Potential Symbiotic Effects of β-1,3 Glucan, and Fructooligosaccharides on the Growth Performance, Immune Response, Redox Status, and Resistance of Pacific White Shrimp, Litopenaeus vannamei to Fusarium solani Infection

Round 1

Reviewer 1 Report

Fishes-2168428

The manuscript by Eissa et al. entitled “Potential symbiotic effects of β-1,3 glucan and  fructooligosaccharides on the growth performance, immune response, redox status, and resistance of Pacific white shrimp, Litopenaeus vannamei to Fusarium solani infection” shows the results of the enhancing various functionalities for mixed prebiotics.

Based on scientific consideration, the manuscript contains some interesting findings. Still, it has many critical problems that the authors must carefully consider to correct and improve the manuscript's quality before the next review round. The major and minor concerns are as follows;

[Materials and Methods]

Line 112 (Preparation of experimental diets): Why was the experimental diet with prebiotics stored frozen at -4°C? Doesn't it have to do with the activity of prebiotics?

Line 118 (Shrimp rearing and feeding trial): Please write the size of experiment tanks in commonly used units. For example, how many liters? And what is the salinity of pond water?

Line 142 (Sample collection): Why was the hemolymph sample overnight at 4°C?

Line 152 (Digestive enzymes analysis): Can you typically collect 1g of intestine from 13-18g of shrimp? Is it pooling weight?

Line 183 (Fusarium solani challenge trial): Where are the clinical and mycological examination results for moribund and freshly dead shrimp?

[Results]

Line 252 (Fungal challenge trial): Please add images of shrimp with infection symptoms as supplemental data. Also, were collapsed, severe necrosis, and atrophy of gill lamellae observed by histopathological analysis? Authors need to attach images.

Summarizing all the analysis results, I think that the economic aspect should also be considered in order to use mixed prebiotics in an actual feed company. Among the various analysis results, there seems to be no significant difference between the 1.0 g/kg added group and the 1.5 g/kg added group. Why was the optimum level set at 1.5 g/kg?

Table 1 : Please inform the manuscript or foot note before using the abbreviation. What is Aquastem? Does "min mix" mean mineral mixture? Why did you add "min mix" when you already added "vit+min mix"?

Table 3 : Please indicate the unit of each analysis parameter.

Author Response

A point-by-point response to reviewers’ comments

Dear editors and reviewers,

Thanks very much for the comments and suggestions, which are all valuable and very helpful to revise and improve our manuscript. We have studied the comments carefully and have made corrections which we hope meet with approval. These revisions are marked with yellow color in the revised manuscript. Looking forward to your positive response. The point-by-point responses:

Reviewer 1:

[Materials and Methods]

Line 112 (Preparation of experimental diets): Why was the experimental diet with prebiotics stored frozen at -4°C? Doesn't it have to do with the activity of prebiotics?

Thanks for the grateful comment. We apologize for the oversight. This was a mistake on typing; the temperature that used is the temperature of refrigerator (4 °C). Considering the health benefits of prebiotics and their safety, as well as their production and storage advantages compared to probiotics, most prebiotics do not require any type of refrigeration. Since low temperature has no serious effect on prebiotic activity, it is still preferable to keep the commercial diet in a cool dry place to avoid unwanted growth of fungi and other marble pathogens in particular when the experiment lasts for several weeks as in our case 75 days.

Line 118 (Shrimp rearing and feeding trial): Please write the size of experiment tanks in commonly used units. For example, how many liters? And what is the salinity of pond water?

Thanks for the precious comment. The hapas size was represented by liter (175-liter holding capacity) and the water salinity was also provided as requested at 22.36‰ .

Line 142 (Sample collection): Why was the hemolymph sample overnight at 4°C?

Thanks for the reviewer’s comment. We apologize for the typing error.  Muscles are the only samples stored for further analysis, while the withdrawn hemolymph samples are analyzed immediately after collection. The sentence was rephrased as follows:

The collected samples were centrifuged at 3000 x g for 10 min, and the supernatants were separated and kept at -80 °C for further analyses.

Line 152 (Digestive enzymes analysis): Can you typically collect 1g of intestine from 13-18g of shrimp? Is it pooling weight?

Thanks for the grateful comment and suggestion. Indeed, the weight that was represented is not for the individual measure, but for pooling samples (samples pooled from six biological replicates).

