Effects of Natural and Synthetic Astaxanthin on Growth, Body Color, and Transcriptome and Metabolome Profiles in the Leopard Coralgrouper (Plectropomus leopardus)

Round 1

Reviewer 1 Report

The study describes the effects of natural and synthetic astaxanthin on growth, body color and transcriptome and metabolome profiles of leopard coral grouper (Plectropomus leopardus). The study is novel as it applies modern transcriptome and metabolome approaches to delineate the physiological; pathways involved, and therefore of greater interest to the future researchers. The methods and its explanation thereof are well defined and qualify a concrete study. Conclusions are solid. My comments for improving the work are appended below:

Introduction: Looks fine. A paragraph about the synthetic carotenoids used here may be incorporated.

Methods:

Section 2.2.: How were the different doses of the natural vs synthetic carotenoids levels derived? Was there any preliminary study or earlier research referred? How was it supplied to the diet? Does extrusion processing damage the carotenoids? Please explain?

Section 2.3. The experiment was carried out in pond system. Why was the water flow initiated, and how? What was the purpose?

Data analysis section is missing.

Discussion

Needs clarity and cite recent works. You mentioned the controversial role of astaxanthin. Clarify through your results how it exactly confirms such gaps. Then only the work sounds very novel and interesting. 

Author Response

Response to Reviewer 1

The study describes the effects of natural and synthetic astaxanthin on growth, body color and transcriptome and metabolome profiles of leopard coral grouper (Plectropomus leopardus). The study is novel as it applies modern transcriptome and metabolome approaches to delineate the physiological; pathways involved, and therefore of greater interest to the future researchers. The methods and its explanation thereof are well defined and qualify a concrete study. Conclusions are solid. My comments for improving the work are appended below:

Introduction: Looks fine. A paragraph about the synthetic carotenoids used here may be incorporated.

Response: Thank you very much for your comment. We have added to the description of carotenoids used in aquaculture as follows: Carotenoids commonly used in aquaculture include synthetic beta-carotene, astaxanthin, cantaxanthin, zeaxanthin and lutein [23].

Methods:

Section 2.2.: How were the different doses of the natural vs synthetic carotenoids levels derived? Was there any preliminary study or earlier research referred? How was it supplied to the diet? Does extrusion processing damage the carotenoids? Please explain?

Response: Thank you. The synthetic astaxanthin powder (Carophyll^® pink) contains 10% astaxanthin and the natural astaxanthin powder (Haematococcus lacustris) contains 3% astaxanthin. The addition of 0.2% synthetic astaxanthin and 0.667% natural astaxanthin is based on our desire to keep the actual astaxanthin content in the feed consistent to ensure that the actual astaxanthin content is 0.02%. We designed the actual content of astaxanthin to be 0.02% based on the fact that the 0.2% synthetic astaxanthin powder (astaxanthin content accounts for 10%) can promote the red coloration of Plectropomus leopardus (Guan et al., 2018). We weighed the astaxanthin accurately according to the feed formula, then used wheat gluten flour to mil the extruder has cooled sufficiently naturally. Moreover, the extrusion process takes less than 15 minutes to avoid damage to astaxanthin from prolonged high temperature. Most importantly, it is known from the study that the actual content of astaxanthin after the extrusion process remains essentially the same as the amount of astaxanthin added in the experimental formulation (Zhu et al., 2022; Micah et al., 2022). 

Section 2.3. The experiment was carried out in pond system. Why was the water flow initiated, and how? What was the purpose?

Response: Thank you. The experiment was not conducted in a pond system, but in an indoor cement pool. We have revised it. Experimental fish are more sensitive to the level of oxygen. Flowing water is used to ensure that the water contains sufficient oxygen while avoiding stress from low oxygen levels.

Data analysis section is missing.

Response: Thank you. We have supplemented the data analysis content in the materials and methods. The content are as follow:

2.8 Data calculation and statistical analysis

The growth performance parameters of adult P. leopardus such as weight gain rate (WGR) and specific growth rate (SGR) were calculated as follows:

WGR (%) = (W_t - W₀)/W₀ × 100

SGR (%/day) = (ln W_t - ln W₀)/t ×100

where W_t: mean value of final fish body weight (g), W₀: mean value of initial fish body weight (g), t: number of feeding days (days).

All data are expressed as mean ± standard deviation (SD). Experimental data were subjected to one-way ANOVA using IBM SPSS Statistics 24.0 (SPSS Inc., Chicago, IL, USA). Differences between group means were determined by Duncan's multiple range tests. P < 0.05 was considered statistically significant.

Discussion:

Needs clarity and cite recent works. You mentioned the controversial role of astaxanthin. Clarify through your results how it exactly confirms such gaps. Then only the work sounds very novel and interesting.

Response: Thank you. We have made corrections as follows: These results suggest that P. leopardus may have a higher absorption and utilization efficiency for natural astaxanthin. Previous studies have found that astaxanthin can enhance the intermediate metabolism to promote the absorption and utilization of nutrients, thus improving the growth performance of rainbow trout [48]. However, in our study, synthetic astaxanthin did not have a significant effect on the growth performance of P. leopardus. Besides, natural astaxanthin did not show a growth promoting effect on the species at the seventh week. The growth of fish may also be related to the development stage, breeding environment, and breeding time. It is important to note that other ingredients in the supplement may also provide a supporting role on growth, even though we have kept the consistency of the astaxanthin, protein and fat content respectively. Hence, the mechanism by which astaxanthin may affect the growth performance of fish needs to be further investigated.

Author Response File: Author Response.docx

Reviewer 2 Report

This manuscript entitled "Effects of natural and synthetic astaxanthin on growth, body color and transcriptome and metabolome profiles of leopard coral grouper (Plectropomus leopardus) " by Zhang et al. is an interesting manuscript focused in evaluate differences between natural and synthetic asaxanthin. However, there are some concerns to may be explain by the authors that correspond to methodology to adjust the same asaxanthin levels between the two sources and the implications in the results.

Line 107-109. The aim of the study needs to be punchy, by including the response parameters that will be report.

