Molecular Characterization and Expression Response of Ghrelin, GLP-1 and PYY to Fasting, Dietary Lipid, and Fatty Acids in Silver Pomfret (Pampus argenteus)

Round 1

Reviewer 1 Report (Previous Reviewer 1)

The present manuscript is a revised version of a manuscript previously submitted to the journal. In my second review, I suggested the authors to include a proximate analysis of the HFD. In their response, the authors told they did it and that the result was that lipid composition was lower than expected, 14%, but still higher than the control. They also provided a description of the methodology used. This information has not been included in the present version and it should be.

Author Response

Response to Reviewer 1

The present manuscript is a revised version of a manuscript previously submitted to the journal. In my second review, I suggested the authors to include a proximate analysis of the HFD. In their response, the authors told they did it and that the result was that lipid composition was lower than expected, 14%, but still higher than the control. They also provided a description of the methodology used. This information has not been included in the present version and it should be.

 

Thanks for your suggestion. We updated the proximate composition of the diets after the actual measurements. We also supplemented and described the methods of analysis as following:

‘The CON diet was commercial feed contained 5.0% carbohydrate, 52.1% protein, and 7.9% lipid. Diet with14% lipid was made by mixing 91.3% (w/w) CON diet with 8.7% (w/w) soybean oil. It finally contained 4.6% carbohydrate, 49.4% protein, and 14.4% lipid. Crude protein, crude lipid and ash content in diets were determined according to the methods of AOAC. Briefly, crude protein content was determined via the Dumas combustion methods with a protein analyzer (FP-528, Leco, USA). Crude lipid contents were determined by the ether extraction method using a Soxtec System HT (Soxtec System HT6, Tecator, Sweden). Ash contents were determined using a muffle furnace run at 550 °C for 8 h.’ Line134-140

Reviewer 2 Report (Previous Reviewer 3)

I recommend accept MS. minor revision. i found only a few minor editorial mistakes which I marked in the text. To see all of them, please open the file in Acrobat Reader.

Comments for author File: Comments.pdf

Author Response

Response to Reviewer 2

I recommend accept MS. minor revision. i found only a few minor editorial mistakes which I marked in the text. To see all of them, please open the file in Acrobat Reader.

 

Thanks for your advices. We have read the file and adjusted the content of the manuscript as your suggestions.

Line 71: ‘our change to present’

Revised. ‘to present knowledge’ Line 70

 

Line 237, 249, 259: ‘Change uppercase letters to lowercase letters’

Revised. We changed ‘Silver Pomfret’ to ‘silver pomfret’ in all legends of figures you marked.

Reviewer 3 Report (New Reviewer)

The findings of the study added value to the existing scientific knowledge as revealed the beneficial effects of fasting in fish. Overall the paper is interesting and can contribute significant findings in the literature. however, there are some issues as follow:

Title: accepted

 Abstract: well described

Introduction

Accepted

Materials & Methods: if possible, add references in every section

Results and discussion: accepted

Conclusion: Authors may put more efforts in this section by providing research gaps, future works and etc 

References: Please provide more recent references. At least 80% of the references are within 5 years

Author Response

Response to Reviewer 3

Title: accepted

 Abstract: well described

Introduction: Accepted

Materials & Methods: if possible, add references in every section

Answer: We had added references in the Materials & Methods section.

2.1. Fish：The experimental condition was controlled according to previous study. Line 95

2.2. Open reading frame (ORF) cloning of ghrelin, glp-1, pyya, and pyyb：The ORF cloning process was conducted as previous studies. Line 102

2.3. Sequence analysis：We had references before. Line 120 and 121

2.4. Feeding experiment：no references

2.5. Lipid concentrition experiment：We had supplemented references. Line 134 and 136

2.6. FAs incubation: We had references before. Line 151 and 161

2.7. Quantitative real-time PCR (qPCR): We had references before. Line 168

2.8. Data analysis and statistics: no references

Results and discussion: accepted

Conclusion: Authors may put more efforts in this section by providing research gaps, future works and etc 

We have supplemented the discussion section as following:

‘However, the regulation of external stimuli on fish feeding depends on the gut-brain axis. It is important and necessary to take further research on how ghrelin, glp-1, and pyy response to different nutritional situation in brain and its underlying mechanisms.’ Line 395-399

References: Please provide more recent references. At least 80% of the references are within 5 years

We had updated and added more recent references, but it is hard to reach 80% of the references within 5 years. We have done our best to find recent references, but there are only few researches mentioned with our point and some of the articles are very classic and original. 

Reviewer 4 Report (New Reviewer)

line 93-4 ; manually fed 

in methods, please describe the cellcultures and media with different FA

line 291 :the claim that s. pomfret has a specific function of ghrelin in tissues is doubtful. I suggest you replace with : in fish.

line 384 are probabaly involved

line 387 in response to different fatty acids, which may affect their utilization in s pomfret.

Author Response

Response to Reviewer 4

line 93-4 ; Change manual feeding to manually fed 

Revised.

‘Fish were manually fed two times a day at 8:00 and 17:00 with commercial feed (larvae love 6#, Japan) until satiety.’ Line 94-95

in methods, please describe the cell cultures and media with different FA

Preparation of different fatty acid mother liquor: ‘Six FAs were separately dissolved into bovine serum albumin (BSA, Sigma, USA) and configured into 6 different FA-BSA mother liquors with a concentration of 10 mM.’ Line 148-150

Incubation with different FA: ‘6 FA-BSAs and serum (as control) were added to the L-15 culture mediums respectively, and the final concentration of 6 FA-BSAs was 200 μM and serum was 0.4%.’ Line 159-160

line 291 :the claim that s. pomfret has a specific function of ghrelin in tissues is doubtful. I suggest you replace with : in fish.