Line 183 (Fusarium solani challenge trial): Where are the clinical and mycological examination results for moribund and freshly dead shrimp?

Thanks for the great suggestion. The following paragraph was added to this section

Moribund and freshly dead shrimp were regularly harvested for clinical and mycological examinations to ascertain the cause of death. Mortality was only considered when the injected strain was retrieved from experimentally infected shrimp (Koch’s postulates). Moreover, figure 3 representing the most common clinical signs was added to the result section

[Results]

Line 252 (Fungal challenge trial): Please add images of shrimp with infection symptoms as supplemental data. Also, were collapsed, severe necrosis, and atrophy of gill lamellae observed by histopathological analysis? Authors need to attach images.

Thanks for the precious comments. A figure representing the most clinical findings observed post-infection was added. Unfortunately, no histological study was performed on the collected gills. Hence, for further clarity, the paragraph has been rephrased as follows:

Within a few days of the challenge, the infected shrimp showed black to brown collapsed gills with severe necrosis and atrophy (Figure 2); whereas a negative control group displayed normal dusky white gills. Nonetheless, the fungal isolate was successfully recovered on PDA from the gills and muscles of infected shrimp and seen under a microscope in wet mount preparations from the same specimens.

Summarizing all the analysis results, I think that the economic aspect should also be considered in order to use mixed prebiotics in an actual feed company. Among the various analysis results, there seems to be no significant difference between the 1.0 g/kg added group and the 1.5 g/kg added group. Why was the optimum level set at 1.5 g/kg?

Thanks for the grateful comment. At first glance, it appears that there are no significant differences between the two doses. However, upon examination, several tested parameters such as trypsin, amylase, SOD, lysozyme, THC, and post-challenge survival rate were significantly improved after the addition of 1.5 g/kg compared to others. Therefore, we recommend using this dosage for maximum benefits and high performance regardless of price value

Table 1 : Please inform the manuscript or footnote before using the abbreviation. What is Aquastem? Does "min mix" mean mineral mixture? Why did you add "min mix" when you already added "vit+min mix"?

Thank you for this comment. Footnotes were added to describe the content that was written in abbreviation. Aquastem is the trade name of β-1,3 glucan and fructooligosaccharides. However, we prefer to write the active principle. So it was changed to β-1,3 GF. Further, brief description of the entire components and the methods of analysis and calculation were added as follows:

Analysis was performed according to Official Methods of Analysis, Association of Official Analytical Chemists, Arlington [83]. *CMC, carboxymethylcellulose as a thickening agent. **Vit & Min Mix, Vitamin and Mineral Mix; each 100 g contained minerals (Zn, 2.50 mg; Mn, 16.00 mg; Fe, 31.50 mg; Cu, 5.50; I, 0.55 mg; Ca, 1.15 gm and P, 450 mg). Vitamins (Vit A, 7500000 Iu; Bi, 100 mg; B3, 500 mg; B6, 150 mg; B12, 2.5 mg; E, 100 mg; K, 100 mg; pantothenic acid, 275 mg; folic acid, 100 mg; vit. D3, 7500 Iu). ***GE (kcal/100 g DM) = CP x 5.64 + EE x 9.44 + NFE x 4.11 calculated according to [84].  NFE = 100 - [% Ash + % lipid + % protein].

Table 3: Please indicate the unit of each analysis parameter.

Thanks for the valuable suggestion. All the chemical analyses were performed in triplicate, and the data were expressed in a dry matter a basis as percentage (%). It was added to the table.

Author Response File: Author Response.pdf

Reviewer 2 Report

The experiments are meticulously performed in this work. The information and findings on improved growth-related traits and elevated activities of antioxidants are valuable and informative. The long-term supplementation with β-1,3 GF significantly improved shrimp weight gain (WG), feed conversion ratio (FCR), and digestive enzyme profiles with no considerable variations in the contents of moisture, crude protein, total lipids, and ash in the muscles of shrimp fed on different diets. Additionally, the reduced mortality of shrimps under infection adds value to the work. All these valuable findings suggest the need for this work to be in the public domain. However, I have some suggestions for improvements, particularly in disease resistance-related components

1.      The title indicates the resistance of Pacific white shrimp to Fusarium solani infection which is not introduced in the abstract part. Please incorporate the findings of infection-related resistance responses.