In the summary it is mentioned that natural asaxanthin and synthetic asaxanthin were added at 0.02%, however in table 1, it is shown that synthetic asaxanthin was added at 0.2% while natural asaxanthin was added at 0.667 % (three times more). There is an error in how it is reported in the summary or in table 1. In material and methods, the amount is reported as 2.0 g/kg and 6.67 g /kg that correspond to 0.2% and 0.667%, as reported in the table. The authors need to homologate how is going to be reported, as g/Kg or %.

In methodology between Line 117-119:The basal diet was not supplemented with astaxanthin (C group) and the experimental diets were supplemented with 2.0 g/kg of synthetic astaxanthin (AS group, Carophyll® pink, 10% astaxanthin) and 6.67 g/kg of Haematococcus pluvialis powder (HP group, 3% astaxanthin) to ensure the consistency of astaxanthin content.

Linked with the previous comment, The authors do not explain the adjust performed to ensure the 0.02% of each asaxanthin source, considering that are not pure sources (AS at 10% and HP at 3%), is confusing the table values, please explain in this section de adjust to ensure an equal amount of asaxanthin. This point is important, since the components that are not asaxanthin in each of the sources can also generate an effect at the transcriptomic and metabolomic level. If we consider that Hematococcus is a microalga, and 97% of the input is not the pigment, all the alternate components of the microalgae can generate an adjunct effect that can be confounded! There are many studies with microalgae supplementation that has been associated with strengthening immune systems, lipid metabolism, antiviral and antibacterial action, improved gut function, stress resistance besides providing a source of protein, amino acids, fatty acids, vitamins and minerals, and other biologically active phytochemicals and maybe the results of higher SGR and WG as well to differential transcriptomic and metabolomic results could be related to that. This is my main concern, that the manuscript is focused only on the pigment as the only factor that could generate the reported and differential results.

2.3. Experimental fish and sample collection

It is not mentioned whether the fish were satiety or ration fed.

Discussion between 365-368: These results suggest that P. leopardus has better absorption and utilization efficiency of natural astaxanthin than synthetic astaxanthin. Natural astaxanthin may enhance intermediate metabolism, promote the absorption and utilization of nutrients and promote the production of digestive enzymes in fish. It promotes the use of fats for growth and development rather than storage.

I am not sure that asaxhanthin is the main effector of all results, as previous mentioned, many other factors are being included by the pigments sources, are not pure sources, and Hematococus was added in higher % than synthetic source!!

Overall, the paper demonstrates a lot of analytical work from the response parameters used, including pigmentation, transcriptomic and metabolomic effects. However, the authors need to present the idea of using different asaxanthin-rich sources and not pure asaxanthin from natural or synthetic sources. Even in the discussion there is no mention of the possible effects of using unpurified sources and all the possible elements that may be causing the effects. The work needs to be strengthened in these aspects and not assume that the effects are only from synthetic or natural asaxanthin.

Question: what is the chemical difference between a synthetic asaxanthin and one produced by hematococcus?

Author Response

Response to Reviewer 2

This manuscript entitled "Effects of natural and synthetic astaxanthin on growth, body color and transcriptome and metabolome profiles of leopard coral grouper (Plectropomus leopardus) " by Zhang et al. is an interesting manuscript focused in evaluate differences between natural and synthetic asaxanthin. However, there are some concerns to may be explain by the authors that correspond to methodology to adjust the same asaxanthin levels between the two sources and the implications in the results.

Line 107-109. The aim of the study needs to be punchy, by including the response parameters that will be report.

Response: Thank you. We have made modifications according to your suggestions, which are as follows: In the present study, we compared the effects of natural astaxanthin and synthetic astaxanthin on the growth and pigmentation of P. leopardus. Meanwhile, we used transcriptomic and metabolomic approaches to investigate the candidate genes and metabolites that may be involved in the formation of body color, providing a theoretical basis for the mechanism of coloration in P. leopardus.

In the summary it is mentioned that natural asaxanthin and synthetic asaxanthin were added at 0.02%, however in table 1, it is shown that synthetic asaxanthin was added at 0.2% while natural asaxanthin was added at 0.667 % (three times more). There is an error in how it is reported in the summary or in table 1. In material and methods, the amount is reported as 2.0 g/kg and 6.67 g /kg that correspond to 0.2% and 0.667%, as reported in the table. The authors need to homologate how is going to be reported, as g/Kg or %.

Response: Thank you. We have deleted g/kg and used % to represent the amount. Besides, we have corrected the amount of astaxanthin supplementation at Experimental diets as follows: The basal diet was not supplemented with astaxanthin (C group) and the experimental diets were supplemented with 0.2% of Carophyll^® pink powder (AS group, 10% astaxanthin) and 0.667% of Haematococcus lacustris powder (HP group, 3% astaxanthin) to ensure that the actual content of natural astaxanthin and synthetic astaxanthin is consistent, both at 0.02%.

In methodology between Line 117-119:The basal diet was not supplemented with astaxanthin (C group) and the experimental diets were supplemented with 2.0 g/kg of synthetic astaxanthin (AS group, Carophyll^® pink, 10% astaxanthin) and 6.67 g/kg of Haematococcus pluvialis powder (HP group, 3% astaxanthin) to ensure the consistency of astaxanthin content.

Linked with the previous comment, The authors do not explain the adjust performed to ensure the 0.02% of each asaxanthin source, considering that are not pure sources (AS at 10% and HP at 3%), is confusing the table values, please explain in this section de adjust to ensure an equal amount of asaxanthin. This point is important, since the components that are not asaxanthin in each of the sources can also generate an effect at the transcriptomic and metabolomic level. If we consider that Hematococcus is a microalga, and 97% of the input is not the pigment, all the alternate components of the microalgae can generate an adjunct effect that can be confounded! There are many studies with microalgae supplementation that has been associated with strengthening immune systems, lipid metabolism, antiviral and antibacterial action, improved gut function, stress resistance besides providing a source of protein, amino acids, fatty acids, vitamins and minerals, and other biologically active phytochemicals and maybe the results of higher SGR and WG as well to differential transcriptomic and metabolomic results could be related to that. This is my main concern, that the manuscript is focused only on the pigment as the only factor that could generate the reported and differential results.