‘The widespread distribution of ghrelin mRNA in silver pomfret, as in many other fish, suggests that ghrelin may have multiple tissue-specific activities in fish.’ Line 296-298

line 384 are probabaly involved

Revised. ‘In conclusion, ghrelin, glp-1, and pyya are probably involved in the regulation of appetite by fasting.’ Line 392-393

line 387 in response to different fatty acids, which may affect their utilization in s pomfret.

‘In addition, the expression of intestinal endocrine factors varied in response to different fatty acids, which may affect their utilization in silver pomfret.’ Line 394-396

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.

Round 1

Reviewer 1 Report

The present manuscript, submitted by Zhang et al., aimed to clone several genes involved in feeding regulation in a fish species, the silver pomfret, study their tissue distribution and how these genes are affected by fasting and fatty acids. In general, the work is interesting and the authors provide a good array of experiments. I thoroughly revised the manuscript and have several concerns that should be considered by the authors:

 

1)      One of the main criticisms is the lack of information in M&Ms section. The paradigm if this is the description of the experiment on tissue culture (section 2.6). For instance, how many fish were used in this experiment? How many replicates were used? How many tissue replicates were obtained from each fish? Were fish fed or fasted prior to sacrifice and tissue collection? In addition, one can guess that each FA was tested separately, but this should be clearly indicated here.

2)      My other main criticism is related to the HFD experiment. Although an effect was observed, the authors should provide a proximate analysis on the composition, in order to check that HFD feed had the expected amount of lipids. In addition, if possible, it would be good to perform also an analysis of the lipid profile of both diets for one main reason: the authors used a vegetable source for the extra lipid content (soybean oil) in the HFD feed. Do they know the lipid source for the control diet? Was it fish or vegetable oil? Are they sure that the differences observed between CON and HFD are due to the different lipid percentage and not to a different composition in the lipid profile of each diet?

 

Other comments:

 

3)      Throughout the manuscript, the authors use the term “starvation”. However, a more adequate term is “fasting” so I suggest replacing “starvation” with “fasting” in the whole manuscript.

4)      The scientific name of all fish should be included, at least the first time each species is mentioned.

5)      In general, the manuscript would benefit from an English revision. I found many grammar mistakes that, although small, in some cases they make the sentence confusing. For example, see: line 50, “resulted”; line 66, who purchased the fish? (“purchased by” or “purchased at”?); line 117, “contained with” or “containing”?; line 251, “similar to”, not “similar with”; etc.

6)      Line 47, not clear what authors mean with “sustainable aquaculture practices” in this context.

7)      Line 55, which neuropeptides? It would be good to list them here.

8)      Line 64, in the ethics section, is it possible to include here a license or authorization number?

9)      Line 137, amino acid sequence?

10)  Figure 4: the authors used the description “after short-term starvation” for the X-axis. This is confusing, I would say better something like “time after feeding” or “fasting time”.

11)  In general, the Discussion is too focused on comparing the author’s results with the results in other species and I missed more discussion on further implications of their results. For instance, in the first paragraph, may the different results on the sequences of pyya and pyyb be due to a difference in their functions? Could this be related with the differences observed in their distribution (e.g. expression in the intestine)?

Author Response

Dear Reviewer 1,

Thank you very much for giving us an opportunity to revise our manuscript. We appreciate for your constructive comments and suggestions on our manuscript entitled “Molecular Characterization and Expression Response of Ghrelin, GLP-1 and PYY to Starvation, Dietary Lipid, and Fatty Acids in Silver Pomfret (Pampus argenteus)”. We have carefully considered your comments and made revision, which is marked in red color in the latest manuscript. We appreciated your careful reading and efforts for our manuscript.

Responds to comments:

Comment 1: One of the main criticisms is the lack of information in M&Ms section. The paradigm if this is the description of the experiment on tissue culture (section 2.6). For instance, how many fish and replicates were used in this experiment? How many tissue replicates were obtained from each fish? Were fish fed or fasted prior to sacrifice and tissue collection? In addition, one can guess that each FA was tested separately, but this should be clearly indicated here.

Response: Thanks for the useful suggestion, more details have been added and revised as following:

Each FA was separately dissolved into bovine serum albumin (BSA, Sigma, USA) and configured into a mixture with a concentration of 10 mM. Intestinal tissue was cultured as described previously in goldfish with slight modifications. Briefly, the foregut of 24 h-fasted silver pomfret (n = 12) was dissected out and cut into small pieces of approximately 1–2 mm width. The intestinal pieces were washed and placed in Leibovitz’s L-15 medium (L-15, Hyclone, USA) containing 200 U/mL penicillin-streptomycin (Solarbio, China), then distributed the tissue (20 mg pieces per well) in 6-well culture plates (Jet Biofil, China). Tissues were preincubated for an hour in L-15 medium containing 44 mM sodium bicarbonate, 1% penicillin-streptomycin, and 0.05% gentamicin at 23 °C under an air environment containing 5% CO₂ and 95% O₂. Then medium was separately replaced by the different FA-BSA mixture at a concentration of 50 μM or BAS. (Line 117-125 in revised manuscript)

Comment 2: My other main criticism is related to the HFD experiment. Although an effect was observed, the authors should provide a proximate analysis on the composition, in order to check that HFD feed had the expected amount of lipids. In addition, if possible, it would be good to perform also an analysis of the lipid profile of both diets for one main reason: the authors used a vegetable source for the extra lipid content (soybean oil) in the HFD feed. Do they know the lipid source for the control diet? Was it fish or vegetable oil? Are they sure that the differences observed between CON and HFD are due to the different lipid percentage and not to a different composition in the lipid profile of each diet?