2.      The disease assessment part has only been correlated with mortality but disease index, symptoms, and lesions have not been included. Please Justify.

3.      Why the data on mortality has been kept up to 14 days and not beyond up to 30 days? The previous report suggests up to 88.6% mortality in shrimps during 30 days of infection

4.      The mode of infection was intravenous injection but the spore suspension exposure would have given a more realistic infectious environment. Please comment.

5.      The manuscript is lacking in providing one or two good figures. 

Author Response

Reviewer 2:

1. The title indicates the resistance of Pacific white shrimp to Fusarium solaniinfection which is not introduced in the abstract part. Please incorporate the findings of infection-related resistance responses.

Thank you and we appreciate the reviewer’s constructive comments. We have carefully reviewed the abstract and the findings of infection-related resistance responses have introduced in the abstract part. The following statement was added

Challenge test results revealed that F. solani could cause a high mortality rate (86.7%) in a group fed a normal basal diet within 14 days at a dose of 5 x 10⁴ conidia mL^-1. Surprisingly, all dietary treated groups with different doses of β-1,3- GF showed high resistance against  F. solani, represented by lower cumulative mortality rates (20-43.3%) compared to the control group. Moreover, most of the infected shrimp showed a typical black to brown gill lesion similar to that observed in the natural infection, where an identical fungus was successfully re-isolated from infected gills and muscles.

2. The disease assessment part has only been correlated with mortality but disease index, symptoms, and lesions have not been included. Please Justify.

We acknowledge the reviewer’s concerns, so the symptoms and the lesions were added to the results section and supported by figure 2.

3. Why the data on mortality has been kept up to 14 days and not beyond up to 30 days? The previous report suggests up to 88.6% mortality in shrimps during 30 days of infection

Thank you for this comment. Mostly, the previous studies calculate the cumulative mortality within 7-14 days, which is quite enough to figure out any lesions and recording a cumulative mortality. Thus, due to the limitations following prolonged culture in comparative rearing system.

Here are some references that support our present study:.

Hoseinifar, S. H., Sohrabi, A., Paknejad, H., Jafari, V., Paolucci, M., & Van Doan, H. (2019). Enrichment of common carp (Cyprinus carpio) fingerlings diet with Psidium guajava: The effects on cutaneous mucosal and serum immune parameters and immune-related genes expression. Fish Shellfish Immunology, 86, 688–694.

Mostafa Mahmoud, M. (2019). Fusarium solani infection of red swamp crayfish (Procambarus clarkii). Assiut Veterinary Medical Journal, 65(161), 50-59.‏

Le, V. K., Hatai, K., Yuasa, A., & Sawada, K. (2005). Morphology and molecular phylogeny of Fusarium solani isolated from kuruma prawn Penaeus japonicus with black gills. Fish Pathology, 40(3), 103-109.‏

Egusa, S. (1972). A Fusarium sp. associated with black gill disease of the kuruma prawn, Penaeus japonicus Bate.‏

4. The mode of infection was intravenous injection but the spore suspension exposure would have given a more realistic infectious environment. Please comment.

Thank you for this comment. The injection was via intramuscular that is the most suitable rout for disease induction for this pathogen.  The experiment was conducted in specific pond designed for that purpose under complete a septic condition. And after the challenge both pond and water are treated with strong disinfectants and hygienically drained.

5. The manuscript is lacking in providing one or two good figures. 

Thank you for the valuable comment. Two figures were added, one represents clinical finding post challenge (Figures 2), and the other (Figure 3) substitute the table 4 of intestinal enzymes activity.vv

Author Response File: Author Response.pdf

Reviewer 3 Report

This works are interesting and could be a good data base for development in prebiotics for Pacific white shrimp feed. However, there are some errors throughout the manuscript. The manuscript should be completely checked in detail for the errors.

1. In this manuscript, English language is sometimes cumbersome or imprecise. The manuscript requires linguistic changes and corrections. Also, it is better to use general terms commonly used in aquacultural journal.

L34: Remove the ‘(WG)’ and ‘(FCR)’.

L38: total hemocyte count phenol oxidase → total hemocyte count, phenol oxidase

L51-52: In what year was the shrimp production more than 58% ?

L62-64: Bian et al. (1981) studied kuruma prawn (Penaeus japonicus), not litopenaeus vannamei.

L67: Abbreviations must be defined at their first mention in the text. You need to check ‘P. japonicus’.

