Molecular Characterization and Nutrition Regulation of the Neutral Amino Acid Transporter ASCT2 in Triploid Crucian Carp

Round 1

Reviewer 1 Report

 This manuscript, “Molecular Characterization and Nutrition Regulation of the Neutral Amino Acid Transporter ASCT2 in Triploid Crucian Carp”, reported about the tissue expression levels of cloned ASCT2, relationship between the expression levels of ASCT2 in muscle and circadian rhythm, and the induction of ASCT2 expression in vivo and vitro by glutamate and glutamine. The information of ASCT2 is very important for research on feed formulation in aquaculture. After minor-revision, I recommend accept the manuscript on Fishs. I write my comments below.

Lane 100, please rewrite genome DNA to cDNA.

Lane 110, please add the version of MEGA-X.

Lane 111, please add the reference about Neighbor-Joining method.

K.Atteson  The performance of Neighbor-Joining Mrthods of phylogenetic reconstruction. Alugorithmica; 25, 251-278 (1999)

Lane 126. please add the reference about 2(-delta delta CT) methods. 

Xiayu Rao, Xuelin Huang, Zhicheng Zhou, and Xin Lin　An improvement of the 2ˆ(–delta delta CT) method for quantitative real-time polymerase chain reaction data analysis　Biostat Bioinforma Biomath.; 3(3): 71–85.(2013)

Lane 252, please add the reference that explain the difference in life habits between crucian carp and grass carp.

Author Response

Dear reviewer of Fishes,

 

Please find our revised manuscript “Molecular Characterization and Nutrition Regulation of the neutral amino acid transporter ASCT2 in Triploid Crucian Carp" (fishes-1448742), which we would like to resubmit for publication as an original research in Fishes. 

Your suggestions were very useful to our manuscript. We have revised this manuscript carefully according to all the suggestions. The following pages are our point-by-point responses to each of your suggestions. We hope that the revisions in the manuscript will be sufficient to make our manuscript suitable for publication in Fishes.

We look forward to hearing from you at your earliest convenience.

 

Yours sincerely,

Jianzhou Tang

—————————————————

Professor Jianzhou Tang (PhD)

Department of Biological and Environmental Science Changsha University,

Changsha, P.R. China, 410003

E-mail: Jianzhou Tang: [email protected]

Responses to reviewer-1

Suggestion 1:

Lane 100, please rewrite genome DNA to cDNA.

Response:

Thank you very much for your good suggestions. The “genomic DNA” in Lane 100 has been rewrited to “cDNA” in the revised version.

The revised version is as follows:

2.3. Cloning of cDNA of ASCT2

The primers of SLC1A5 were designed based on conserved sequences of ASCT2/SLC1A5 in other teleosts (Table 1). Polymerase chain reactions (PCRs) were carried out using a volume of 20 μL with 1 μL of cDNA (approximately 20 ng), 10 μL of 2 × Taq PCR Master Mix (Tiangen, Beijing, China), 8 μL of ddH2O, and 0.5 μL of each primer (100 μM).

Suggestion 2-4:

Lane 110, please add the version of MEGA-X.

Lane 111, please add the reference about Neighbor-Joining method.

Lane 126. please add the reference about 2(-delta delta CT) methods. 

Response:

Thank you very much for your good suggestions. We have inserted references based on your suggestions.

The related comtents is as follows: Evolutionary analyses were conducted in MEGA-X [23]. Evolutionary history was inferred using the Neighbor-Joining method [24].

The efficiency of the assay was determined by the amplification of a dilution series of cDNA and the analysis of relative mRNA expression was performed using the 2-ΔΔCt method [25].

The related confererce is as follows:

[23] Sudhir, K.; Glen, S.; Li, M.; Christina, K.; Koichiro, T. MEGA X: Molecular Evolutionary Genetics Analysis across computing platforms. Mol. Biol. Evol. 2018, 35, 1547-1549.

[24] Atteson, K. The Performance of Neighbor-Joining Methods of Phylogenetic Reconstruction. Algorithmica 1999, 25, 251-278.

[25] Rao, X.; Huang, X.; Zhou, Z.; Lin, X. An improvement of the 2(-delta delta CT) method for quantitative real-time polymerase chain reaction data analysis. Biostat Bioinforma Biomath 2013, 3, 71-85.

Suggestion 5:

Lane 252, please add the reference that explain the difference in life habits between crucian carp and grass carp.

Response:

Thank you very much for your good suggestions. As far as we know, crucian carp is an omnivorous fish and grass carp is a herbivorous fish. However, after the author's argument, we believe that the behavior of crucian carp is complicated by multiple factors, and the data in this article is not enough to support the original conclusion, so we revised the relevant discussion of the manuscript.

Author Response File: Author Response.pdf

Reviewer 2 Report

The manuscript submitted by Mao et al. investigated the characterization of the Alanine, Serine, Cysteine Transporter 2 of the triploid crucian carp. ASCT2 also plays an important role in glutamine and glutamate absorption. Authors elucidated the dynamics of mRNA expression levels of asct2 as the feed glutamine and glutamate concentration changes. Also, focusing on the diurnal variation and the effects of feed protein levels are good attempt at understanding of ASCT2. This study provides an important contribution to the development of the aquaculture technologies. While the contents are sound and present well, I have concerns about the discussion and experimental design. There are some improvements that should be made before publication, which I believe will improve the readability of the paper.