Response: Thanks for your valuable advice. Nutritional composition of diets has been added and shown in the following table. The lipid source for the control diet is shrimp oil and squid oil. The diets we formulated was referred to previous study (Meguro et al. 2019). This study reported damage to zebrafish caused by high-fat diet and the high-fat diet was made by directly adding oil in the control diet. Lipid level of diets was our primary concern in this experiment, and we have indeed observed the effect of lipid level on genes expression. Different lipid sources may affect the expression of these appetite factors, which is also the reason why we further study different fatty acids.

Nutritional composition of the CON and HFD.

	CON	HFD
Ingredient (% w/w)
Soybean oil	0	8.7
Commercial feed	100	91.3
Nutrient (% w/w)
Carbohydrate	5.2	4.6
Protein	52.1	47.4
Crude lipid	7.9	15.8
Ash	34.8	32.2

 

Comment 3: Throughout the manuscript, the authors use the term “starvation”. However, a more adequate term is “fasting” so I suggest replacing “starvation” with “fasting” in the whole manuscript.

Response: We sincerely appreciated your considerable suggestion and revised ‘starvation’ to ‘fasting’ in the full text and all figures with this new version.

Comment 4: The scientific name of all fish should be included, at least the first time each species is mentioned.

Response: Thank you for your kind suggestion. The scientific name of all fish has been added in the latest manuscript.

Comment 5: In general, the manuscript would benefit from an English revision. I found many grammar mistakes that, although small, in some cases they make the sentence confusing. For example, see: line 50, “resulted”; line 66, who purchased the fish? (“purchased by” or “purchased at”?); line 117, “contained with” or “containing”?; line 251, “similar to”, not “similar with”; etc.

Response: We apologized that the grammar of this manuscript is not good. Subsequently, every word and sentence have been checked and revised to ensure accuracy in the latest manuscript.

Comment 6: Line 47, not clear what authors mean with “sustainable aquaculture practices” in this context.

Response: Thanks for your kind suggestion and we carefully revised as following:

High fat diets (HFD) are extensively used in aquaculture. (Line 49 in revised manuscript)

Comment 7: Line 55, which neuropeptides? It would be good to list them here.

Response: We agreed with your considerable suggestion and the sentence is revised as following:

OA, linoleic acid (LA), and α-linolenate (ALA) regulated the agouti-related peptide 2, neuropeptide Y, and proopiomelanocortin genes expression and thus affected appetite in Chinese perch. (Line 57-59 in revised manuscript)

Comment 8: Line 64, in the ethics section, is it possible to include here a license or authorization number?

Response: Thanks for your suggestion. The authorization number have been supplemented in this part as following:

(Approval numbers: NBU20220079, approved on 7 March 2022). (Line 69-70 in revised manuscript)

Comment 9: Line 137, amino acid sequence?

Response: Thanks for your suggestion, and we revised as following:

The amino acid sequence of the silver pomfret ghrelin showed high identity with ghrelin from Pacific bluefin tuna (Thunnus orientalis) (83%) and largemouth bass (Micropterus salmoides) (83%) (Line 148 in revised manuscript)

Comment 10: Figure 4: the authors used the description “after short-term starvation” for the X-axis. This is confusing, I would say better something like “time after feeding” or “fasting time”.

Response: Thanks for your constructive suggestion, and we revised as following:

Relative expression levels of (A) ghrelin, (B) glp-1, (C) pyya, and (D) pyyb mRNA at different fasting time. (Line 216-217 in revised manuscript)

Comment 11: In general, the Discussion is too focused on comparing the author’s results with the results in other species and I missed more discussion on further implications of their results. For instance, in the first paragraph, may the different results on the sequences of pyya and pyyb be due to a difference in their functions? Could this be related with the differences observed in their distribution (e.g. expression in the intestine)?

Response: Thanks for your suggestion. We would pay more attention to our own results and explore the potential causes of this results. the specific revisions are as follows:

Considering the differences between silver pomfret PYYa and PYYb, one can speculate that PYYa and PYYb may response for different metabolic process and it might be reflected in their tissue distribution. (Line 253-256 in revised manuscript)

Refrences:

Meguro, S., Hosoi, S., & Hasumura, T. (2019). High-fat diet impairs cognitive function of zebrafish. Scientific Reports, 9(1), 1-9.

 

Reviewer 2 Report

The manuscript fishes-2004861 « Molecular Characterization and Expression Response of 2 Ghrelin, GLP-1 and PYY to Starvation, Dietary Lipid, and Fatty Acids in Silver Pomfret (Pampus argenteus) » by Zhang et al., evaluated the impact nutritional status in the mRNA level and expression of Ghrelin, glucagon-like peptide-1 (GLP-1), and peptide YY (PYY) in silver pomfret (Pampus argenteus). For this They observed the impact of fasting, high fat diet (16%) and different fatty acid incubation.

 

The topic of the presented manuscript is easy to understand, insteresting, very and succint with easy hypothesis to understand. Also, the topic is clearly in adequation with Fishes journal. Abstract is well written and clear even some main result are missing. Authors have realized a phylogenetic analysis that should be mentioned in the abstract.