L89: The notation is different (β-1,3 / β1,3 glucan)

L89: fructooligosaccharide → FOS

L95: β-1,3-glucan and fructooligosaccharide → β-1,3-glucan and fructooligosaccharide (β-1,3 GF)

L96: What is ‘ARRIVE’? Abbreviations must be defined at their first mention in the text. Also, it would be better to unify the word referring to L. vannamei in the manuscript with the same word as Pacific white shrimp instead of ‘white leg shrimp’.

L105: β-1,3-glucan and fructooligosaccharides (β-1,3 GF) →  β-1,3 GF

L105-106: I think it would be good to write the purpose of the study.

L107-109: Write what β-1,3 GF is. It should be clearly stated that aquastem is a β-1,3 GF. Also, for the reproducibility of the experiment, the purity and composition of the prebiotic products used in the study should be provided!!!!

L112: Pellet size and the source of the meat grinder should be identified.

L116-120: Indicate the initial weight at the beginning of the shrimp tiral. It is necessary to check whether the weight was 3g before acclimatization for 3 weeks or 3g after placing 30 animals.

L117: Litopenaeus vannamei → L. vannamei

L117: Litopenaeus vannamei is divided into postlarva, juvenile, sub-adults, and adults according to the growing season. In this study, you said Litopenaeus vannamei juvenile. What is the standard for distinguishing postlarva, juvenile, sub-adults, and adutls?

L119: 40 CP kg^-1 diet → 38.8% or 39% CP kg^-1 diet

L123: Fed  2 times a day for 6% of shrimp body weight. It means you fed 3% of feed for one time. Usually for shrimp trial, the shrimp is fed at least 3-4 times. Is there any reason you fed the shrimp 2 times a day?

L125-128: The ammonia concentration in the culture water can be greatly increased by the high crude protein of the feeds and the feeding habit of the shrimp that slowly nibbles its feed. Therefore, shrimp nutrition studies, especially pond culture, should check the ammonia concentration in the experimental water tank. Did you perform an analysis on this? Please mention how you measured the pH and dissolved oxygen level. Did you not measure salinity during the trial?

L129: The calculation formula for weight gain rate and SGR is missing.

L134: You need to check calculation formula ‘final weight (g ∕ shrimp) − initial weight (g)’. It should be changed to (g/shrimp) → (g).

L136: feed intake (g) → dry feed intake (g) & Feed conversation ratio → Feed conversion ratio. In the case of FCR, it should be accurately stated whether the moisture value was included or not.

L139: Remove the bracket. (- 4 °C) → - 4 °C

L140: 'extracting' doesn't seem appropriate. Try using a word like 'dissecting'

L142: Why was the hemolymph not immediately centrifuged and stored at 4°C overnight?

L145-146: There is no information on the method of analyzing the composition of feed. (e.g. carbohydrates, gross energy)

L152-156: Are the intestines of several shrimps pooled and used?  How were the five digestive enzymes activities analyzed? Sample preparation procedures were mentioned, but procedures and references about enzyme analysis were not.

L158-161: SOD, CAT, GPx, MDA analyzed on hemolymph? Or is it the hepatopancreas? Additional and detailed writing is required in materials and methods.

L159: 'whereas' doesn't seem appropriate.

L168: Total hemocyte counts → THC

L169-170: Fusarium solani → F. solani

L173: In PDA, other fungal can grow including F. solani. How can you prove if there is no other microorganism in media?

L186-188: Are percentage data converted to arcsine before statistical comparison?

L191-193: There is no difference between the 1.5 and 1.0 treatments. Please write clearly.

L191-194: The C4 group showed no significant difference from the C3 group in FW and WG. Write correct sentences.

L192: weight gain (WG) → WG

L193: final weight (FW) → final body weight (FBW)

L195-196: feed conversion ratio (FCR) → FCR

L197-198: Survival rate → SR

L198 & 200: (P>.05) → (P≥0.05)

L203: Shrimp → Pacific white shrimp

L203: survival of Shrimp, → survival of Pacific white shrimp,

L219 & 257: white shrimp → Pacific white shrimp

L230: Total hemocyte counts → THC

L251: survival rate → SR

L261: at p ≤ 0.05 → p < 0.05

L273: β glucan and fructooligosaccharide → β glucan and FOS

Line 274: SGRs → SGR

L279: β glucan and fructooligosaccharide (FOS) are → β glucan and FOS are

L291: Litopenaeus vannamei → L. vannamei

L294: fructooligosaccharide → FOS

L318-320: Because it is the same result as other studies, you claimed that it improves antioxidant enzyme ability. However, it is not convincing enough to simply obtain the same results as studies with other raw materials and shrimp species. Please consider why β-1,3 GF enhances non-specific immunity.