 

Major comments

・With regard to content, there appears to be a lack of introductory material. I would anticipate that adding this more complete introduction would also require to clearly articulate why these experiments were undertaken.

・It seems inadequate to conclude that ASCT2 exhibits the circadian rhythm from those experimental designs and results. I recommend to use the word “diurnal variation” instead of “circadian rhythm”. Please be careful about the word usage “circadian rhythm”.

・Authors contrasted in vivo and in vitro experiments. However, the intestine was used for in vivo experiment and the muscle was used for in vitro experiment. It would be difficult to compare those two results equally. It could be worth checking the muscular mRNA expression levels as in vivo experiment.

・ASCT2 are known to be a Na⁺ and pH-dependent transport. Feeding condition changes pH in the gastrointestinal tract. Moreover, medium pH is important. Please mention the pH condition and discuss carefully.

 ・In discussion, the interpretation of obtained results was not enough. Particularly, increases and decreases of mRNA expression levels.  

 

Minor comments

Overall

・”in vivo” and “in vitro” should be italicized.

・Please confirm the significant differences. Some points were needs to re-exam as seen in the size of error bars.

・I recommend that authors give the information which tissue used in the M&M section, although authors put the information in the results section.

 

Materials and Methods

・Please include enough information of fish such as the body sizes and weight.

・(line 93) please provide the company’s location.

・(line 100)　Is “genomic” DNA correct?  It would be “complementary” DNA?

・(line 101) please mention the concentration of primes.

・(section 2.3.) Sequencing methods are lacked.

・(section 2.5. and other qPCR) Did authors confirm the single amplification?

For example, a dissociation curve method or electrophoresis.

・(section 2.6.-2.8) the biological replication numbers is required.

・(section 2.6.-2.8) please give the information of feeding conditions.

Feeding or fasting ? (How long ? ).  Satiation or constant volume?

・(section 2.8.) please explain why authors put the additional methionine into feeds.

・(lines 160 and 161) Remove the location of a company.

・(section 2.8.) please describe the method for the statistical analysis. If authors used Duncan as described in reference No.26, I recommend to use the other methods. The multiplicity problem has remained in the Duncan.

Results and Discussions

・(section 3.3.) please describe the results of feeding experiments, such as daily feed intake, daily growth rate, and feed efficiency .

・(section 3.4.) How many percent of the protein was required in this species basically?

・(line 284) spelling “groups”

 

Figures

 ・(Overall) The error bars indicate SE of SD?

・(Overall) Please explain the y axis of “relative expression”. The numbers were divided by the expression levels of beta-actin?

・(lines 327 for 005455767.2) the name of fish is missed. Please confirm the accession number.

・(Figure 2A) Why authors expressed “gut“ only this area. Other parts are expressed as “intestine”

Tables

・(Table. 1) Please confirm the sequences of ASCT2 and RT-ASCT2 primers. I could not find the nucleotide sequences in MW4355568.1.

 

References

・(Overall) The page numbers are lacked in most references.

・(Overall) Some references are incorrect. Please confirm all the references.

   No.9 : plos journal

Author Response

Dear reviewer of Fishes,

 

Please find our revised manuscript “Molecular Characterization and Nutrition Regulation of the neutral amino acid transporter ASCT2 in Triploid Crucian Carp" (fishes-1448742), which we would like to resubmit for publication as an original research in Fishes. 

Your suggestions were very useful to our manuscript. We have revised this manuscript carefully according to all the suggestions. The following pages are our point-by-point responses to each of your suggestions. We hope that the revisions in the manuscript will be sufficient to make our manuscript suitable for publication in Fishes.

We look forward to hearing from you at your earliest convenience.

 

Yours sincerely,

Jianzhou Tang

—————————————————

Professor Jianzhou Tang (PhD)

Department of Biological and Environmental Science Changsha University,

Changsha, P.R. China, 410003

E-mail: Jianzhou Tang: [email protected]

Responses to reviewer-2

Major comments

Suggestion 1:

With regard to content, there appears to be a lack of introductory material. I would anticipate that adding this more complete introduction would also require to clearly articulate why these experiments were undertaken.

Response:

Thank you very much for your good suggestions. We have added the introductory material in the background based on your modification suggestions.

The related confererce is as follows:

Glutamine is a vehicle for nitrogen transfer between tissues, which plays an im-portant role in cell growth [14]. Hu et al. reported that 7.5 g/kg glutamine and 2.5 g/kg glutamine dipeptide could remarkably improve the expression of ASCT2 in grass carp (Ctenopharyngodon idelus) [15]. Therefore, it is necessary to study the transport mechanism of glutamine in ASCT2. In the intestine, glutamate, an important nutrient in the maintenance of intestinal mucosal activity, is taken up from the lumen or is produced in the epithelium from glutamine through glutaminase activity [16]. Moreover, Kanai et al. reported that glutamate was also one of the transport substrates of ASCT2 [17]. Thus, the regulatory effect of glutamate on ASCT2 is also worthy of our attention.