The Background is succint introduction to the studies with adequate and recents references (even some is missing).

However, the authors list one by one the different points addressed for their analysis but without really understanding why. Authors should explain why they focus on these 3 nutritional points in their manuscripts and not others. Why they are looking at these particular fatty acids and not others. Why this species and not other (the explication is very succint and poor). And finally why the authors have focused to ghrelin, glp-1, pyya, and pyyb a and not others hormones. Why they do not observe the Leptin to compare in the view of ghrelin expression ?

 

The materials and methods section is not clear and a lot of information are missing.

All the legends of the figures are messed up. the discussion section is very poor with not really explanations of the results.

Overall, the article gives the impression of four different experiments without scientific links.

A lot of work is needed for this article before a possible acceptance

Major/Minors considerations:

- a lot of information about the construction of phylogenectic tree are missing. The authors have to add all information about this.

- The composition of diet have to be add by table section.

- why authors used 10mM for FA concentration ?

- why authors dissolved the FA in BSA ? not in ethanol ?

- why authors used these FA in this experiment ? if there is no reason, authors have to check the composition of FA profiles in their feeding experiment and to make the parralel with FA incubation.

- In table 1, the accession number have to be specify for each sequence.

- Statistical Analysis : authors have to mention the criteria of eligibility (levene and shapiro test are mandatory). Did they used a post-hoc test ?

- in results sections, authors showed that 3 to 4 hormones were very low (mRNA level expression) in intestine (figure 3) but continue their analysis on this organ in figure 4 . it’s seem not relevant. In opposite 3 to 4 were very expressed in the brain and brain is also known to play fundamental role in regulatio of apetite. I could suggest to also observed the effect of fasting in brain tissue. Concerning starvation figure 4 ; for me it is more a feeding experiment. The effect of the feeding in the expression of these 4 hormones. Because authors fed the fish until satiation and take samples. For me, it was a feeding experiment. The regulation of the expression of these 4 hormones after last feedieng. Or for sure time 0, 1h, 3h, 6h were clearly not the effect of « starvation » and very limit for 12h. only 72h could be represented to starvation effect.

- figure 5, the effect was very important for PYYb but, in figure 3 this gene was very very low in instetine and not effect of the feeding (figure 4). Authors have to discussed this.

- figure 6 : Why the authors have observd 2h after incubation ? Why authors have isolated the gut using in vitro method ?

Author Response

Dear Reviewer 2,

Thank you very much for giving us an opportunity to revise our manuscript. We appreciate for your constructive comments and suggestions on our manuscript entitled “Molecular Characterization and Expression Response of Ghrelin, GLP-1 and PYY to Starvation, Dietary Lipid, and Fatty Acids in Silver Pomfret (Pampus argenteus)”. We have carefully considered your comments and made revision, which is marked in red color in the latest manuscript. We appreciated your careful reading and efforts for our manuscript.

Responds to comments:

Comment 1: The topic of the presented manuscript is easy to understand, interesting, very and succinct with easy hypothesis to understand. Also, the topic is clearly in adequation with Fishes journal. Abstract is well written and clear even some main results are missing. Authors have realized a phylogenetic analysis that should be mentioned in the abstract.

Response: Thanks for your suggestion. We have made a more complete and description of the abstract, and the revisions were made as following:

Ghrelin, glucagon-like peptide-1 (GLP-1), and peptide YY (PYY) are potent hormones mediating food intake according to the nutritional status in fish. However, limited information is available on these genes and their expression in response to nutrition in silver pomfret (Pampus argenteus). Comparison analysis revealed that ghrelin and GLP-1 were relatively conserved in marine fish. PYYa and PYYb shared low identity and clustered to different PYY branches. Ghrelin, pyya, and pyyb mRNAs were highly expressed in the brain, while glp-1 showed high transcriptional level in the gill and liver. The ghrelin mRNA expression was relatively high at an hour post-feeding and decreased after 3 to 72 hours of fasting. The glp-1 mRNA expression was increased after 6 to 24 hours of fasting. The pyya mRNA expression was increased after 72 hours of fasting, while the pyyb expression remained stable during the fasting. Diet with 16% lipid promoted the expressions of glp-1, pyya, and pyyb but inhibited ghrelin expression. The ghrelin expression in the intestine was increased after palmitic acid, oleic acid (OA), linoleic acid (LA), α-linolenic acid, eicosapentaenoic acid (EPA), and docosahexaenoic acid (DHA) incubation. OA decreased the expression of glp-1 and increased the expression of pyya. The expression of pyyb was downregulated by LA, DHA, and EPA. These results suggested the importance of ghrelin, glp-1, and pyy in coordinating food intake in response to fasting, dietary lipid concentration and fatty acids in silver pomfret. (Line 9-24 in revised manuscript)

Comment 2: The Background is succinct introduction to the studies with adequate and recent references (even some is missing).

Response: Thank you for your suggestion. More details have been provided in the manuscript., and the specific revisions are as follows:

High fat diets (HFD) are extensively used in aquaculture. (Line 49 in revised manuscript)

OA, linoleic acid (LA), and α-linolenate (ALA) regulated the agouti-related peptide 2, neuropeptide Y, and proopiomelanocortin genes expression and thus affected appetite in Chinese perch (Siniperca chuatsi). (Line 56-59 in revised manuscript)

Studies indicated that the consumer demand for silver pomfret fish has increased in recent years. (Line 62-63 in revised manuscript)

Comment 3: However, the authors list one by one the different points addressed for their analysis but without really understanding why. Authors should explain why they focus on these 3 nutritional points in their manuscripts and not others. Why they are looking at these particular fatty acids and not others. Why this species and not other (the explication is very succinct and poor). Finally, why the authors have focused to ghrelin, glp-1, pyya, and pyyb a and not others hormones. Why they do not observe the leptin to compare in the view of ghrelin expression?