L326: What is LYZ?

L327: Please write a review of your own study, not the results of other studies (how did lysozyme improve)

L348: total hemocyte counts → THC

L356: survival rate → SR

L362: Litopenaeus vannamei → L. vannamei

L363-367: All the data shows linear trend to enhancing growth, immunity and Fusarium resistanve of shrimp in this study. However, we cannot identify the effect of β-1,3 glucan supplementation above 1.5 g/kg diet from the results in this study. Thus, we recommend to you to make a conclusion as 'further study is recommended under effect of dietary supplementation above 1.5 g/kg diet'.

L365: [52] is a thesis not related to Pacific white shrimp. Need to be checked again.

Table 1: What is the ‘Aquastem’, ‘CMC’, ‘Vit.&Min.Mix’ and ‘min mix’? Abbreviations must be defined at their first mention in the text. Also, be clear about the name and components of ingredients in your formulation. Insufficient description of Ingredients. Fill in details about fish meal, fish oil, soybean meal, wheat flour, vitamin and mineral mix, etc..

- You should mention the units of anlyzed data (Ex: %).  You have to check the total amount of ingredients (0.5 diet: total 997g / 1.0 diet: total 996g / 1.5 diet: total 997g). Why content of wheat flour is different in 0.5, 1.0, 1.5 g kg^-1 groups? Also, why does wheat flour increase in the 1.5 group?  Why was the min mix added again even though Vit&Min mix was added to the feed?

- The crude protein of the experimental diets used in this study is significantly higher than that of other recent shrimp nutrition researches, especially in pond culture. Why did you set the crude protein in the experimental feed such high?  Change the experimental group in the dietary formulation (Ex; 0.0, 0.5, 1.0, 1.5 → C1, C2, C3, C4).

Table 2: You should change the significant figures of all parameter values correctly. Weight gain rate should be expressed as %, not g [Weight gain rate (g) → Weight gain rate (%)]. Also, there is no calculation of weight gain rate.  Why is there a significant difference in mean weight in the initial setting?

Table 3: You have to check the errors of array and significant digits of the table. The values of Con and 1.5 treatment are not sorted. Is it correct that there is no significant difference between the 0.5 test group and the other test groups in fat content?

Table 4:  Indicate the units for data.

Table 5: You have to check the errors of array and significant digits of the table. Unify the format of the table. There is a lot of unnecessary spaces (Ex: Line 85, 485 and so on). SOD (U/L) → SOD (U/mg protein) or SOD (% inhibition). Remove the space in [MDA (nmol/ml ) → MDA (nmol/ml)] & [Respiaratory burstactivity ( mg/ml ) → Respiaratory burstactivity (mg/ml)]. It is better to unify the unit (ml → mL). Phenol oxidase → PO

Author Response

Reviewer 3

1. In this manuscript, English language is sometimes cumbersome or imprecise. The manuscript requires linguistic changes and corrections. Also, it is better to use general terms commonly used in aquacultural journal.

Thank you for valuble comment, linguistic changes and corrections were done.

L34: Remove the ‘(WG)’ and ‘(FCR)’.

Done as requested.

L38: total hemocyte count phenol oxidase → total hemocyte count, phenol oxidase

Done as requested.

L51-52: In what year was the shrimp production more than 58% ?

Thank you for this comment. It was in 2016 as indicated in the provided reference. We added the year and highlighted it with yellow colour.

L62-64: Bian et al. (1981) studied kuruma prawn (Penaeus japonicus), not litopenaeus vannamei.

Thank you for this comment. It was replaced by other recent reference related to litopenaeus vannamei.

L67: Abbreviations must be defined at their first mention in the text. You need to check ‘P. japonicus’.

Done as requested .

L89: The notation is different (β-1,3 / β1,3 glucan)

Done as requested in the entire manuscript.

L89: fructooligosaccharide → FOS

Done as requested.