Protein is the core component in fish feed, so researchers improve the feed formula by adjusting the protein level and composition of the diet and the use of feed additives. In our previous research, we reported the effects of different dietary protein levels on the expression levels of Peptide transporter 1, L-Type amino acid transporter 2 (LAT2), and Glutamate dehydrogenase (GDH) in crucian carp [18-20]. These proteins showed different preferences for dietary protein levels. For example, LAT2 of crucian carp showed high expression levels after fed with diets of 37 and 42% protein levels [19]. GDH of crucian carp showed high expression levels after fed with diets of 22% [20]. Therefore, studying the expression of crucian carp ASCT2 under the different protein levels will help to deepen the understanding of ASCT2 and improve the dietary formula.

Crucian carp is one of the most important freshwater species for Chinese aquacul-ture. Triploid crucian carp (3n = 150) has the characteristics of resilience, sterile traits and fast growth rate [21]. However, the molecular mechanism of the rapid growth of triploid crucian carp is still unclear. Hence, we cloned ASCT2 cDNA from triploid crucian carp and studied the expression pattern in different tissues and diurnal variation. Furthermore, we analyzed the regulation of ASCT2 by dietary protein levels and protein source, as well as feed additives (glutamate and glutamine) in triploid crucian carp. The results of this study will contribute to enrich the knowledge of fish ASCT2, and have a potential application in improving fish feed formulation in aquaculture.

 

Suggestion 2:

It seems inadequate to conclude that ASCT2 exhibits the circadian rhythm from those experimental designs and results. I recommend to use the word “diurnal variation” instead of “circadian rhythm”. Please be careful about the word usage “circadian rhythm”.

Response:

Thank you very much for your good suggestions. We have revised the related content in the revised manuscript.

 

Suggestion 3:

Authors contrasted in vivo and in vitro experiments. However, the intestine was used for in vivo experiment and the muscle was used for in vitro experiment. It would be difficult to compare those two results equally. It could be worth checking the muscular mRNA expression levels as in vivo experiment.

Response:

Thank you very much for your good suggestions. We have added resluts of muscle in vivo in the modified version, and analyzed/discussed the added data.

 

Suggestion 4:

ASCT2 are known to be a Na⁺ and pH-dependent transport. Feeding condition changes pH in the gastrointestinal tract. Moreover, medium pH is important. Please mention the pH condition and discuss carefully.

Response:

Thank you very much for your good suggestions. Based on your suggestion, we carefully discussed the effect of pH on the results of the experiment (in vivo and in vitro) in the experiment. In addition, in the in vitro experiment, due to the presence of HEPES in the medium, The pH in the medium is stable （pH = 7.2).

 

Suggestion 5:

In discussion, the interpretation of obtained results was not enough. Particularly, increases and decreases of mRNA expression levels.

Response:

Thank you very much for your good suggestions. In the revised version, we have added relevant discussions on changes in experimental results.

The partial related confererce is as follows:

In the present study, we found that FCR and FR of triploid crucian carp were increased with dietary supplementation with gluatamate or glutamine, which were consistent with the research in Atlantic salmon (Salmo salar L.) and grass carp [37,38]. Glutamine is the main respiratory substrate of intestinal cells, which can significantly improve the morphology and function of the intestine [6]. In intestine, it is transported by ASCT2. Besides, ASCT2 was also reported as the transporter of glutamate, despite with low af-finity compared to that of glutamine [17]. By comparing the effect of glutamate additive on expression level of ASCT2 between intestine and muscle in vivo, we found they appeared the similar trend. When the additive of glutamate in the feed was low, the expression level of ASCT2 decreased with the increase of glutamate additives. We speculate it may be related the change of pH, as ASCT2 is a Na+-dependent neutral amino acid transporter. Then, the expression level of ASCT2 increased with the increase of glutamate additives, which indicated that glutamate could improve the expression level of ASCT2. Later, the expression level of ASCT2 decreased significantly with the increase of glutamate additives. We speculated that it was a self-protection mechanism of ASCT2. By comparing the effects of glutamate on muscle in vivo and in vitro, we found that they also shared the similar trend. It is worth noting that ASCT2 is less sensitive to glutamate in vitro compared to in vitro, which may be due to the neutral pH in the medium. Compared to the glutamate group, the expression level of ASCT2 in the glutamine group showed the more complex trend. It indicated that ASCT2 was more sensitive to glutamine compared to glutamate. When the additive of glutamine in the feed was low, the expression level of ASCT2 in the intestines and muscles shows oscillating changes, although the changes are not obvious. When the glutamine additive reached 2.0% and 2.5%, the expression level of ASCT2 in the intestine and muscles increased significantly, respectively. It indicated that glutamine could also promote the expression of ASCT2. In vitro, the expression levels of ASCT2 showed a sharp decline in the glutamine additive groups, which might be related to a certain concentration of glutamine in medium. It also indicated that ASCT2 was more sensitive to glutamine compared to glutamate.

 

Minor comments

Suggestion 6:

“in vivo” and “in vitro” should be italicized.

Response:

Thank you very much for your good suggestions. According to your suggestion, we revised the corresponding content in the article.

 

Suggestion 7:

Please confirm the significant differences. Some points were needs to re-exam as seen in the size of error bars.

Response:

Thank you very much for your good suggestions. In response to the data in the article, we restarted the calculation and made corrections.

 

Suggestion 8:

I recommend that authors give the information which tissue used in the M&M section, although authors put the information in the results section.