Response: Thank you for your advice and we carefully considered. The answers to your questions have been listed as following:

why they focus on these 3 nutritional points in their manuscripts and not others?

Fasting, dietary lipid concentration and dietary fatty acids different fatty acids chosen as the discussion points, because they are closely related to the aquaculture and directly affected to the production of fish.

Why they are looking at these particular fatty acids and not others?

Potential candidates for substitution fish oil are vegetable oils (VO) produced in excess of fish oil. OA, LA, PA and ALA are common composition of daily VO in fish diet. EPA and EPA are components which are mainly exist in fish oil. The influence of oil on appetite changes with the source of oil. Different oils have their main fatty acids, thus fatty acids, to some extent, help us to explain the difference between fat and appetite regulation.

Why this species and not other?

Silver pomfret is a common marine fish with high commercial and ecological value in China, India, Japan, and Kuwait. Because of its high nutritive value and edibility, the consumer demand for silver pomfret fish has increased in recent years (Alipour et al. 2021). With the development of artificial culture of silver pomfret, more attention should be paid to its nutritional requirement.

Finally, why the authors have focused to ghrelin, glp-1, pyya, and pyyb a and not others hormones?

Ghrelin, glp-1, pyya, and pyyb representative appetite regulating genes. Ghrelin is the only known peripheral appetite stimulating hormone, and it acts as a potential orexigenic signal in the nutritional status of fish. On the contrary, glp-1, pyya, and pyyb all have the function of inhibiting appetite, and they are selected as signals reflecting anorexia.

Why they do not observe the Leptin to compare in the view of ghrelin expression?

Leptin has been identified in several fish species and appears to act as an anorexigenic factor in fish examined to date. Therefore, its expression level also reflects the inhibition of fish appetite, which is similar to the role played by glp-1, pyya and pyyb. Further research on Leptin in silver pomfret is necessary in the near future.

Comment 4: The materials and methods section are not clear and a lot of information are missing.

Response: Thanks for your valuable suggestion, more details have been added and revised as following:

Each FA was dissolved into bovine serum albumin (BSA, Sigma, USA) separately and configured into a mixture with a concentration of 10 mM. Intestinal tissue was cultured as described previously in goldfish with slight modifications. Briefly, the foregut of 24 h-fasted silver pomfret (n = 12) was dissected out and cut into small pieces of approximately 1–2mm width. The intestinal pieces were washed and placed in Leibovitz’s L-15 medium (L-15, Hyclone, USA) containing 200 U/mL penicillin-streptomycin (Solarbio, China), then distributed the tissue (20 mg pieces per well) in 6-well culture plates (Jet Biofil, China). Tissues were preincubated for an hour in L-15 medium containing 44 mM sodium bicarbonate, 1% penicillin-streptomycin, and 0.05% gentamicin at 23 °C under an air environment containing 5% CO₂ and 95% O₂. Then, medium was replaced by the different FA-BSA mixture at a concentration of 50 μM or BAS separately. (Line 117-124 in revised manuscript)

In addition, we have supplemented and modified the sequence analysis part (Line 92-99 in revised manuscript) and the feeding experiment part (Line 107-113 in revised manuscript).

Comment 5: All the legends of the figures are messed up. the discussion section is very poor with not really explanations of the results.

Response: Thanks for your suggestion, we have carefully checked and revised all the illustrations of legends. We have further analyzed the results and explored the potential causes of this results as following:

Considering the differences between silver pomfret PYYa and PYYb, one can speculate that PYYa and PYYb may response for different metabolic process and it might be reflected in their tissue distribution. (Line 253-256 in revised manuscript)

Considering that these genes have obvious response to lipid concentration, fatty acid concentration may also be an important factor affecting gene expression. Thus, the effect of fatty acid concentration on the appetite regulation of fish remains to be elucidated further. (Line 322-325 in revised manuscript)

We have supplemented and modified legends of the figure 4 (Line 216 in revised manuscript), figure 5 (Line 225 in revised manuscript) and figure 6 (Line 235 in revised manuscript).

Comment 6: Overall, the article gives the impression of four different experiments without scientific links.

Response: Fasting, high-fat diet and different fatty acids are very important parts of the aquaculture industry. We aim to explore ghrelin, glp-1 and pyy genes and their expression in response to nutrition in silver pomfret. The possible connection between these experiments needs further study.

A lot of work is needed for this article before a possible acceptance.

Major/Minors considerations:

Comment 6: A lot of information about the construction of phylogenetic tree are missing. The authors have to add all information about this.

Response: Thanks for your suggestion, more details were added and the revisions were made as following:

Multiple sequence alignments of deduced amino acid sequences from four different species were performed using DNAMAN software. A phylogenetic tree based on the amino acid sequences was constructed by the neighbor-joining method of the Clustal W (http://www.ddbj.nig.ac.jp/search/clustalw-e.html) and MEGA 7 program (http://www.megasoftware.net/index.html). 1000 bootstraps were performed to check the credibility of each branch. The accession number of amino acid sequences used for alignments and phylogenetic analysis were listed in the legends of the figure1 and figure 2. (Line 92-99 in revised manuscript)

Comment 7: The composition of diet has to be added by table section.

