497-501 - rephrase there two sentence and combine them as one
Response 50: Thank you very much for your comments, we have made the following changes in response to this. [It is further explained that the clearhead icefish mainly produces a large amount of energy through aerobic metabolism at this stage, and the oxygen demand increases, which may be related to the increase in swimming intensity]
Point 51: Lines 505 - What does the Chinese words mean?
Response 51: We're sorry that this is a mistake we made when we cross-referenced, and it has been modified.
Point 52: Lines 509, 512 - were
Response 52: We have fixed the error.
Point 53: Lines 520-3 - rewrite the sentence!
Response 53: We have rewrited the sentence.[ In addition, studies have shown that the visual, olfactory, and lateral line systems play an important role in fish predation, for example, zebrafish predominantly use vision systems to locate food, and juvenile American redfish (Sciaenops ocellatus) predominantly use lateral line systems (mechanical sensing) to locate food]
Point 54: Lines 528 - the larva begins
Response 54: We have fixed the error.
Point 55: Lines 529 - were
Response 55: We have fixed the error.
Point 56: Lines 538 - “And” can be omitted!
Response 56: We have delected the“And”.
Point 57: Lines 551, 553 - was
Response 57: We have fixed the error.
Point 58: Lines 553-4 - “The trend analysis and enrichment analysis indicated that” can be omitted!
Response 58: We have delected this.
Point 59: 557-9 - this statement is not a conclusion, It is just general information. The authors should specify this information based on the results of study.
Response 59: Thank you very much for your suggestion, we have made the following changes for this part [In addition, we used WGCNA method to describe the main biological functions of this species at various stages of early development and to identify hub genes that play an important role in this process. In the embryonic stage, the development of the eyes, bones and heart is mainly carried out through cell proliferation and differentiation, in which ccnd2, seh1l, kdm6a, arf4 and ankrd28 play important roles; after hatching, the tissue system and digestive capacity of the larvae are gradually enhanced. In the endogenous nutrient stage, the larvae mainly uses the lipids in the yolk sac to synthesize triglycerides and store them in the body; in the mouth-opening stage, the larvae mainly uses amino acids and fatty acids for aerobic metabolism, and it will synthesizes unsaturated fatty acids and stores them in the body; after entering the exogenous nutrition, cholesterol, lipids and proteins are the main nutritional sources of the larvae.]
We would like to thank the referee again for taking the time to review our manuscript,
All authors
Author Response File: Author Response.pdf
Reviewer 2 Report
This is an interesting manuscript that studied the dynamic changes in clear-head icefish during their early development using transcriptome sequencing analysis. The authors identify genes that were enriched during early developments of clear-head icefish. The manuscript is a good fit for the journal, I have few minor suggestions that are missing or not clear in the method that the authors may consider.
Minor concerns:
- It is not clear to me what the composition of the diet used in this investigation
- How much feed was fed to the fish daily?
- At what temperature were the fish maintained post-hatching?
- The duration of the entire experiment was missing or not clearly mentioned.
Author Response
Thank you for taking the time to revise our manuscript and provide valuable comments. The responses to the comments made are as follows:
Point 1: It is not clear to me what the composition of the diet used in this investigation
Response 1: From the third day after hatching, the larvae are fed twice a day, with egg yolks, rotifers, copepods and cladocerans. Before our experiment,there was no initial feed for the clearhead icefish and the mouth-open time point was not determined , so we wanted to see the dietary preferences of the larvae, and the other was to see when the larvae started the first feeding. Therefore, according to your suggestion, we have made some changes in the manuscript.
Point 2: How much feed was fed to the fish daily?
Response 2: It was an oversight of us in that we did not mention this in our article. We unfortunately did not weigh the food we are feeding in this study, so we are not sure about the specific feeding amount. But we deal with food residues、change the water and microscopic examination every time after half an hour, so we modify it as follows [daily microscopy is performed after half an hour of feeding each day to see if the larvae had eaten].
Point 3: At what temperature were the fish maintained post-hatching?
Response 3: We thank the reviewer for pointing this out. We have revised. [ Fertilized eggs hatching and larval rearing were all done in a plastic box (length, width and height: 0.4 m×0.3 m×0.2 m) with fresh water at 9.2±0.5°C].
Point 4: The duration of the entire experiment was missing or not clearly mentioned.
Response 4: It was our negligence not to explicitly mention the duration of the experiment in the manuscript. In response to this comment, we have added the time of the third day after hatching [18 January 2019], then it was supplemented that the time to collect the fertilized eggs was within 24h after fertilization.
We would like to thank the referee again for taking the time to review our manuscript,
All authors
Round 2
Reviewer 1 Report
Comments-fishes-1708583-v2
Manuscript fishes-1708583 has been significantly improved by the authors according to the previous comments. However, not all the issues are completely addressed and further improvement should be done to increase the quality of revised ms. Many grammatical errors are still found in the revised ms which needs a serious attention by the authors for the next submission. In addition, the authors have to include the line number for each comment addressed on the response letter in order to make easier in reviewing process.
Introduction
Lines 48-55 - this sentence is too long which makes it difficult to follow. I would recommend modifying it again! It can be broken down into two or more sentences.
52 - 5100 h℃??? What is this? I think it is not common. Could the authors explain this with another way!?
69-73 - this does not fully address the previous comment! then, what is the association between nutritional requirements and early development of the clearhead icefish which deserves to investigate in the present study as stated on lines 94-6. State this!