Response:

Thank you very much for your good suggestions. We have added relevant organization information to M&M section.

 

Suggestion 9:

Please include enough information of fish such as the body sizes and weight.\

Response:

Thank you very much for your good suggestions. Compared with volume, we believe that weight can better reflect the effect of dietary levels on fish nutrition regulation, so we only added weight-related information in the revised version.

 

Suggestion 10:

(line 93) please provide the company’s location.

Response:

Thank you very much for your good suggestions. We have added relevant information in the revised version.

The related confererce is as follows:

The integrity of RNA samples were tested by agarose gel electrophoresis and the quality of RNA samples were determined by calculating through the A260/A280 and A260/A230 ratios spectrophotometer (BioPhotometer Eppendorf, Germany).

 

Suggestion 11:

(line 100) Is “genomic” DNA correct? It would be “complementary” DNA?

Response:

Thank you very much for your good suggestions. The “genomic DNA” in Lane 100 has been rewrited to “cDNA” in the revised version.

The revised version is as follows:

2.3. Cloning of cDNA of ASCT2

The primers of SLC1A5 were designed based on conserved sequences of ASCT2/SLC1A5 in other teleosts (Table 1). Polymerase chain reactions (PCRs) were carried out using a volume of 20 μL with 1 μL of cDNA (approximately 20 ng), 10 μL of 2 × Taq PCR Master Mix (Tiangen, Beijing, China), 8 μL of ddH2O, and 0.5 μL of each primer (100 μM).

 

Suggestion 12:

(line 101) please mention the concentration of primes

Response:

Thank you very much for your good suggestions. We described the concentration of primers in the related content.

The revised version is as follows:

Polymerase chain reactions (PCRs) were carried out using a volume of 20 μL with 1 μL of cDNA (approximately 20 ng), 10 μL of 2 × Taq PCR Master Mix (Tiangen, Beijing, China), 8 μL of ddH2O, and 0.5 μL of each primer (100 μM).

 

Suggestion 13:

(section 2.3.) Sequencing methods are lacked

Response:

Thank you very much for your good suggestions. We have added related sequencing methods in 2.3.

The revised version is as follows:

The temperature profile during amplification was as follows: initial denaturation at 94°C for 5 min, followed by 35 cycles at 94°C for 30 s, 55°C for 1 min, and 72°C for 2 min. A final extension step was performed at 72°C for 10 min. The PCR products were separated by polyacrylamide gel electrophoresis (PAGE). The DNA fragments were purified using a gel extraction kit (UNIQ-10 Spin Column DNA Gel Extraction Kit for PAGE, Sangon) and ligated into the pMD19-T vector. The plasmids were transformed into E.coli DH5α and purified. The inserted DNA fragments in the pMD19-T vector were sequenced using an automated DNA sequencer (ABI PRISM 3730, Applied Biosystems, Carlsbad, CA). All the PCRs in this study were repeated, and the results were consistent.

 

Suggestion 14:

(section 2.5. and other qPCR) Did authors confirm the single amplification?

For example, a dissociation curve method or electrophoresis.

Response:

Thank you very much for your good suggestions. Before the qPCR experiment, we tested the specificity of the primers by agarose gel electrophoresis.

The revised version is as follows:

The ASCT2 mRNA levels were determined by qRT-PCR in a Prism 7500 Sequence Detection System (Applied Biosystems, Foster City, CA, USA). The primers were de-signed using Primer Premier 5.0 software (Table 1) and the single band of amplification is determined by agarose gel electrophoresis.

 

Suggestion 15:

(section 2.6.-2.8) the biological replication numbers is required.

Response:

Thank you very much for your good suggestions. We have added the relevant data of biological replication numbers (n=3) in the revised version.

The revised version is as follows:

2.6. ASCT2 expression in various tissues and diurnal variation

Before the experience, the fish (body weight 11.72 ± 0.11 g) were acclimatized for two weeks to indoor recirculating aquaculture system comprising 4 glass fiber tanks (1500 L). They were fed twice daily at 8:30 a.m. and 2:30 p.m. respectively. In order to explore the expression differences of ASCT2 among different tissues, different tissues were collected from triploid crucian carp, including heart, spleen, brain, intestine, liver, kidney, and muscle. Later, the samples of muscle in triploid crucian carp were collected at eight different timing (00:00, 03:00, 06:00, 09:00, 12:00, 15:00, 18:00, 21:00, and 24:00). Three replicates for each experimental group were designed. Total RNA was isolated from these samples, after synthesis of cDNAs, ASCT2 expressions were assayed by qRT-PCR.