Response: Thank you. As mentioned in the previous response, we have made corrections at Experimental diets. In order to ensure the logical consistency of the context, synthetic astaxanthin and natural astaxanthin in Table 1 have also been replaced by Carophyll^® pink powder and Haematococcus lacustris powder, respectively.

We prepare the diets with microcrystalline cellulose as a filler. For example, 5% microcrystalline cellulose was added to the basal diet without astaxanthin (C). microcrystalline celluloses are a non-nutritional ingredient. For treatments that included synthetic astaxanthin (AS) or natural astaxanthin (HP), we replaced equal amounts of microcrystalline cellulose with Carophyll^® pink powder and Haematococcus lacustris powder, respectively. The addition of Carophyll^® pink powder and H. lacustris powder was calculated based on the concentration of astaxanthin in both sources. In summary, we added 4.8% microcrystalline cellulose and 0.2% Carophyll^® pink powder to the AS group diet, while we added 4.333% microcrystalline cellulose and 0.667% H. lacustris powder to the HP group diet. Hence, it was hoped to achieve the quantification of astaxanthin content so that the actual astaxanthin content in both treatments would be consistent. The diets were prepared following the method of Jiang et al (Jiang et al., 2019).

In designing the experimental protocol, we were inspired by Jiang et al.(Jiang et al., 2019) and wanted to investigate the effect of different sources of astaxanthin on the coloration of Plectropomus leopardus by using quantified astaxanthin concentrations. We did not exclude the interference of other components of Carophyll^® pink powder and H. lacustris powder in the experiment, as we considered their addition to the whole diet to be negligible. Thank you for your valuable comments. Indeed, the auxiliary effects formed by the alternation of other ingredients in the additive with each other could also interfere with our results to some extent. We will explore and further verify in the next study as you suggested.

Jiang, J., Nuez-Ortin, W., Angell, A., Zeng, C., de Nys, R., Vucko, M.J., 2019. Enhancing the colouration of the marine ornamental fish Pseudochromis fridmani using natural and synthetic sources of astaxanthin. Algal Research. 42, 101596. http://doi.org/10.1016/j.algal.2019.101596.

2.3. Experimental fish and sample collection

It is not mentioned whether the fish were satiety or ration fed.

Response: Thank you. Now the sentence has been revised as follow: The fish were cultured in flowing water and fed twice a day (8 am and 4 pm) at 5% of the their body weight for 14 weeks.

Discussion between 365-368: These results suggest that P. leopardus has better absorption and utilization efficiency of natural astaxanthin than synthetic astaxanthin. Natural astaxanthin may enhance intermediate metabolism, promote the absorption and utilization of nutrients and promote the production of digestive enzymes in fish. It promotes the use of fats for growth and development rather than storage.

I am not sure that asaxhanthin is the main effector of all results, as previous mentioned, many other factors are being included by the pigments sources, are not pure sources, and Hematococus was added in higher % than synthetic source!!

Response: Thank you. We have made corrections as follows: These results suggest that P. leopardus may have a higher absorption and utilization efficiency for natural astaxanthin. Previous studies have found that astaxanthin can enhance the intermediate metabolism to promote the absorption and utilization of nutrients, thus improving the growth performance of rainbow trout [48]. However, in our study, synthetic astaxanthin did not have a significant effect on the growth performance of P. leopardus. Besides, natural astaxanthin did not show a growth promoting effect on the species at the seventh week. The growth of fish may also be related to the development stage, breeding environment, and breeding time. It is important to note that other ingredients in the supplement may also provide a supporting role on growth, even though we have kept the consistency of the astaxanthin, protein and fat content respectively. Hence, the mechanism by which astaxanthin may affect the growth performance of fish needs to be further investigated.

Overall, the paper demonstrates a lot of analytical work from the response parameters used, including pigmentation, transcriptomic and metabolomic effects. However, the authors need to present the idea of using different astaxanthin-rich sources and not pure astaxanthin from natural or synthetic sources. Even in the discussion there is no mention of the possible effects of using unpurified sources and all the possible elements that may be causing the effects. The work needs to be strengthened in these aspects and not assume that the effects are only from synthetic or natural astaxanthin.

Response: Thank you. We wanted to study the effect of natural and synthetic astaxanthin on the colouration of P. leopardus and the changes at the genetic level during this process, while controlling for consistent concentrations of astaxanthin. The experimental design refers to the article by Jiang et al (Jiang et al., 2019). You have made a valuable suggestion that we should choose equal amounts of Carophyll^® pink powder and Haematococcus lacustris powder instead of considering the consistency of the astaxanthin content. However, as mentioned by Li et al., the degree of redness in blood parrot was strongly consistent with the concentration of astaxanthin in the diet. The coloration of blood parrot in the synthetic astaxanthin group than in the natural astaxanthin group, which is based on the fact that the content of the synthetic astaxanthin is much higher than natural astaxanthin (Li et al., 2016). Both of these methods may have some unavoidable problems. In the next study, we will choose the latter method for the experiments as a way of comparing them with each other for analysis.

Jiang, J., Nuez-Ortin, W., Angell, A., Zeng, C., de Nys, R., Vucko, M.J., 2019. Enhancing the colouration of the marine ornamental fish Pseudochromis fridmani using natural and synthetic sources of astaxanthin. Algal Research. 42, 101596. http://doi.org/10.1016/j.algal.2019.101596.

Li, T., He, C., Xing, W., Ma, Z., Jiang, N., Li, W., Sun, X., Luo, L., 2016. Effects of different carotenoids on pigmentation of blood parrot (Cichlasoma synspilum × Cichlasoma citrinellum). Journal of Aquaculture Research and Development. 7(3). http://doi.org/10.4172/2155-9546.1000414.

Question: what is the chemical difference between a synthetic asaxanthin and one produced by hematococcus?