L95: β-1,3-glucan and fructooligosaccharide → β-1,3-glucan and fructooligosaccharide (β-1,3 GF)

Done as requested.

L96: What is ‘ARRIVE’? Abbreviations must be defined at their first mention in the text. Also, it would be better to unify the word referring to L. vannamei in the manuscript with the same word as Pacific white shrimp instead of ‘white leg shrimp’.

Thank you for this comment. A new Institutional Review Board Statement (Animal Ethics) was added following the journal guidelines and based on the suggestion of the Editor as follows:

The study was conducted according to the guidelines of the Declaration of Helsinki and approved by the Animal Ethics Review Committee of Suez Canal University (AERC-SCU), Egypt with approved No. 2022037 on 9 November 2022).

L105: β-1,3-glucan and fructooligosaccharides (β-1,3 GF) →  β-1,3 GF

Done as requested.

L107-109: Write what β-1,3 GF is. It should be clearly stated that aquastem is a β-1,3 GF. Also, for the reproducibility of the experiment, the purity and composition of the prebiotic products used in the study should be provided!!!!

Thanks for the comment. AquastemTM V is a unique combination of algal source of linear 1,3 - beta glucan, enriched with vitamin C and fructooligosaccharides. Brief information about the main compositions, features, and instructions for use are available in the company website (https://www.kemin.com/content/dam/kemin/aquaculture/product/pdfs/Aquastem%20V_1pager_EN_v2020.pdf).

L112: Pellet size and the source of the meat grinder should be identified.

The pellet size was 1.5-2 mm in diameter.  We added a full information about diet preparation in preparation section as follows:

AquastemTM V is a unique combination of an algal source of linear 1,3 - beta glucan, enriched with vitamin C and fructooligosaccharides. Brief information about the main compositions, features, and instructions for use are available on the company website (https://www.kemin.com/content/dam/kemin/aquaculture/product/pdfs/Aquastem%20V_1pager_EN_v2020.pdf). The compound was diluted in 100 mL water and thoroughly mixed with the basic components of the control diet for 30 min. The feed is formulated from commercial ingredients (shrimp meal, fishmeal, rice bran, wheat flour, rice bran, soybean meal, fish oil, vitamin and mineral mixture). The composition and chemical analysis of the experimental diets are shown in Table (1). The dry ingredients were passed through a sieve (aperture of 1.5 - 2.0 mm in diameter) before being mixed into the diet. Emulsified oil was added with equal quantity of water with 0.7% phosphatidylcholine (lecithin) according to [34], to the experimental rations. Mixtures were homogenized in a model SNFGA forage mixer (St. Joseph Kitchen Assistant, M149085 USA). Then the boiling water was mixed with the mixtures at a rate of 50% for pelleting. Diets were pelleted (1.5-2.0 mm in diameter) using a Kitchen Assistant meat mincers and stored in refrigerator (4 °C) until used.

L116-120: Indicate the initial weight at the beginning of the shrimp tiral. It is necessary to check whether the weight was 3g before acclimatization for 3 weeks or 3g after placing 30 animals.

Thank you for this comment, 3 gm after acclimatization.

L117: Litopenaeus vannamei → L. vannamei

Done as requested.

L117: Litopenaeus vannamei is divided into postlarva, juvenile, sub-adults, and adults according to the growing season. In this study, you said Litopenaeus vannamei juvenile. What is the standard for distinguishing postlarva, juvenile, sub-adults, and adutls? Thank you

Thank you for this comment, Depending on its size (3 gm ), it is classified as juvenile, the best on growing stage.

L119: 40 CP kg^-1 diet → 38.8% or 39% CP kg^-1 diet

Thanks for the comment. It was revised as ~39%, based on the chemical composition analysis.

L123: Fed  2 times a day for 6% of shrimp body weight. It means you fed 3% of feed for one time. Usually for shrimp trial, the shrimp is fed at least 3-4 times. Is there any reason you fed the shrimp 2 times a day?

Thank you for this comment, Feed are given at fixed rate 5-10 & of the estimated shrimp biomass per day and the common feeding frequency adopted is 2-3 times a day. Most culturists feed their stock every morning and afternoon only. We conduct our experiment in private farming indoor and follow the protocol implemented by this farm and both feeding rate and frequency are matched with FAO standard guidelines.  https://www.fao.org/3/ac210e/AC210E10.htm .