2.7. The effect of glutamate and glutamine on ASCT2

In order to analyze the effects of different proportions of dietary glutamate and glutamine additives on ASCT2, the fish were randomly divided into thirteen groups (n = 15 for each group) including control group (body weight of fish is 300.00 ± 0.00 g), six groups (body weight of fish is 300.63 ± 1.34 g) of glutamate and six groups (body weight of fish is 300.00 ± 0.00 g) of glutamine. The formula for the control group contained 32% CP, while the formula for the glutamate/glutamine groups were supplemented with 0.5, 1, 1.5, 2.0, 2.5, 3.0 g/kg glutamate or glutamine (Table 2 and 3). The fish were acclimatized for two weeks to outdoor cage (1.2 m × 1.2 m × 2.0 m, water depth: 1.8 m). They were fed on the control diet twice daily at 8:30 a.m. and 2:30 p.m., respectively. Five hundred and eighty-five acclimatized fish of the same age and size (each weighing 300.00 ± 10.15 g) were batch weighed and randomly distributed into 39 cages. For each of the 39 diet treatments, triplicate cages were set up with each cage having 15 fish. The fish were fed manually to apparent satiation twice daily, at 8:30 a.m. and 2:30 p.m. Feeding was done for 60 days with each of the 13 diets to the allocated cages. After 60 d, the fish were fasted for 24 h and then sacrificed with 50 mg L 1 MS-222 (tricaine methane sulfonate, SigmaAldrich, USA) before sampling and the ASCT2 expression levels of intestine and muscle in triploid crucian carp (n = 3 for each group) were assayed by qRT-PCR.

In order to deepen explore the effects of additive of glutamate and glutamine on ASCT2, in vitro, muscle cell of triploid crucian carp was used to carry out the experiment. During the experience, the muscle was rapidly separated from triploid crucian carp (25 g) after anesthetized with 2-phenoxyethanol. The muscle was cut into pieces by using scissors. After three washes with PBS containing 500 U/ml penicillin (Gibco, USA) and 500 U/ml streptomycin (Gibco), minced muscle tissues were seeded in dishes with DMEM/F12 containing 20% FBS (Gibco), 500 U/ml penicillin, 500 U/ml streptomycin and 15 ng/ml fibroblast growth factor (FGF, PEPROTECH, USA) in 28 ℃. Afterward, culture medium was replaced with fresh medium by half every 3 days. Subculture was carried out at a split ratio of 1:2 when primary cell cultures grew to above 90% of confluence. After subculture of 3 passages, the cells were inoculated into 6-well plates and randomly divided into 6 groups of 4 wells. After passaging, the cells had adhered well and were normally extended (3rd day). The original culture solution was aspirated using a sterile pipette and gently washed with 2 mL PBS. Complete cell medium containing different concentrations of glutamate/glutamine (0, 0.10, 0.25, 0.50, 1.00 and 2.00 mg/mL) were added to each group of cells. Because HEPES is added to DMEM/F12 medium, the pH of the medium remains stable (pH = 7.2). After 8 h of culture, removing the cell culture medium and collecting the cells. The relative mRNA expression levels of ASCT2 were detected by qRT-PCR.

2.8. Regulation of dietary protein levels and protein sources on ASCT2

To reveal the effects of dietary protein level on triploid crucian carp intestine ASCT2 expression, six isocaloric diets with five different dietary protein levels at 26, 29, 32, 35, 38 and 41%, respectively were formulated (Table 4). To reveal the effects of different protein sources on triploid crucian carp intestine ASCT2 expression, two protein diets of fish meal and soybean meal were formulated (Table 5). The diets of different protein levels and sources were prepared by thoroughly mixing dry ingredients with oil and then water until a stiff dough is obtained. The dough was then passed through a meat-mincer equipped with a 2 mm diameter, and the products were dried using an electrical fan at 28 ℃. After drying, the material was broken up into regular pieces sieved to a convenient pellet size and stored at 20 ℃. Juvenile triploids (body weight 11.72 ± 0.11 g) were cultured in eight fiberglass tanks (1.2 m height 0.8 m depth; n = 25 per tank). Before the experience, the fish were fasted for 24 h and then fed with different diets for 60 d. The fish were fed manually to apparent satiation twice daily, at 8:30 a.m. and 2:30 p.m. After 60 d, the fish were fasted for 24 h and then sacrificed with 50 mg L 1 MS-222 (tricaine methane sulfonate, SigmaAldrich, USA) before sampling. Finally, the intestine of triploid crucian carp (n = 3 for each group) was collected for gene expression analysis by qRT-PCR.

 

Suggestion 16:

(section 2.6.-2.8) please give the information of feeding conditions.

Feeding or fasting ? (How long ? ). Satiation or constant volume?

Response:

Thank you very much for your good suggestions. We have added information about breeding conditions in the modified version. Before the experience, the fish were fasted for 24 h and then fed with different diets for 60 d. The fish were fed manually to apparent satiation twice daily, at 8:30 a.m. and 2:30 p.m.

 

Suggestion 17:

(section 2.8.) please explain why authors put the additional methionine into feeds.

Response:

Thank you very much for your good suggestions. Methionine was added to the feed formula in this experiment to meet the nutritional requirements of crucian carp feed for methionine.

 

Suggestion 18:

(lines 160 and 161) Remove the location of a company.

Response:

Thank you very much for your good suggestions. We have remove the location of a company in the related lines.

The revised version is as follows:

After three washes with PBS containing 500 U/ml penicillin (Gibco, USA) and 500 U/ml streptomycin (Gibco), minced muscle tissues were seeded in dishes with DMEM/F12 containing 20% FBS (Gibco), 500 U/ml penicillin, 500 U/ml streptomycin and 15 ng/ml fibroblast growth factor (FGF, PEPROTECH, USA) in 28 ℃.

 

Suggestion 19:

(section 2.8.) please describe the method for the statistical analysis. If authors used Duncan as described in reference No.26, I recommend to use the other methods. The multiplicity problem has remained in the Duncan.

