Three Types of Enteromorpha prolifera Bio-Products Based on Different Processing Procedures as Feed Additives in the Diets of Pacific White Shrimp (Litopenaeus vannamei)
, Mengqing Liang 1 and Yuliang Wei 1,2,*
Round 1
Reviewer 1 Report
Comments and Suggestions for AuthorsThe paper “Three Types of Enteromorpha prolifera Bio-products Based on 2 Different Processing Procedures as Feed Additives in The Diets 3 of Pacific White Shrimp (Litopenaeus vannamei)” presents novel information on the use of additives in aquaculture to increase resistance to stress. However, some clarifications and specifications must be carried out.
Do not use ambiguous terms like "obviously" (L33). use values and terms from the statistical analyzes carried out
Throughout the text, use the full name of the enzymes and standardize their use in different paragraphs (i.e. glutathione is used several times instead of glutathione peroxidase). It is recommended to use abbreviations (i.e. gpx). Make the necessary changes throughout the text
Name scientific names with the appropriate nomenclature (Key Contribution section)
Was any buffer or reagent used to preserve the molecular biology samples (RNalater, trizol, nitrogen)?
How many sections were made for each hepatopancreas, how many readings were made for each histological section? The conclusions drawn were based on the average of the values obtained?
briefly describe the calculations carried out to obtain the final values of relative expression from 2 different genes (were the averages obtained for each gene?)
What statistical test was used for the cumulative mortality values?
how many numbers of drops of fat (L287)??, add a table with the values of the counts to make a real decision. Avoid using ambiguities, you must put the values (and thus know what is less than...)
Hypothermia tests are mentioned when they have not been carried out in the experiment (L363)
The discussion should be improved, in certain sections only what is compared in fish or even mammals is discussed. You should look for information related to crustaceans (look for information on similar compounds that produce a similar response in shrimp, so you can have a better reference parameter)
Comments on the Quality of English LanguageIn general good quality of English in the paper
Author Response
- The paper “Three Types of Enteromorpha prolifera Bio-products Based on Different Processing Procedures as Feed Additives in The Diets of Pacific White Shrimp (Litopenaeus vannamei)” presents novel information on the use of additives in aquaculture to increase resistance to stress. However, some clarifications and specifications must be carried out.
Response: Thank you very much for the reviewer's comments. We will try our best to revise it and hope to meet your expectations.
- Do not use ambiguous terms like "obviously" (L33). use values and terms from the statistical analyzes carried out
Response: Thank you for your kind reminder. It was a description of qualitative observations of the amounts of lipid droplets from histological analysis in the hepatopancreas of shrimp. Thus, it had been revised as following: “Gross qualitative observation showed that the amounts of lipid droplets decreased in hepatopancreas of the EPH0.4% and EPM3% groups”.
- Throughout the text, use the full name of the enzymes and standardize their use in different paragraphs (i.e. glutathione is used several times instead of glutathione peroxidase). It is recommended to use abbreviations (i.e. gpx). Make the necessary changes throughout the text
Response: Thanks for your recommendation. The abbreviations had been used throughout the text and the necessary changes had also been made.
- Name scientific names with the appropriate nomenclature (Key Contribution section)
Response: Thanks for your comments. The scientific names had been revised.
- Was any buffer or reagent used to preserve the molecular biology samples (RNalater, trizol, nitrogen)?
Response: The molecular biology samples was frozen in liquid nitrogen and stored at -80 ℃ until subsequent analysis. It had been supplemented in the manuscript.
- How many sections were made for each hepatopancreas, how many readings were made for each histological section? The conclusions drawn were based on the average of the values obtained?
Response: We apologized for the misunderstanding caused by our description. Hepatopancreas was collected from 6 shrimp per tank after hemolymph samples were collected. The hepatopancreas of three of them were used for gene expression analysis, and the hepatopancreas of the other three were used for histological analysis.
“After hemolymph sampling, six shrimp were euthanized with lethal dose of MS-222 (500 mg / L) and dissected to collect hepatopancreas. Hepatopancreas sample from three of them was collected, frozen in liquid nitrogen and stored at -80 ℃ for the analysis of gene expression and triglycerides contents, and the other three was fixed in bouin's solution for histological processing.”
- briefly describe the calculations carried out to obtain the final values of relative expression from 2 different genes (were the averages obtained for each gene?)
Response: Thanks for your suggestion. Firstly, the Ct values of β-actin and gapdh were used to calculate the geometric mean of the Ct values. And then, gene expression was normalized relative to the geometric mean of the Ct values of two reference genes (Vandesompele et al., 2002). The description of the calculations had been supplemented in the manuscript.
“β-actin and glyceraldehyde-3-phosphate dehydrogenase (gapdh) were used as reference genes in this study. Relative mRNA expression was calculated by 2-ΔΔCt method [22]. Target expression was examined with normalization to the geometric mean of the Ct values from two reference genes.”