Response: According to your valuable suggestion, nutritional composition of diets was added and shown as following:

Nutritional composition of the CON and HFD.

	CON	HFD
Ingredient (% w/w)
Soybean oil	0	8.7
Commercial feed	100	91.3
Nutrient (% w/w)
Carbohydrate	5.2	4.6
Protein	52.1	47.4
Crude lipid	7.9	15.8
Ash	34.8	32.2

 

Comment 8: Why authors used 10 mM for FA concentration?

Response: Thanks for your question. Ten mM is a mother liquor, which will be diluted into 50 μM working solution during the experiment. This concentration of mother liquor is based on previous studies (Li et al.,2013; Shi et al., 2022).

Comment 9: Why authors dissolved the FA in BSA? not in ethanol?

Response: It’s a good question. We use BSA to dissolve FA referred to previous studies (Li et al.,2013; Shi et al., 2022).

Comment 10: Why authors used these FA in this experiment? if there is no reason, authors have to check the composition of FA profiles in their feeding experiment and to make the parallel with FA incubation.

Response: Thanks for the useful suggestion. All fatty acids used in our experiment are long-chain fatty acids (LCFAs). High levels of LCFA in mammals, such as mice, can activate the hypothalamic FA sensing systems to reduce food intake (Feng et al, 2022). Food intake in rainbow trout could be modulated by LCFAs (Calo et al, 2022). Therefore, these LCFAs may cause the response of intestinal appetite factors.

Comment 11: In table 1, the accession number have to be specified for each sequence.

Response: Thanks for your valuable advice. The nucleotide sequence of the silver pomfret gene in our study based on our previous transcriptome data (unpublished), so the primers sequences were designed by ourselves.

Comment 12: Statistical Analysis: authors have to mention the criteria of eligibility (levene and shapiro test are mandatory). Did they used a post-hoc test?

Response: Thanks for your question. Data analysis and statistics are corrected and the revisions were made as following:

FAs incubation assay and feeding experiment were analyzed by one-way analysis of variance (ANOVA) followed by Duncan's multiple range tests. Tissue distribution and fasting experiment were examined with Levene's test, followed by Tukey's multiple range test. Statistical analysis was performed using SPSS 20.0 statistical software package (SPSS, Inc., USA). Data were log-transformed when equal variances were not assumed. The data are presented as means ± SEM, and differences were considered statistically significant at the *p < 0.05 level. (Line139-145 in revised manuscript)

Comment 13: In results sections, authors showed that 3 to 4 hormones were very low (mRNA level expression) in intestine (figure 3) but continue their analysis on this organ in figure 4. it’s seemed not relevant. In opposite 3 to 4 were very expressed in the brain and brain is also known to play fundamental role in regulation of appetite. I could suggest to also observed the effect of fasting in brain tissue. Concerning starvation figure 4; for me it is more a feeding experiment. The effect of the feeding in the expression of these 4 hormones. Because authors fed the fish until satiation and take samples. For me, it was a feeding experiment. The regulation of the expression of these 4 hormones after last feeding. Or for sure time 0, 1h, 3h, 6h were clearly not the effect of « starvation » and very limit for 12h. only 72h could be represented to starvation effect.

Response: Thanks for your suggestion. What you mentioned is definitely right that most genes are abundantly expressed in the brain. The intestine is the main organ for nutrient absorption and transport. Because our study focuses on whether nutrients can activate the expression of appetite factors to regulate appetite, we chose the intestine as the research object. The dynamic changes of appetite factors in the brain are indeed worthy of further study.

Starvation may not be a suitable word here, so we have replaced starvation with a more suitable word, fasting. After the last feeding, the response of appetite factors of silver pomfret to time is not obvious, but the fish did not take any food during this period. We aim to explore the dynamic changes of appetite factors after ingestion, so I think fasting may also be a reasonable description.

Comment 14: Figure 5, the effect was very important for PYYb but, in figure 3 this gene was very very low in intestine and not effect of the feeding (figure 4). Authors have to discussed this.

Response: Thanks for your question. Pyyb was low expressed in intestine, and its expression was not affected of the feeding. However, the ghrelin mRNA expression in fish fed the 16% lipid diet was significantly higher than fish fed the 8% lipid diet. This may be because pyyb is more sensitive to lipid level. Fatty acid treatment does not affect the expression of pyy, which may be related to the concentration of fatty acids.

Comment 15: Figure 6: Why the authors have observed 2h after incubation? Why authors have isolated the gut using in vitro method?

Response: Thanks for your question. The incubation time has been chosen after a preliminary experiment. The time we incubate tissues with fatty acids is consistent with the previous studies (Blanco et al., 2017). In vitro experiment can reduce interference factors, and it can reflect the intestinal response to fatty acids in a more direct way.

References:

Alipour, M., Sarafraz, M., Chavoshi, H., Bay, A., Nematollahi, A., Sadani, M. & Khaneghah, A. M. (2021). The concentration and probabilistic risk assessment of potentially toxic elements in fillets of silver pomfret (Pampus argenteus): A global systematic review and meta-analysis. Journal of Environmental Sciences, 100, 167-180.

Blanco, A. M., Bertucci, J. I., Valenciano, A. I., Delgado, M. J. & Unniappan, S. (2017). Ghrelin suppresses cholecystokinin (CCK), peptide YY (PYY) and glucagon-like peptide-1 (GLP-1) in the intestine, and attenuates the anorectic effects of CCK, PYY and GLP-1 in goldfish (Carassius auratus). Hormones and behavior, 93, 62-71.