Response:

Thank you very much for your good suggestions. We have received your advice and described the new method for the statistical analysis in the revised version

The revised version is as follows:

2.9. Statistical analysis

The data analysis was measured by using SPSS 18 analysis program and represented as means ± standard error (SE) [26]. The significant difference between groups was confirmed using one-way ANOVA analysis. Differences between mean values of individual treatments were separated using the Least Significance Difference (LSD) test at P < 0.05.

 

Suggestion 20:

(section 3.3.) please describe the results of feeding experiments, such as daily feed intake, daily growth rate, and feed efficiency

Thank you very much for your good suggestions. We have added the results of the daily growth coefficient (DGC), feed conversion efficiency (FCR) and feeding rate (FR) in the revised version.

The revised version is as follows:

After 60 days of feeding trial, the daily growth coefficient (DGC), feed conversion efficiency (FCR) and feeding rate (FR) of experimental fish fed with different levels of glutamate and glutamine are shown in Table 3. DGC of fish fed diet with 1% glutamate, as well as 1.0%, 1.5%, and 2.5% glutamine, were significantly lower than fish fed control diet (p < .05). FCR of fish fed 1.0% glutamate diet and 2.5% diet glutamine were significantly higher than fish fed control diet (p < .05). Additionally, FR of fish fed 3.0% glutamate was significantly higher than fish fed control diet (p < .05).

 

Suggestion 21:

(section 3.4.) How many percent of the protein was required in this species basically?

Response:

Thank you very much for your question. According to the standard of crucian carp diet, the protein feed requirement for adult crucian carp is between 30 and 35.

 

Suggestion 22:

(line 284) spelling “groups”

Response:

Thank you very much for your reminding. Due to the addition of the content in the results, we have revised the related contents in the discussion. The spelling error you pointed out has been removed in the new version.

 

Suggestion 23:

(Overall) The error bars indicate SE of SD?

Response:

Thank you very much for your question. In the previous version, the error bars we used is SD. However, in our modified version, we re-stated all the data and used a new error bar (SE).

 

Suggestion 24:

(Overall) Please explain the y axis of “relative expression”. The numbers were divided by the expression levels of beta-actin?

Response:

Thank you very much for your question. The “Related expression” in y axis indicated the relative expression level between data. Beta-actin was used as an internal reference gene.

 

Suggestion 25:

(lines 327 for 005455767.2) the name of fish is missed. Please confirm the accession number.

Response:

Thank you very much for your reminding. We have added the missing content in the revised version.

The related content in the revised version is as follows:

Figure 1. Phylogenetic tree of ASCT2 constructed using the neighbor-joining method. The accession numbers are as below: Oreochromis niloticus XM_005455767.2.

 

Suggestion 26:

(Figure 2A) Why authors expressed “gut“ only this area. Other parts are expressed as “intestine”

Response:

Thank you very much for your reminding. We have unified the wording of the intestine in the revised version

 

Suggestion 27:

(Table. 1) Please confirm the sequences of ASCT2 and RT-ASCT2 primers. I could not find the nucleotide sequences in MW4355568.1.

Response:

Thank you very much for your reminding. The GenBank Accession number is MW435568.1.

The related content in the manuscript is as follows:

3. Results

3.1. Isolation and Sequence Analysis of ASCT2 cDNA from Triploid Crucian Carp

The cDNA of ASCT2 (GenBank Accession No. MW435568.1) was isolated from the cDNA library of triploid crucian carp intestine.

 

Suggestion 28:

(Overall) The page numbers are lacked in most references.

Response:

Thank you very much for your reminding. We have added the page numbers in the related reference.

 

Suggestion 29:

(Overall) Some references are incorrect. Please confirm all the references

Response:

Thank you very much for your reminding. In the revised version, we have checked all the references.

Author Response File: Author Response.docx

Reviewer 3 Report

Mao et al present an interesting analysis about asct2 in triploid crucian carp. Authors determined the sequence of asct2 and found characteristic expressional patterns of asct2 in triploid crucian carp tissues. I listed some points to care about below.

 

First of all, throughout the text, please use accurate nomenclature, ex. “asct2” for gene and mRNA, and “Asct2” for protein.

 

Introduction

• Line54-55: Please add line 51.
• Line 55-56: Not necessary.
• Line57-58: If any reports about the relationship between asct2 and glutamine in fish, please add the details.
• Line62-63: Redundant.

 

Materials

• Line 90: “integrality” means “integrity”?
• Line 110: Please add the reference of MEGA-X.
• Please explain the section 2.8 before the section 2.7, as the same order with result section.

 

Results

• Please add the accurate p-values.
• Please add which tissues (intestine or muscle) you used for glutamate/glutamine analysis in section 3.3 and 3.4.
• In Figure 2A, why the expression level of intestine was not included? If any expression data of intestine, please add in Figure 2A.
• In Figure 3, why you do not use the same kind of tissues? If any asct2 expression data in muscle tissue (in vivo) or in intestine cells (in vitro), please add in Figure 3.
• In Figure4, please change the y-axis label from “relative expression” to “Relative expression”.

 

Discussion

• Please use “in intestine tissues” and “in muscle cells”, instead of “in vivo” and “in vitro” to avoid confusion.
• Line 251; Please explain the details of how the difference in life habits effects on the expressional difference of asct2 between grass carp between triploid crucian carp.
• Line259: “zabrafish” means “zebrafish”?
• Line271: “on the other hand” is not necessary.