Response: Astaxanthin produced by the microalgae Haematococcus lacustris (formerly Haematococcus pluvialis) is a kind of natural astaxanthin. There are fundamental differences in stereochemistry and esterification between natural and synthetic astaxanthin. For stereochemistry, ratios of the (3R,3′R), (3R,3′S), and (3S,3′S) optical isomers are different between the sources of astaxanthin, where natural astaxanthin has a ratio of 1:2:22, while synthetic astaxanthin has a ratio of 1:2:1 of these isomers (Mimoun-Benarroch et al., 2016). For esterification, more than 95% of the natural astaxanthin molecules are esterified, while synthetic astaxanthin synthetic astaxanthin is not esterified and is in the free form (Capelli et al., 2013).

Author Response File: Author Response.docx

Reviewer 3 Report

REVIEW OF THE ARTICLE BY JUNPENG ZHANG ET AL. ENTITLED “EFFECTS OF NATURAL AND SYNTHETIC ASTAXANTHIN ON GROWTH, BODY COLOR AND TRANSCRIPTOME AND METABOLOME PROFILES OF LEOPARD CORAL GROUPER (PLECTROPOMUS LEOPARDUS)” (animals-2200820) 

The article aims to describe an effect of supplementation of synthetic and natural astaxanthin on color,growth, gene expression and metabolic profile of the fish Plectropomus leopardus (Perciformes, Serranidae). Animals (with one exclusion) cannot synthesize carotenoids, such as astaxanthin, de novo, however the pigment determines skin and meat color, which is an essential parameter for assessment of quality. Hence, astaxanthin is an essential part of a fish diet. Thus, the work is actual and in scope of the journal. State of the art of the work is well described. It corresponds to current technologies in the field. Zhang et al. used UPLC-MS, RNA-seq, and qPCR. Color of fish was assessed in the CIE L*a*b* color space. Required ethical statement is provided for the work with animals. Results are new and valuable. Discussion is addressed to interesting concepts of carotenoid effect on fish and their transport. The work may be accepted after some corrections (see specific comments below).  

GENERAL COMMENTS

-Haematococcus pluvialis is a wrong obsolete synonym. Haematococcus lacustris is correct (doi:10.12705/652.11).

-FC, fold change and foldchange should be unified through the text.

-RT-PCR and qPCR are synonyms. It is better to use one of these terms instead RT-qPCR.

INTRODUCTION, ABSTRACT AND SUMMARY

-l. 22-23. Astaxanthin is a red colorant rather than color enhancer.

-Abstract is good but inconclusive. Do not introduce unused symbols (L*, a*, b*).l suggest adding the conclusion about which astaxanthin (synthetic or natural) is better for fish feeding.

-l. 59. Which melanin do you mean? Eumelanin?

-l. 73. “β,β-carotene” should be “β-carotene”.

-l. 83. “Astaxanthin, a natural red carotenoid,” - it is wrong and is in contradiction with the information below. There are natural and synthetic astaxanthin.

-l. 91-92. Microalgae are also microorganisms. Please, rephrase.

-l. 91-95. It is also important to say that there are other sources of astaxanthin (doi: 10.3390/biology10070643).

-l. 105. Spirulina is not a dye. It is an alga. Please, rephrase.

MATERIALS AND METHODS

-l. 111-114. Were the animals killed in the experiments? If yes, the procedure of eutanasia should be described according to AVMA guidelines (Underwood, W., Anthony, R., Cartner, S., Corey, D., Grandin, T., Greenacre, C., ... & Yanong, R. (2013) AVMA guidelines for the euthanasia of animals: 2013 edition. Schaumburg, IL: American Veterinary Medical Association). For fish immersion into 200 mg/L benzocaine is recommended.

-Table 1. All abbreviations (C, AS, HP) must be explained in the legend.

-l. 119. Please, indicate the manufacturer and country for Carophyll® pink. What was the origin of Haematococcus astaxanthin?

-l. 142-155. Please, describe the dataset in more detail in terms of its uniformity. Was sex of the fish determined? Indicate age of the fish (young/adult/old). Were they healthy or ill?

-l. 175-176. Indicate sequenator and reagents for sequencing.

-l. 157-164. Please, describe briefly in several sentences the CIEL*a*b* space concept (doi:org/10.1016/j.talanta.2012.11.026).

-l. 178-183. Which metric was used for the measurement of absolute expression level? FKPM? Were replications in each experimental group?

-l. 200. m/z should be italicized through the text.

-The procedures of statistical treatment should be indicated in a separate subsection.

-l. 433. And leukotrienes also.

-l. 441-452. It should be stated here, that astaxanthin is not a provitamin A, therefore its effect on the retinol metabolism is questionable (with corresponding reference(s)).

-l. 454. Tyrosine metabolism is associated with the synthesis of eumelanin.

RESULTS

-Table 2. All abbreviations (C, AS, HP, WGR, SGR) must be explained in the legend. What do letters near the numbers mean? What do ± mean? Is it standard error or standard deviation? It also should be explained. 

-Figure 1. All abbreviations (C, AS, HP) must be explained in the legend. What do errors and letters mean?

-l. 250-251. It is repetition of methods. Remove.

-l. 261. “Interestingly” - sounds unscientific. Remove.

-Figure 2. All abbreviations (C, AS, HP) must be explained in the legend.

-Table 3. All abbreviations (C, AS, HP) must be explained in the legend. ID should be an Identifier. Identifier in which database?

-l. 287, 324, 336. “alpha-linolenic” should be “α-linolenic”.

-Figure 3. All abbreviations (C, AS, HP) must be explained in the legend.

-l. 299-217. I advise against the terms “metabolite upregulation/downregulation”. They refer to gene expression or metabolic pathways. It is rather increasing/decreasing the content of a metabolite.

-Table 4. All abbreviations (C, AS, HP, POS, NEG) must be explained in the legend.

-Table 5. All abbreviations (C, AS, HP, RT) must be explained in the legend. ID should be an Identifier. Identifier in which database?