Reference:
Vandesompele, J., De Preter, K., Pattyn, F., Poppe, B., Van Roy, N., De Paepe, A., Speleman, F., 2002. Accurate normalization of real-time quantitative RT-PCR data by geometric averaging of multiple internal control genes. Genome Biol. 3 (7), 0034.1.
- What statistical test was used for the cumulative mortality values?
Response: Cumulative mortality was expressed using arithmetic mean of three tank. Due to the significant difference in mortality rates among the three tanks of the same treatment at the same time after hypoxic stress, reliable statistical analysis was not possible. Therefore, we compared the arithmetic mean values between treatments. Given that the arithmetic mean can also provide the trend of mortality rate over time after hypoxic stress, the data of cumulative mortality was provided in this study.
- how many numbers of drops of fat (L287)??, add a table with the values of the counts to make a real decision. Avoid using ambiguities, you must put the values (and thus know what is less than...)
Response: Thanks for your suggestion. We also hope to be able to count the number of lipid droplets. However, many lipid droplets are fused together, as shown in Figure 1A, which seriously interferes with the counts of the number of lipid droplets. In addition, because the size of each lipid droplet is different, simply counting the number of lipid droplets cannot truly reflect the content of lipid droplets in the hepatopancreas. In fact, the results of hepatopancreatic lipid droplets in this study were evaluated in conjunction with the contents of triglycerides in the hepatopancreas.
- Hypothermia tests are mentioned when they have not been carried out in the experiment (L363)
Response: Hypothermia stress had been conducted in this experiment and described in the section of Hypoxia stress and hypothermia stress 2.7.
“At the end of sampling, remaining shrimp from each tank were refed their allocated diets, and then average daily feed intake of each shrimp were measured. After that, compared to normal temperature water (about 26 ℃), lower temperature water (21 ℃) were added to the tanks to perform the hypothermia challenge tests. Average daily feed intake per shrimp were measured again to calculate decreased percentage of feed intake, which would be used to evaluate the response of shrimp to hypothermia stress.”
- The discussion should be improved, in certain sections only what is compared in fish or even mammals is discussed. You should look for information related to crustaceans (look for information on similar compounds that produce a similar response in shrimp, so you can have a better reference parameter)
Response: Thanks for your suggestion. In this study, we mainly discussed the research results of Enteromorpha prolifera in aquatic animals and mammals. Due to the limited research of E. prolifera on crustaceans, the information related to crustaceans provided in the discussion is also relatively limited. This also indicates the necessity of this study. In addition, we also searched the research progress of E. prolifera on crustaceans again, and did not find more new references.
Author Response File: 
Author Response.docx
Reviewer 2 Report
Comments and Suggestions for AuthorsInteresting work that seeks to solve the problem of excess E. prolifera algae on some Asian coasts, improving its use in feed for Pacific white shrimp.
Materials y methods
2.1. Preparation of bio-products of Enteromorpha prolifera
The data on the composition of the bio-products used from E. prolifera are assumed to be expressed in dry substance, since the production process involves drying. So, what other components are there until reaching 100% of the material obtained?
2.3. Experimental shrimp and feeding trial
The temperature variation is excessively high. 9°C difference may cause changes in results. What are these temperature changes due to?
2.4. Sample collection
An anticoagulant is added to the hemolymph, however, the text says that centrifugation is carried out after coagulation. What explanation does this have?
Sampling of shrimp for body composition is not specified. It is not known how many specimens are used for each experimental situation.
2.5.2. Serum or hepatopancreas biochemical, antioxidant and immunological parameters
Triglycerides of hepatopancreas: in the previous section it is specified that the hepatopancreas is divided into 2 portions: for gene expression and histological study, where are triglycerides analyzed then?
The measurement of malondialdehyde and reduced glutathione is included as if they were serum enzymes related to antioxidant capacity.
Results
Table 6. The errors of the average triglycerides in hepatopancreas are very large, in some cases 20-25%, which makes the results of this parameter not very credible.
Conclusions
It would be interesting to know how much E. prolifera is needed to obtain a certain amount of hydrolyzate or polysaccharides and the cost of these processes. This way, it could really be evaluated whether it is interesting to carry out this process or whether to include a certain amount of E. prolifera flour directly in the feed.
Author Response
- Interesting work that seeks to solve the problem of excess E. prolifera algae on some Asian coasts, improving its use in feed for Pacific white shrimp.
Response: Thanks for your comments.
Materials y methods
- Preparation of bio-products of Enteromorpha prolifera
The data on the composition of the bio-products used from E. prolifera are assumed to be expressed in dry substance, since the production process involves drying. So, what other components are there until reaching 100% of the material obtained?
Response: It is a good question. The data on the composition of E. prolifera bio-products was provided by their production enterprise (Qingdao Seawin Biotech Group Co., Ltd.). Although the content of ash has not been determined, according to the ash content of E. prolifera reported by Asino et al. (2010), we believe that the remaining components should be mainly ash. In addition, given that ash is not biologically active molecules, it was not specifically measured in this study.