Author Response

Dear reviewer of Fishes,

 

Please find our revised manuscript “Molecular Characterization and Nutrition Regulation of the neutral amino acid transporter ASCT2 in Triploid Crucian Carp" (fishes-1448742), which we would like to resubmit for publication as an original research in Fishes. 

Your suggestions were very useful to our manuscript. We have revised this manuscript carefully according to all the suggestions. The following pages are our point-by-point responses to each of your suggestions. We hope that the revisions in the manuscript will be sufficient to make our manuscript suitable for publication in Fishes.

We look forward to hearing from you at your earliest convenience.

 

Yours sincerely,

Jianzhou Tang

—————————————————

Professor Jianzhou Tang (PhD)

Department of Biological and Environmental Science Changsha University,

Changsha, P.R. China, 410003

E-mail: Jianzhou Tang: [email protected]

Responses to reviewer-3

Suggestion 1:

First of all, throughout the text, please use accurate nomenclature, ex. “asct2” for gene and mRNA, and “Asct2” for protein.

Response:

Thank you very much for your good suggestions. In our revised version, we use SLC1A5 to represent gene and ASCT2 to represent protein. We think this revision is appropriate.

 

Suggestion 2:

Introduction

Line54-55: Please add line 51.

Response:

Thank you very much for your kindly suggestions. In our revised version, we have adjusted the location of related content.

The related confererce is as follows:

In contrast to numerous research hotspots in human physiology and pathology, the researches in aquaculture regarding ASCT2 molecular characterization and nutrition regulation, which could contribute to the researches on the improvement of feed formula, were sparse.

 

Suggestion 3:

• Line 55-56: Not necessary.

Response:

Thank you very much for your kindly suggestions. We have removed the related content in the revised version.

 

Suggestion 4:

• Line57-58: If any reports about the relationship between asct2 and glutamine in fish, please add the details.

Response:

Thank you very much for your good suggestions. In the revised version, we have described the reports on the relationship between glutamate and ASCT2 in fish.

The related confererce is as follows:

Hu et al. reported that 7.5 g/kg glutamine and 2.5 g/kg glutamine dipeptide could remarkably improve the expression of ASCT2 in grass carp (Ctenopharyngodon idelus) [15].

 

Suggestion 5:

• Line62-63: Redundant.

Response:

Thank you very much for your kindly suggestions. We have removed the related content in the revised version.

 

Suggestion 6:

• Line 90: “integrality” means “integrity”?

Response:

Thank you very much for your good suggestions. We replaced integrity with integrity in the modified version

The related confererce is as follows:

The integrity of RNA samples were tested by agarose gel electrophoresis and the quality of RNA samples were determined by calculating through the A260/A280 and A260/A230 ratios spectrophotometer (BioPhotometer Eppendorf, Germany).

 

Suggestion 7:

Line 110: Please add the reference of MEGA-X.

Response:

Thank you very much for your good suggestions. We have added the reference of MEGA-X in the revised version.

The related confererce is as follows:

Evolutionary analyses were conducted in MEGA-X [23].

• Sudhir, K.; Glen, S.; Li, M.; Christina, K.; Koichiro, T. MEGA X: Molecular Evolutionary Genetics Analysis across computing platforms. Mol. Biol. Evol. 2018, 35, 1547-1549.

 

Suggestion 8:

• Please explain the section 2.8 before the section 2.7, as the same order with result section.

Response:

Thank you very much for your good suggestions. We have adjusted the location of related content.

 

Suggestion 9:

Please add the accurate p-values.

Response:

Thank you very much for your suggestions. In the revised version, in order to ensure the accuracy of the p-value, we recalculated all the experimental data in the article, and used a new statistical method (LSD) and error bars (SE). We make sure that the p-values in the revised version is accurate.

 

Suggestion 10:

• Please add which tissues (intestine or muscle) you used for glutamate/glutamine analysis in section 3.3 and 3.4.

Response:

Thank you very much for your suggestions. Followed your suggestion, we add the tissues information in the sectin 3.3 and 3.4.

The related confererce is as follows:

The expression levels in intestine and muscle of triploid crucian carp fed with dif-ferent concentrations of glutamate were showed in Figure 3A and B.

After feeding for 60 d, the expression levels of ASCT2 in triploids (intestine and muscle) were assayed by qRT-PCR. The results were showed in Figure 4.

 

Suggestion 11:

In Figure 2A, why the expression level of intestine was not included? If any expression data of intestine, please add in Figure 2A.

Response:

Thank you very much for your suggestions. In Figure 2, we have used “gut” to represent intestine. thus, in the revised version, we revised “gut” into “intestine”.

 

Suggestion 12:

• In Figure 3, why you do not use the same kind of tissues? If any asct2 expression data in muscle tissue (in vivo) or in intestine cells (in vitro), please add in Figure 3.

Response:

Thank you very much for your suggestions. We have added the data in muscle tissue in vivo in Figure 3, and carried out discussion/analysis on the added data.

 

Suggestion 13:

• In Figure4, please change the y-axis label from “relative expression” to “Relative expression”.

Response:

Thank you very much for your suggestions. We have revised the mistake in the revised version.