Calo, J., Conde-Sieira, M., Comesaña, S., Soengas, J. L. & Blanco, A. M. (2022). Fatty acids of different nature differentially modulate feed intake in rainbow trout. Aquaculture, 738961.

Feng, H., Peng, D., Liang, X. F., Li, J., Luo, H., Tang, S. & Chai, F. (2022). Intracerebroventricular injection with octanoic acid activates hypothalamic fatty acid sensing systems and regulates appetite in Chinese perch Siniperca chuatsi. Fisheries Science, 88(1), 83-90.

Li, Q., Ai, Q., Mai, K., Xu, W. & Zheng, Y. (2013). A comparative study: in vitro effects of EPA and DHA on immune functions of head-kidney macrophages isolated from large yellow croaker (Larmichthys crocea). Fish & shellfish immunology, 35(3), 933-940.

Shi, P., Liao, K., Xu, J., Wang, Y., Xu, S. & Yan, X. (2022). Eicosapentaenoic acid mitigates palmitic acid-induced heat shock response, inflammation and repair processes in fish intestine. Fish & Shellfish Immunology, 124, 362-371.

 

Reviewer 3 Report

A very interesting manuscript. In my opinion, it requires minor revision before acceptance. My detailed comments are included in the text. To see them all, open the file in Acrobat Reader.

Comments for author File: Comments.pdf

Author Response

Dear Reviewer 3,

Thank you very much for giving us an opportunity to revise our manuscript. We appreciate for your constructive comments and suggestions on our manuscript entitled “Molecular Characterization and Expression Response of Ghrelin, GLP-1 and PYY to Fasting, Dietary Lipid, and Fatty Acids in Silver Pomfret (Pampus argenteus)”. We have carefully considered your comments and made revision, which is marked in red color in the latest manuscript. We appreciated your careful reading and efforts for our manuscript.

 

1.Line 76: ‘add country’

Country has been added. line 82

 

2. Line 78: ‘number of fish’

The statement is changed to ‘Total RNA was extracted from tissues of three silver pomfret’. line 82

 

3. Line 100:’deleted s’

It has been deleted. line 107

 

4. Line 108 and 113: ‘table 1 changed to table 2’

Revised. line 121 and 127

 

5. Line 82, 133 and137: ‘table 2 changed to table 1’

Revised. line 90,151 and 147

 

6.Line 110: ‘Are the conditions the same as described earlier?’

The body weight of the fish and the feeding conditions in the present study were the same as mentioned above unless otherwise stated. All fish were acclimatized to the aquarium conditions for at least 2 weeks before the experiment. line79 to 82

 

7.Line 110: ‘how many days’

This was an acute feeding experiment in which the fish were fed only three times. line 124

 

8. Line 113: ‘add English and Latin names of the spcecies’

The statement is changed to ‘Nutritional composition of the CON and HFD for Silver Pomfret (Pampus argenteus).’ line 127

 

9. Line 153: ‘add European’

European has been added. line 167

 

10. Line 166: ‘Crass carp change to Grass carp’

Revised. line 182

 

11. Figure 2: ‘It should be corrected. The lines and number are on top of each other’

Figure 2 has been modified.

 

12. Line 193: ‘which frog’

Frog has been changed to African clawed frog. Line 206,208

 

10. Line 197, 239, 246, 254, 278, 290 and 312: ‘our change to present’

Revised. line 312, 255, 262, 270, 302, 310, 311and 332

 

11.Line 205, 216, 225 and 235: ‘add English and Latin names of the species’

English and Latin names of the species have been added. Line 220, 233, 241 and 251

 

12. Line 201: ‘Fig. 1C changed toFig. 3C’

Revised. line 216

 

13. Line 264: ‘deleted enter’

It has been deleted. line 280

 

Round 2

Reviewer 1 Report

The present manuscript is a revised version of a previously submitted manuscript. Although the manuscript has improved with the changes, there are still some comments that I made to the first version and have not been tackled by the authors:

1) Regarding the diet composition provided in the new Table 2, are those results from a proximate analysis or just the expected values calculated by the authors? As I mentioned in my first revision, a proximate analysis of the main composition of the diet is needed to be sure that the amount of each macronutrient, especially lipids, is as expected. If it is the proximate analysis, then the authors should include a brief description of the analysis performed in the methodology (e.g. protein, lipid and ash determination).

2) The manuscript was not revised for grammar mistakes. Actually, the authors did not even change the mistakes I pointed out in my first revision as examples…

 

Author Response

Dear Reviewer 1,

Thank you very much for your review of our manuscript. We appreciate for your constructive comments and suggestions on our manuscript entitled “Molecular Characterization and Expression Response of Ghrelin, GLP-1 and PYY to Fasting, Dietary Lipid, and Fatty Acids in Silver Pomfret (Pampus argenteus)”. We have carefully considered your comments and made a new revision, which is marked in red color in the latest manuscript. We appreciated your careful reading and efforts for our manuscript.

Responds to comments:

Comment 1: Regarding the diet composition provided in the new Table 2, are those results from a proximate analysis or just the expected values calculated by the authors? As I mentioned in my first revision, a proximate analysis of the main composition of the diet is needed to be sure that the amount of each macronutrient, especially lipids, is as expected. If it is the proximate analysis, then the authors should include a brief description of the analysis performed in the methodology (e.g. protein, lipid and ash determination).