-Table 5. There is an issue of accuracy of measurements. Namely, what is the justification of the number of significant figures after a point? Why is it different in all cases? This data should be revised.

-l. 323-328, 333-343. In English, names of metabolites and processes should not be written with capital letters in the middle of the sentences, e.g. “Vitamin” should be “vitamin”, “Nonribosomal peptide” should be “nonribosomal peptide”, “Ferroptosis” should be “ferroptosis”, etc.

-Table 6. All abbreviations (C, AS, HP, POS, NEG) must be explained in the legend. ID should be an Identifier. Identifier in which database? “alpha-Linolenic” should be “α-Linolenic”. What is the justification of the number of significant figures after a point in the p-value column?

-Figure 4. All abbreviations (C, AS, HP, RT-qPCR) must be explained in the legend. What do errors mean? For the foldchange the symbol FC was introduced in the work. It should be used through the text.

DISCUSSION

-l. 362-363. What is “NADPH dehydrogenase (ubiquinone)”? Is it NADPH:ubiquinone oxidoreductase?

-l. 363. beta should be β.

-In the results, you compare a* and b* in different parts of the fish body (l. 230-236), Figure 1), whereas in Discussion you compare the parameter for different experimental groups (l. 370-374). It would be nice to have in Results a table/figure with this comparison.

-l. 370-374. How was explained the difference in the L*?

-l. 381-383. Rephrase to “Angell et al. found that the esterified astaxanthin was better absorbed than the free pigment, resulting in better coloration compared to that of the free form of synthetic astaxanthin”.

-l. 394. “hydrophobic and fat-soluble.” - it is the same!

-l. 394-395. Carotenoids are also lipids. Rephrase to “Carotenoid transport processes is closely associated with the transport of other lipids”.

-l. 473-476. May be melanin content can be assessed by L* or ITA values. For example, see e.g. doi:10.1111/jocd.14356. ITA values gives an opportunity to remove redness from melanin assessment. Number and size of black spots also can be considered as a feature. Is it possible to do these simple analyses?

-l. 486. Names of metabolites and processes should not be written with capital letters in the middle of the sentences.

Author Response

Response to Reviewer 3

The article aims to describe an effect of supplementation of synthetic and natural astaxanthin on color,growth, gene expression and metabolic profile of the fish Plectropomus leopardus (Perciformes, Serranidae). Animals (with one exclusion) cannot synthesize carotenoids, such as astaxanthin, de novo, however the pigment determines skin and meat color, which is an essential parameter for assessment of quality. Hence, astaxanthin is an essential part of a fish diet. Thus, the work is actual and in scope of the journal. State of the art of the work is well described. It corresponds to current technologies in the field. Zhang et al. used UPLC-MS, RNA-seq, and qPCR. Color of fish was assessed in the CIE L*a*b* color space. Required ethical statement is provided for the work with animals. Results are new and valuable. Discussion is addressed to interesting concepts of carotenoid effect on fish and their transport. The work may be accepted after some corrections (see specific comments below).

GENERAL COMMENTS

-Haematococcus pluvialis is a wrong obsolete synonym. Haematococcus lacustris is correct (doi:10.12705/652.11).

Response: Thank you so much for your comment and suggestion. We have double-checked the full text and replaced Haematococcus pluvialis with Haematococcus lacustris.

-FC, fold change and foldchange should be unified through the text.

Response: Thank you. We have double-checked the full text and used FC consistently.

-RT-PCR and qPCR are synonyms. It is better to use one of these terms instead RT-qPCR.

Response: Thank you. We have double-checked the full text and used qPCR consistently.

INTRODUCTION, ABSTRACT AND SUMMARY

-l. 22-23. Astaxanthin is a red colorant rather than color enhancer.

Response: Thank you. Now the sentence has been revised as follow: Astaxanthin is commonly used as a red colorant in aquaculture.

-Abstract is good but inconclusive. Do not introduce unused symbols (L*, a*, b*).l suggest adding the conclusion about which astaxanthin (synthetic or natural) is better for fish feeding.

Response: Thank you. We have described CIE L*a*b* at Materials and Methods. We have added conclusions to the abstract, which reads as follows: In conclusion, natural astaxanthin has better coloration effect on P. leopardus, which is more suitable as red colorant in aquaculture.

-l. 59. Which melanin do you mean? Eumelanin?

Response: Thank you. We have revised it..

-l. 73. “β,β-carotene” should be “β-carotene”.

Response: Thank you for your suggestion. We have made the correction.

-l. 83. “Astaxanthin, a natural red carotenoid,” - it is wrong and is in contradiction with the information below. There are natural and synthetic astaxanthin.

Response: Thank you. Now the sentence has been revised as follow: Astaxanthin, a keto-carotenoids, promotes coloration [25] and improves antioxidant activity [26] and immunity [27].

-l. 91-92. Microalgae are also microorganisms. Please, rephrase.

Response: Thank you. Now the sentence has been revised as follow: Natural astaxanthin is mainly obtained from microalgae, yeasts or crustacean, such as Bracteacoccus aggregatus BM5/15, Phaffia rhodozyma, shrimp and crabs [30,31]. The microalgae Haematococcus lacustris (formerly Haematococcus pluvialis) was reported to have abundant astaxanthin [32].

-l. 91-95. It is also important to say that there are other sources of astaxanthin (doi: 10.3390/biology10070643).

Response: Thank you. Now the sentence has been revised as follow: Natural astaxanthin is mainly obtained from microalgae, yeasts or crustacean, such as Bracteacoccus aggregatus BM5/15, Phaffia rhodozyma, shrimp and crabs [30,31]. The microalgae Haematococcus lacustris (formerly Haematococcus pluvialis) was reported to have abundant astaxanthin [32].

-l. 105. Spirulina is not a dye. It is an alga. Please, rephrase.

Response: Thank you. Now the sentence has been revised as follow: Astaxanthin has been shown to be more effective in coloring P. leopardus than other carotenoid sources, such as spirulina [39,40].