 

Suggestion 14:

Please use “in intestine tissues” and “in muscle cells”, instead of “in vivo” and “in vitro” to avoid confusion.

Response:

Thank you very much for your suggestions. In order to avoid confusion, in the results and discussion of revised version, we have described the experimental materials in detail.

 

Suggestion 15:

Line 251; Please explain the details of how the difference in life habits effects on the expressional difference of asct2 between grass carp between triploid crucian carp.

Response:

Thank you very much for your suggestions. Due to the changes in the data structure of the manmuscript, we have made some modifications in the discussion. After our discussion, we think that there is a complex regulation mechanism for fish behavior. The data in this study is not enough to support the original conclusion, so we remove it from the revised version.

 

Suggestion 16:

• Line259: “zabrafish” means “zebrafish”?

Response:

Thank you very much for your suggestions. We have corrected the spelling in the modified version.

 

Suggestion 17:

Line271: “on the other hand” is not necessary

Response:

Thank you very much for your suggestions. The related content was removed in the revised version.

Author Response File: Author Response.docx

Round 2

Reviewer 2 Report

This second version of the paper is a great improvement. I think this manuscript will be acceptable after some corrections have been done.

1. The representation of p-value should be unified throughout the manuscript.
2. (Section 2.7) Approximately 300 g fish were used for this experiment. In other section，less than one-tenth (11 g-25 g) of fish were used. Moreover, the body size of fish (300.00 ±0.00) is correct?
3. (Lines 363 and 486) Please make corrections for “50 mg L 1 MS-222”

Author Response

Dear reviewer of Fishes,

 

Please find our revised manuscript “Molecular Characterization and Nutrition Regulation of the neutral amino acid transporter ASCT2 in Triploid Crucian Carp" (fishes-1448742), which we would like to resubmit for publication as an original research in Fishes. 

Thank you very much for your valuable suggestions on the revision of the article. We have further revised the manuscript according to your suggestions. The following pages are our point-by-point responses to each of your suggestions. We hope that the revisions in the manuscript will be sufficient to make our manuscript suitable for publication in Fishes.

We look forward to hearing from you at your earliest convenience.

 

Yours sincerely,

Jianzhou Tang

—————————————————

Professor Jianzhou Tang (PhD)

Department of Biological and Environmental Science Changsha University,

Changsha, P.R. China, 410003

E-mail: Jianzhou Tang: [email protected]

Point-by-point responses:

Suggestion 1:

The representation of p-value should be unified throughout the manuscript.

Response:

Thank you very much for your good suggestions. We have unified the representation of p-value in the revised version, and unified the writing of 0.05 to .05.

 

 

Suggestion 2:

(Section 2.7) Approximately 300 g fish were used for this experiment. In other section，less than one-tenth (11 g-25 g) of fish were used. Moreover, the body size of fish (300.00 ±0.00) is correct?

Response:

Thank you very much for your good suggestions. First, the reason for the use of small-sized fish in the experiment is the number of large-sized fish was not enough to complete the other experiment, so we used small-sized fish with the same specification. In addition, we determined that the weight of the fish in some experimental groups was 300±0.00 g. We have also considered such a coincidental number. In the previous version, we respected the original data and described the weight of fish truthfully-"In order to analyze the effects of different proportions of dietary glutamate and glutamine additives on ASCT2, the fish were randomly divided into thirteen groups (n = 15 for each group) including control group (body weight of fish is 300.00 ± 0.00 g), six groups (body weight of fish is 300.63 ± 1.34 g) of glutamate and six groups (body weight of fish is 300.00 ± 0.00 g) of glutamine." Most of the fish weights are exactly 300, so in order to avoid readers' doubts, we provide accurate fish weights for each group (as shown in our previous version). However, according to your doubts, we believe that this description does not achieve our expected results, so we decided to aggregate the weights of these groups of fish to avoid a coincidence number such as 300.00 ± 0.00 g.

The revised version is as follows:

In order to analyze the effects of different proportions of dietary glutamate and glutamine additives on ASCT2, the fish were randomly divided into thirteen groups (n = 15 for each group, body weight of fish is 300.35 ± 0.04 g).

 

 

Suggestion 3:

(Lines 363 and 486) Please make corrections for “50 mg L 1 MS-222”

Response:

Thank you very much for your good suggestions. We have made corrections for “50 mg L 1 MS-222” in the revised version.

The revised version is as follows:

After 60 d, the fish were fasted for 24 h and then sacrificed with 50 mg L-1 MS-222 (tricaine methane sulfonate, SigmaAldrich, USA) before sampling and the ASCT2 ex-pression levels of intestine and muscle in triploid crucian carp (n = 3 for each group) were assayed by qRT-PCR.

After 60 d, the fish were fasted for 24 h and then sacrificed with 50 mg L-1 MS-222 (tricaine methane sulfonate, SigmaAldrich, USA) before sampling.

Reviewer 3 Report

Thank you very much for your sincerely revision.

I would like to recommend acceptance of your manuscript.

Author Response

Dear reviewer of Fishes,

      Thank you very much for your affirmation.

Yours sincerely,

Jianzhou Tang

—————————————————

Professor Jianzhou Tang (PhD)

Department of Biological and Environmental Science Changsha University,

Changsha, P.R. China, 410003

E-mail: Jianzhou Tang: [email protected]