Reference:
Asino, H., Ai, Q., & Mai, K. (2011). Evaluation of Enteromorpha prolifera as a feed component in large yellow croaker (Pseudosciaena crocea, Richardson, 1846) diets. Aquaculture Research, 42(4), 525-533.
Experimental shrimp and feeding trial
- The temperature variation is excessively high. 9°C difference may cause changes in results. What are these temperature changes due to?
Response: I am very sorry. It is a mistake. Water temperature should be 26.0-29.2 ℃, not 20.0-29.2 ℃.
Sample collection
- An anticoagulant is added to the hemolymph, however, the text says that centrifugation is carried out after coagulation. What explanation does this have?
Response: The collection of serum samples was based on the procedure reported in previous references (Li et al., 2009). As stated by the reviewer, there are only a very small amount of clots after the centrifugation of hemolymph samples. Therefore, our existing description “the coagulation of hemolymph samples” was inappropriate. The sentence had been revised as following:
“Hemolymph samples were collected and mixed with SIC-EDTA anticoagulant (10 mmol / L of ethylenediaminetetraacetic acid (EDTA)-Na2, 450 mmol / L NaCl, 10 mmol / L KCl, 10 mmol / L 4- (2- hydroxyethyl) piperazine-1-ethanesulfonic acid (HEPES), and pH 7.0) at a 1:2 ratio, and kept at -4 ℃ for 3 h [20]. And then, they were centrifuged at 3000 g for 10 min, and the supernatant was used as serum samples and stored at -80 ℃ for further analysis.”
Reference:
Li, J., Tan, B., & Mai, K. (2009). Dietary probiotic Bacillus OJ and isomaltooligosaccharides influence the intestine microbial populations, immune responses and resistance to white spot syndrome virus in shrimp (Litopenaeus vannamei). Aquaculture, 291(1-2), 35-40.
- Sampling of shrimp for body composition is not specified. It is not known how many specimens are used for each experimental situation.
Response: Thanks for your reminder. Two shrimp from each tank were sampled for the analysis of body composition. The information on sampling of shrimp for body composition had been supplemented in the section of Sample collection (2.4).
“In addition, two shrimp per tank were sampled for the analysis of body composition.”
- Serum or hepatopancreas biochemical, antioxidant and immunological parameters
Triglycerides of hepatopancreas: in the previous section it is specified that the hepatopancreas is divided into 2 portions: for gene expression and histological study, where are triglycerides analyzed then?
Response: Thanks for your comments. The determination of triglycerides in the hepatopancreas was not our initial experimental plan. When we found that lipid droplets in the hepatopancreas of shrimp fed bio-products of E. prolifera had a tendency to decrease compared with control group by oil red O staining. To verify this observation, we measured the triglyceride content in hepatopancreas. The samples used for determining triglycerides contents in the hepatopancreas is the remaining samples after the analysis of gene expression. The sentence had been revised as following:
“Hepatopancreas sample from three of them was collected, frozen in liquid nitrogen and stored at -80 ℃ for the analysis of gene expression and triglycerides contents, and the other three was fixed in bouin's solution for histological processing.”
- The measurement of malondialdehyde and reduced glutathione is included as if they were serum enzymes related to antioxidant capacity.
Response: Thanks for your reminder. In Table 7, it has been marked that superoxide dismutase and glutathione peroxidase are enzymes, while malondialdehyde and reduced glutathione are non-enzyme antioxidants. It had been revised in Table 7.
Results
- Table 6. The errors of the average triglycerides in hepatopancreas are very large, in some cases 20-25%, which makes the results of this parameter not very credible.
Response: Thanks for your reminder. We did not notice this case. But after re-checking the data of the triglycerides in hepatopancreas, we found that the homogeneity of variances for this data were confirmed by Levene’s test (P = 0.162), and met the demand for intra-group differences in statistical analysis. Therefore, although the errors of the average triglycerides in hepatopancreas are large, the results may be reliable.
Conclusions
- It would be interesting to know how much E. prolifera is needed to obtain a certain amount of hydrolyzate or polysaccharides and the cost of these processes. This way, it could really be evaluated whether it is interesting to carry out this process or whether to include a certain amount of E. prolifera flour directly in the feed.
Response: Thanks for your suggestion. The amounts of E. prolifera bio-products in experimental diets were recommended by their production enterprise (Qingdao Seawin Biotech Group Co., Ltd.), and the cost of three types of E. prolifera bio-products had already been considered when providing the recommended amounts. Therefore, the amounts of E. prolifera polysaccharide and E. prolifera hydrolysate provided in this study can be used as a guideline for feed formulation. However, for commercial reasons, the production enterprise did not provide specific prices for E. prolifera polysaccharide and E. prolifera hydrolysate, only knowing that E. prolifera meal is about 6000 RMB per ton, so it is not mentioned herein.
Author Response File: Author Response.docx