MATERIALS AND METHODS

-l. 111-114. Were the animals killed in the experiments? If yes, the procedure of eutanasia should be described according to AVMA guidelines (Underwood, W., Anthony, R., Cartner, S., Corey, D., Grandin, T., Greenacre, C., ... & Yanong, R. (2013) AVMA guidelines for the euthanasia of animals: 2013 edition. Schaumburg, IL: American Veterinary Medical Association). For fish immersion into 200 mg/L benzocaine is recommended.

Response: Thank you. We executed some fish in the course of the experiment. As we mentioned in the Experimental fish and sample collection, we anesthetized the experimental fish with MS-222 (Sigma, USA). As far as we know, MS-222 acting as an anesthetic is more common in fish experiments. Besides, MS-222 causes relatively little stress and other adverse effects on the fish. However, we also agree with your suggestion that the fish will be immersed into 200 mg/L benzocaine for the next study. We have modified the Ethics Statement as follows: All experimental protocols were approved by the Animal Research and Ethics Committee of Guangdong Ocean University (201903003) in this study. The study does not involve endangered or protected species. During the experiments, the experimental fish were treated according to the euthanasia procedures described in the AVMA Guidelines for the Euthanasia of Animals: 2013 Edition.

-Table 1. All abbreviations (C, AS, HP) must be explained in the legend.

Response: Thank you. We have made the changes in Table 1, which reads as follows: Note: 1. Fish meal, wheat gluten flour, soybean meal, Seaweed powder, wheat meal, soybean lecithin, fish oil, Ca(H₂PO₄)₂ were purchased from Guangdong Yuehai Biotechnology Co., Ltd., Zhanjiang, China. 2. Astaxanthin content in Carophyll® pink powder accounted for 10%. Carophyll® pink powder was produced by Guangzhou Leader Biotechnology Co., Ltd., Guangzhou, China. 3. Astaxanthin content in Haematococcus lacustris powder accounted for 3%. The H. lacustris powder was produced by Yunnan Alphy Biotechnology Co., Ltd., Chuxiong, China. 4. The letter C is control, AS is synthetic astaxanthin, HP is natural astaxanthin.

-l. 119. Please, indicate the manufacturer and country for Carophyll^® pink. What was the origin of Haematococcus astaxanthin?

Response: Thank you for your comment. We have illustrated these in the legend in Table 1.

-l. 142-155. Please, describe the dataset in more detail in terms of its uniformity. Was sex of the fish determined? Indicate age of the fish (young/adult/old). Were they healthy or ill?

Response: Thank you for your suggestion. We have made the changes according to your suggestions, which are as follows: The experimental fish (7-month-old adult fish) were purchased from Guangdong Sanhe Lvyuan Aquaculture Co., Ltd., Zhanjiang, China. Fish were placed in an indoor cement pool (3.5 m × 5 m × 0.6 m) and fed basal feed for 14 days to adapt to the experimental environment. After that, 450 healthy adult fish with uniform size (fish average mass: (132.43 ± 6.69) × 10^-3 kg) were selected and then randomly separated into three groups, each of which was placed in three cages (2 m × 1 m × 1 m).

-l. 175-176. Indicate sequenator and reagents for sequencing.

Response: Thank you so much for your suggestion. Now the sentence has been revised as follow: The nine constructed libraries were then sequenced on the Illumina NovaSeq 6000 (Illumina, Inc., San Diego, USA) and paired-end reads were generated.

-l. 157-164. Please, describe briefly in several sentences the CIEL*a*b* space concept (doi:org/10.1016/j.talanta.2012.11.026).

Response: Thank you. We have made the changes according to your suggestions, which are as follows: The Chroma Meter was used to measure in L*, a* and b* measurement modes (CIE1976) with the D65 illuminant. The white plate supplied by the manufacturer was used to calibrate the Chroma Meter. The L*a*b* color space (also referred to as CIELAB) is a system for expressing color digitally developed by the International Commission on Illumination (CIE) in 1976. Colors and standardized L* color space values are matched by this system. Where a point in the L*a*b* color space represents the color [41]. In this space, the color parameter L* represents the brightness, and its value ranges from 0 (black) to 100 (white). The a* and b* both represent the color direction. a* > 0 indicates the red direction, a* < 0 indicates the green direction, b* > 0 indicates the yellow direction, and b* < 0 indicates the blue direction.

-l. 178-183. Which metric was used for the measurement of absolute expression level? FKPM? Were replications in each experimental group?

Response: Thank you. We have made the changes according to your suggestions.

-l. 200. m/z should be italicized through the text.

Response: Thank you. We have double-checked the full text and made corrections.

-The procedures of statistical treatment should be indicated in a separate subsection.

Response: Thank you. We have added the Data calculation and statistical analysis as you suggested.

-l. 433. And leukotrienes also.

Response: Thank you for your comment. Now the sentence has been revised as follow: Moreover, 5(S)-HETE and leukotriene are lipoxygenase metabolites resulting from arachidonic acid metabolism [71,72].

-l. 441-452. It should be stated here, that astaxanthin is not a provitamin A, therefore its effect on the retinol metabolism is questionable (with corresponding reference(s)).

Response: Thank you for your comment. We have removed the paragraph. We also have amended the content as follows: Besides, gene rdh12 related to retinol metabolism using carotenoids as substrates was down-regulated in HP vs C, which may explain the low carotenoid substrate conversion in the fish skin.

-l. 454. Tyrosine metabolism is associated with the synthesis of eumelanin.

Response: Thank you. We have made changes as follows: Tyrosine metabolism is associated with the synthesis of melanin, especially eumelanin.

RESULTS

-Table 2. All abbreviations (C, AS, HP, WGR, SGR) must be explained in the legend. What do letters near the numbers mean? What do ± mean? Is it standard error or standard deviation? It also should be explained.

Response: Thank you so much for your comment. We have revised it according to your comment as follows: Data were presented as mean ± SD (n = 9). Column with different letters indicate difference (P < 0.05) among groups. Where C is control, AS is synthetic astaxanthin, HP is natural astaxanthin, WGR is weight gain rate and SGR is specific growth rate.