97-102 - it would be better to bring all this information to the M&M section as suggested previously!
108 - of clearhead icefish
Materials and methods
Line 112 - should be “clearhead icefish”. See the previous comment regarding this! Once again, apply this for the entire text, for example: line 118 and others (if any)
213-5 - what about the gene ID for internal reference (gapdh)? State this internal reference also in the text that used for normalizing the qPCR data.
222 - 30 s
227 - analyses
Results
Line 242 - the original data with acc. No: PRJNA687639 is still not available in NCBI database. Make sure the Transcriptome data has been already deposited to the public database before the ms is accepted for publication (see the PDF version of comments and suggestions for Authors).
Discussion
Line 581 - larva begins (larvae is plural)
Conclusions
611-3 - delete this sentence!
614-21 - the sentence needs to be rephrase. Build the sentence in the form of past tense!
Comments for author File: Comments.pdf
Author Response
Dear reviewer,
Thank you very much again for your detailed comments and taking the time to read our manuscript. In response to the comments made, we have made detailed changes and marked the relevant line numbers as follows:
Point 1: Lines 48-55 - this sentence is too long which makes it difficult to follow. I would recommend modifying it again! It can be broken down into two or more sentences.
Response 1: To make this sentence a little easier to understand, we divide it into two sentences, line 53-60 [However, at present, the species cannot be artificially cultivated, so that the annual replenishment in natural water can only rely on natural reproduction or artificially stocked fertilized eggs. Clearhead icefish is a rare winter spawning group, the sum of effective temperature (SET) of embryonic development is about 5100 degree C-hours, and the parental generation has no nurturing behavior, so that in the natural environment, the survival rate of the early development stage is very low, and the population number is also prone to large fluctuations due to environmental changes]
Point 2: 52 - 5100 h℃??? What is this? I think it is not common. Could the authors explain this with another way!?
Response 2: Thank you for pointing this out, We have changed this description from 5100 h℃ to 5100 degree C- hours. Line 57. We found that degree-days were used to describe the effective accumulation temperature, degree-hours are similar to degree-days, but the literature found is limited, so it is not known whether this is common, add C in the middle because we are using a degree Celsius.
Point 3: 69-73 - this does not fully address the previous comment! then, what is the association between nutritional requirements and early development of the clearhead icefish which deserves to investigate in the present study as stated on lines 94-6. State this!
Response 3: Reading the full article again, we found that we really didn't put too much emphasis on changes in nutritional needs, so we changed line 74-76 and line 99-105. [but the dynamic changes in the main biological functions as well as the underlying molecular regulatory mechanisms during its early developmental processes remain unexplored] [In order to comprehensively explore the dynamic changes in the main biological functions of the early development of clearhead icefish and the underlying molecular regulatory mechanisms, samples from five key periods were collected for transcriptome sequencing and assembly on the basis of the preliminary research of our group.]
Point 4: 97-102 - it would be better to bring all this information to the M&M section as suggested previously!
Response 4: We have deleted this part.
Point 5: 108 - of clearhead icefish
Response 5: Thanks to the comments of the reviewers, we have changed the content to “clearhead icefish” of the manuscript in this regard.
Point 6: Line 112 - should be “clearhead icefish”. See the previous comment regarding this! Once again, apply this for the entire text, for example: line 118 and others (if any).
Response 6: we have changed the content to “clearhead icefish” of the manuscript in this regard.
Point 7: 213-5 - what about the gene ID for internal reference (gapdh)? State this internal reference also in the text that used for normalizing the qPCR data.
Response 7: It was our negligence to forget to describe the internal reference gene in the manuscript, which we have added to line 226-227. [and gapdh (glyceraldehyde-3-phosphate dehydrogenase) was used as a reference gene to normalize the expression levels between samples [33]]
Point 8: 222 - 30 s
Response 8: we have fixed the error. Line 233.
Point 9: 227 – analyses
Response 9: we have fixed the error. Line 237.
Point 10: Line 242 - the original data with acc. No: PRJNA687639 is still not available in NCBI database. Make sure the Transcriptome data has been already deposited to the public database before the ms is accepted for publication.
Response 10: We're sorry that we set the data release time to January 1, 2023 in the early days, which caused the data to be found online, but now we have released the data.
Point 11: Line 581 - larva begins (larvae is plural)
Response 11: we have fixed the error. Line 608.
Point 12: 611-3 - delete this sentence!
Response 12: we have deleted this sentence.
Point 13: 614-21 - the sentence needs to be rephrase. Build the sentence in the form of past tense!
Response 13: We re-modified this sentence. Line 641-650. [In the embryonic stage, the development of the eyes, bones and heart was mainly carried out through cell proliferation and differentiation, in which ccnd2, seh1l, kdm6a, arf4 and ankrd28 played important roles; after hatching, the tissue system and digestive capacity of the larvae were gradually enhanced. In the endogenous nutrient stage, the larva mainly used the lipids in the yolk sac to synthesize triglycerides and stored them in the body; in the mouth-opening stage, the larva mainly used amino acids and fatty acids for aerobic metabolism, and it could synthesize unsaturated fatty acids and stored them in the body; after entering the exogenous nutrition, cholesterol, lipids and proteins were the main nutritional sources of the larva.]
We would like to thank the referee again for taking the time to review our manuscript,
All authors
Author Response File: Author Response.pdf
This manuscript is a resubmission of an earlier submission. The following is a list of the peer review reports and author responses from that submission.