 

Response: Thank you for your kind suggestion. The diet composition provided in the previous table was a calculated result, but this value is somewhat different from the actual content. The diets lipid levels used in our high-fat diet groups were 6% higher than the lipid levels of the control diet, not the speculative 8%. However, there were still significant differences in this lipid level. We updated the composition of the diets after the actual measurements. We also supplemented and described the methods of analysis as following:

 

The control diet (CON) was commercial feed. Diet with 16% lipid was made by mixing 91.3% (w/w) CON diet with 8.7% (w/w) soybean oil. Crude protein, crude lipid and ash content in diets were determined according to the methods of AOAC. Briefly, crude protein content was determined via the Dumas combustion methods with a protein analyzer (FP-528, Leco, USA). Crude lipid contents were determined by the ether extraction method using a Soxtec System HT (Soxtec System HT6, Tecator, Sweden). Ash contents were determined using a muffle furnace run at 550 °C for 8 h. Table 2 shows the nutritional composition of the two diets. (Line 114-122 in revised manuscript)

 

Nutritional composition of the CON and HFD.

	CON	HFD
Ingredient (% w/w)
Soybean oil	0	8.7
Commercial feed	100	91.3
Nutrient (% w/w)
Protein	52.1	49.4
Crude lipid	7.9	14.4
Ash	14.1	11.9

 

 

Comment 2: The manuscript was not revised for grammar mistakes. Actually, the authors did not even change the mistakes I pointed out in my first revision as examples…

 

Response: We apologize for not correcting those grammatical issues you pointed out, and we have examined the article more closely and marked in red color.

 ‘purchased by’ has been changed to ‘purchased at’ in line 74

 ‘resulted from fish oil substitution’ has been deleted in line 54.

Reviewer 2 Report

The authors have done the bare minimum in answering questions and requests for changes to the document. Most of the requests have been further explained (not necessarily convincingly) in the responses to the reviewer document but not at all in the manuscript and when it is, it is not what is asked (for example the accession number still missing in the primers table).

Thus, the manuscript cannot be published. 

Author Response

Dear Reviewer 2,

Thank you very much for your review of our manuscript. We appreciate for your constructive comments and suggestions on our manuscript entitled “Molecular Characterization and Expression Response of Ghrelin, GLP-1 and PYY to Fasting, Dietary Lipid, and Fatty Acids in Silver Pomfret (Pampus argenteus)”. We have carefully considered your comments and made a new revision, which is marked in red color in the latest manuscript. We appreciated your careful reading and efforts for our manuscript.

Responds to comments:

Comment: The authors have done the bare minimum in answering questions and requests for changes to the document. Most of the requests have been further explained (not necessarily convincingly) in the responses to the reviewer document but not at all in the manuscript and when it is, it is not what is asked (for example the accession number still missing in the primers table).

 

Response: Thank you for your suggestions. We have made explanations according to your suggestions and requirements in the manuscript.

Why they are looking at these particular fatty acids and not others?

High fat diets (HFD) and vegetable oils (VO) are extensively used in aquaculture. Line 51

Oleate acid (OA), linoleic acid (LA), palmitic acid (PA) and α-linolenate (ALA) are common FAs of daily VO in fish diet. Line 54 to 56

Why this species and not other?

Silver pomfret is a common marine fish with high commercial and ecological value in China, India, Japan, and Kuwait. Studies indicated that the consumer demand for silver pomfret fish has increased in recent years. With the development of artificial culture of silver pomfret, more attention should be paid to its nutritional requirement. Line 63 to 66

Figure 5, the effect was very important for PYYb but, in figure 3 this gene was very very low in intestine and not effect of the feeding (figure 4). Authors have to discussed this.

Considering that these genes have obvious response to lipid concentration, fatty acid concentration may also be an important factor affecting gene expression. Thus, the effect of fatty acid concentration on the appetite regulation of fish remains to be elucidated further. Line 342 to 345

In results sections, authors showed that 3 to 4 hormones were very low (mRNA level expression) in intestine (figure 3) but continue their analysis on this organ in figure 4. it’s seemed not relevant.

Gut-brain axis is a complex bidirectional communication system between the gastrointestinal tract and the central nervous system. Within this axis, the gastrointestinal tract sends information to the brain, which transmits feedback to the gastrointestinal tract. The gut was chosen for the experiment because it senses changes in nutritional status more directly than the brain. Line 280 to 284

 

As for the accession number you mentioned, we have made an explanation that the nucleotide sequence of the silver pomfret gene in our study based on our previous transcriptome data (unpublished). Therefore, we did not upload the sequence and could not provide the accession number. Based on previous studies, the primer accession number may not be a mandatory option (He et al, 2021; Gao et al, 2022; de et al, 2022).

 

He, Y., Zhong, Y., Bao, Z., Wang, W., Xu, X., Gai, Y., & Wu, J. (2021). Evaluation of Angelica decursiva reference genes under various stimuli for RT-qPCR data normalization. Scientific reports, 11(1), 1-10.

Gao, K., Khan, W. U., Li, J., Huang, S., Yang, X., Guo, T., ... & An, X. (2022). Identification and Validation of Reliable Reference Genes for Gene Expression Studies in Koelreuteria paniculata. Genes, 13(5), 714.

de Oliveira, L. F., Piovezani, A. R., Ivanov, D. A., Yoshida, L., Floh, E. I. S., & Kato, M. J. (2022). Selection and validation of reference genes for measuring gene expression in Piper species at different life stages using RT-qPCR analysis. Plant Physiology and Biochemistry, 171, 201-212.