-Figure 1. All abbreviations (C, AS, HP) must be explained in the legend. What do errors and letters mean?

Response: Thank you so much for your comment. We have corrected it in Figure 1 following your comment.

-l. 250-251. It is repetition of methods. Remove.

Response: Thank you so much for your comment. We have removed this sentence.

-l. 261. “Interestingly” - sounds unscientific. Remove.

Response: Thank you so much for your suggestion. We have removed this word.

-Figure 2. All abbreviations (C, AS, HP) must be explained in the legend.

Response: Thank you so much for your comment. We have corrected it in Figure 2 following your comment.

-Table 3. All abbreviations (C, AS, HP) must be explained in the legend. ID should be an Identifier. Identifier in which database?

Response: Thank you so much for your comment. We have corrected it in Table 3 following your comment. The ID in the Table 3 means gene ID. You can find them in Table S3-5 of the Supplementary Material.

-l. 287, 324, 336. “alpha-linolenic” should be “α-linolenic”.

Response: Thank you for your suggestion. We have corrected it.

-Figure 3. All abbreviations (C, AS, HP) must be explained in the legend.

Response: Thank you so much for your comment. We have corrected it in Figure 3 following your comment.

-l. 299-217. I advise against the terms “metabolite upregulation/downregulation”. They refer to gene expression or metabolic pathways. It is rather increasing/decreasing the content of a metabolite.

Response: Thank you so much for your suggestion. We have made revisions throughout the text.

-Table 4. All abbreviations (C, AS, HP, POS, NEG) must be explained in the legend.

Response: Thank you so much for your comment. We have corrected it in Table 4 following your comment.

-Table 5. All abbreviations (C, AS, HP, RT) must be explained in the legend. ID should be an Identifier. Identifier in which database?

Response: Thank you for your comment. We have corrected it in Table 5 following your comment.

-Table 5. There is an issue of accuracy of measurements. Namely, what is the justification of the number of significant figures after a point? Why is it different in all cases? This data should be revised.

Response: Thank you for your comment. We have changed all the result values to retain 2 significant figures.

-l. 323-328, 333-343. In English, names of metabolites and processes should not be written with capital letters in the middle of the sentences, e.g. “Vitamin” should be “vitamin”, “Nonribosomal peptide” should be “nonribosomal peptide”, “Ferroptosis” should be “ferroptosis”, etc.

Response: Thank you so much for your suggestion. We have made revisions throughout the text.

-Table 6. All abbreviations (C, AS, HP, POS, NEG) must be explained in the legend. ID should be an Identifier. Identifier in which database? “alpha-Linolenic” should be “α-Linolenic”. What is the justification of the number of significant figures after a point in the p-value column?

Response: Thank you so much for your comment. We have corrected it in Table 6 following your comment. Besides,we have expressed the result values of this column in scientific notation and reserved 2 significant figures. The ID in the Table 6 means metabolite ID. We can find it in Table S6-8 of the Supplementary Material.

-Figure 4. All abbreviations (C, AS, HP, RT-qPCR) must be explained in the legend. What do errors mean? For the foldchange the symbol FC was introduced in the work. It should be used through the text.

Response: Thank you so much for your comment. We have corrected it in Figure 4 following your comment. Data were presented as mean ± SD. Besides, we have consistently used FC instead of foldchange throughout the text.

DISCUSSION

-l. 362-363. What is “NADPH dehydrogenase (ubiquinone)”? Is it NADPH:ubiquinone oxidoreductase?

Response: Thank you for your comment. We have corrected it.

-l. 363. beta should be β.

Response: Thank you for your suggestion. The editor suggest that the Greek letter cannot be used in gene names. We choose to use beta.

-In the results, you compare a* and b* in different parts of the fish body (l. 230-236), Figure 1), whereas in Discussion you compare the parameter for different experimental groups (l. 370-374). It would be nice to have in Results a table/figure with this comparison.

Response: Thank you so much for your comment and suggestion. We have corrected it in the results as follows: For the whole fish coloration, the a* and b* values of HP were higher than those of C and AS.

-l. 370-374. How was explained the difference in the L*?

Response: Thank you so much for your comment. We have supplemented the explanation of the difference in L* as follows: The decrease in fish brightness may be related to astaxanthin. Some studies have shown that astaxanthin supplementation increased redness and yellowness but decreased brightness in blood parrotfish and Colisa lalia [45,50]. This suggests that astaxanthin deposition may promote fish coloration but also lead to a reduction in brightness, resulting in a dark red appearance of the fish.

-l. 381-383. Rephrase to “Angell et al. found that the esterified astaxanthin was better absorbed than the free pigment, resulting in better coloration compared to that of the free form of synthetic astaxanthin”.

Response: Thank you so much for your suggestion. We have made modifications.

-l. 394. “hydrophobic and fat-soluble.” - it is the same!

Response: Thank you so much for your comment. We have made modifications.

-l. 394-395. Carotenoids are also lipids. Rephrase to “Carotenoid transport processes is closely associated with the transport of other lipids”.

Response: Thank you so much for your suggestion. We have made modifications.

-l. 473-476. May be melanin content can be assessed by L* or ITA values. For example, see e.g. doi:10.1111/jocd.14356. ITA values gives an opportunity to remove redness from melanin assessment. Number and size of black spots also can be considered as a feature. Is it possible to do these simple analyses?

Response: Thank you so much for your comment and suggestion. There may be a relationship between brightness and melanin content. Thank you again for your valuable suggestions. The number of samples we have is no longer sufficient to support our measurements of melanin content in the skin. Its results may not be convincing. The same is true for the analysis of ITA values. Thank you again for your valuable suggestions. We will use ITA values for the analysis in our next study.

-l. 486. Names of metabolites and processes should not be written with capital letters in the middle of the sentences.

Response: Thank you so much for your suggestion. We have made revisions throughout the text.

Author Response File: Author Response.docx

Round 2

Reviewer 2 Report

This manuscript entitled "Effects of natural and synthetic astaxanthin on growth, body color and transcriptome and metabolome profiles in the leopard coralgrouper (Plectropomus leopardus) " by Zhang et al. is an excellent work, which makes use of multiple omics tools, which give a deep understanding of the metabolic pathways associated with the effects obtained, providing an understanding of the differences found, as well as questions to continue with future research. The authors made the necessary adjustments to all the reviewers, with this the manuscript increased its quality and is ready to be accepted in a high impact and prestigious journal such as Animals.

Author Response

Thank you very much for your comments.

Reviewer 3 Report

The authors have improved the text. However, there are some minor issues, which should be improved before publsihing.

1. l. 73, 74, 86, 475, 342, 380, 394. These are not gene names, therefore, alpha should be α and beta should be β.

2. l. 92, 95. Why 'mainly'? Synthetic astaxanthin is obtained by chemical synthesis (by definition). Natural is obtained from natural sources.

3. l. 95-97. The newly added sentence is confusing. It is better to rephrase as microalgae, such as Bracteacoccus aggregatus BM5/15, yeasts  Phaffia rhodozyma, or crustaceans, such as  shrimp and crabs [29,30].". Now the reference Chekanov et al. (2021) can be replaced/supplemented by recent work by same authors (https://doi.org/10.3390/md21020108).

4. l. 121-123. The reference for the guidelines should be added to the reference list.

5. l. 194. This description is substandard, remove. Indeed, RNA extraction should be performed. Then, revers transcription should be done. Whas the shotgun method applied for libraries preparation? Please, revise.

6. l. 253. What are M and CV?

7. l. 260. Please, replace = (ln Wt - ln W0)/t ×100 with = (ln (Wt/W0))/t ×100 to remove units of W under logarithm.

8. l. 287-288. Please, suport this statement by some numerical estimations What do you mean on color of the whole fish? is it an average of colors of different parts? somthing other?

Author Response

Response to Reviewer 3

The authors have improved the text. However, there are some minor issues, which should be improved before publsihing.

1. 73, 74, 86, 475, 342, 380, 394. These are not gene names, therefore, alpha should be α and beta should be β.

Response: Thank you so much for your comment and suggestion. We have made corrections in the full text.

2. l. 92, 95. Why 'mainly'? Synthetic astaxanthin is obtained by chemical synthesis (by definition). Natural is obtained from natural sources.

Response: Thank you for your comment. Now the sentence has been revised as follow:  Synthetic astaxanthin is obtained by chemical synthesis using chemical raw materials and is a racemic mixture with an optical isomer ratio of 1:2:1 (3S,3’S:3R,3’S:3R,3’R) [25,28]. Natural astaxanthin is obtained from microalgae, such as Bracteacoccus aggregatus BM5/15, yeasts Phaffia rhodozyma, or crustaceans, such as shrimp and crabs [29-31].

3. l. 95-97. The newly added sentence is confusing. It is better to rephrase as microalgae, such as Bracteacoccus aggregatus BM5/15, yeasts  Phaffia rhodozyma, or crustaceans, such as  shrimp and crabs [29,30].". Now the reference Chekanov et al. (2021) can be replaced/supplemented by recent work by same authors (https://doi.org/10.3390/md21020108).

Response: Thank you. Now the sentence has been revised as follow: Synthetic astaxanthin is obtained by chemical synthesis using chemical raw materials and is a racemic mixture with an optical isomer ratio of 1:2:1 (3S,3’S:3R,3’S:3R,3’R) [25,28]. Natural astaxanthin is obtained from microalgae, such as Bracteacoccus aggregatus BM5/15, yeasts Phaffia rhodozyma, or crustaceans, such as shrimp and crabs [29-31].

4. l. 121-123. The reference for the guidelines should be added to the reference list.

Response: Thank you for your comment. We have made the corrections as you suggested.

5. 194. This description is substandard, remove. Indeed, RNA extraction should be performed. Then, revers transcription should be done. Whas the shotgun method applied for libraries preparation? Please, revise.

Response: Thank you. The library was not prepared using the shotgun method. Now the sentence has been revised as follow: The total RNA of skin was extracted using the TRIzol reagent (Life Technologies, Carlsbad, CA, USA). The DNase I (New England Biolabs, Ipswich, MA, USA) was used to remove genomic DNA from RNA samples. RNA quality was assessed using 1% agarose gel electrophoresis and Nano spectrophotometer (Nanodrop 2000c, Thermo Scientific, Wilmington, DE, USA). High quality RNA samples were sent to Biomarker Technologies (Beijing, China) for subsequent library construction and sequencing. Nine cDNA libraries (three pooled samples per group) were constructed from nine RNA samples. Library construction was performed according to the instructions of the NEBNext^® Ultra™ RNA Library Prep Kit for Illumina^® (NEB, USA). The mRNA was enriched with magnetic beads with Oligo (dT). The mRNA was fragmented by adding fragmentation buffer and the first strand cDNA was synthesised using random hexamer primer. The second strand cDNA was then synthesised using DNA Polymerase I and RNase H. The poly-A base was added to the 3' end of the cDNA fragment, followed by fragment size selection (240 bp range). Finally, the cDNA library was obtained by PCR enrichment.

6. l. 253. What are M and CV?

Response: Thank you for your comment. Now the sentence has been revised as follow: Gene eukaryotic elongation factor 2 (eef2) was used as a reference gene (average expression stability (M) = 0.58, coefficient of variation (CV) = 4.49%).

7. l. 260. Please, replace = (ln Wt - ln W0)/t ×100 with = (ln (Wt/W0))/t ×100 to remove units of W under logarithm.

Response: Thank you so much for your suggestion. We have made the corrections as you suggested.

8. 287-288. Please, suport this statement by some numerical estimations What do you mean on color of the whole fish? is it an average of colors of different parts? somthing other?

Response: Thank you. We have used different lowercase letters such as ‘a, b and c’ to indicate differences between groups in Figure 1. The content of the last sentence may be unreasonable, we have deleted it.

Once again, thank you.

Author Response File: Author Response.docx