L125-128: The ammonia concentration in the culture water can be greatly increased by the high crude protein of the feeds and the feeding habit of the shrimp that slowly nibbles its feed. Therefore, shrimp nutrition studies, especially pond culture, should check the ammonia concentration in the experimental water tank. Did you perform an analysis on this? Please mention how you measured the pH and dissolved oxygen level. Did you not measure salinity during the trial?

Thank you for this comment. Brief data about water characters and measuring parameters were added as follows:

Water temperature, pH, and DO were measured using a handheld digital meter (YSI Pro1020), while salinity was measured by an ATC salinity refractometer. Ammonia and nitrite were evaluated twice per week using API test commercial kits, and never exceeded 0.05 and 0.25 mg L⁻¹, respectively. Periodic water exchange of 20% was performed every two days during the adaptation period to keep ammonia level constant.

L129: The calculation formula for weight gain rate and SGR is missing.

Thank you for valuable comment. The calculation formula for weight gain rate and SGR  were added.

L134: You need to check calculation formula ‘final weight (g ∕ shrimp) − initial weight (g)’. It should be changed to (g/shrimp) → (g).

 Done as requested.

L136: feed intake (g) → dry feed intake (g) & Feed conversation ratio → Feed conversion ratio. In the case of FCR, it should be accurately stated whether the moisture value was included or not.

Thank you this comment. FCR—the amount of aquaculture biomass realized per unit of feed input. The FCR is based on the air dry or “as is” feed weight, and the live weight of aquaculture biomass.

L139: Remove the bracket. (- 4 °C) → - 4 °C

Done as requested.

L140: 'extracting' doesn't seem appropriate. Try using a word like 'dissecting'

Thank you for this suggestion.  We wrote dissecting instead of extracting.

L142: Why was the hemolymph not immediately centrifuged and stored at 4°C overnight?

Thanks for the reviewer comment. We apologize for the typing error.  Muscles are the only samples stored for further analysis, while the withdrawn hemolymph samples are analyzed immediately after collection. The sentence was rephrased as follows:

The collected samples were centrifuged at 3000 x g for 10 min, and the supernatants were separated and kept at -80 °C for further analyses.

L145-146: There is no information on the method of analyzing the composition of feed. (e.g. carbohydrates, gross energy)

Thank you for this, we added some sentences under table (1).

Analysis was performed according to Official Methods of Analysis, Association of Official Analytical Chemists, Arlington (AOAC, 1990). Each 100 gram of vitamin and mineral contained: Mineral: Zn, 2.50 mg; Mn, 16.00 mg; Fe, 31.50 mg; Cu, 5.50; I, 0.55 mg; Ca, 1.15 gm and P, 450 mg.    Vitamins : A, 7500000 Iu; Bi, 100 mg; B3, 500 mg; B6, 150 mg; B12, 2.5 mg; E, 100 mg; K, 100 mg; Pantothenic acid, 275 mg; Folic acid, 100 mg and vit. D3, 7500 Iu.

**GE (kcal/100 g DM) = CP x 5.64 + EE x 9.44 + NFE x 4.11 calculated according to (Macdonald et al., 1973)

*CMC, carboxymethylcellulose as a thickening agent.

NFE = 100 - [% Ash + % lipid + % protein]

L152-156: Are the intestines of several shrimps pooled and used?  How were the five digestive enzymes activities analyzed? Sample preparation procedures were mentioned, but procedures and references about enzyme analysis were not.

Thank you for this comment. Yes it was a pooled sample previously mentioned above. The procedure references and digestive enzymes measuring units were included.

L158-161: SOD, CAT, GPx, MDA analyzed on hemolymph? Or is it the hepatopancreas? Additional and detailed writing is required in materials and methods.

Thank you for this comment, we measure antioxidant enzymes from hemolymph samples. We prefer not to write briefly about the methodology to avoid repetition and plagiarism.

L159: 'whereas' doesn't seem appropriate.

 Thank you for this comment. We remove it.

L168: Total hemocyte counts → THC

Done as requested.

L169-170: Fusarium solani → F. solani

Done as requested.

L173: In PDA, other fungal can grow including F. solani. How can you prove if there is no other microorganism in media?

Thank you for this comment. Regarding the inoculum preparation we used fully identified pure colonies isolates, so there is no possible way for any cross-contamination. In terms of re-isolation, we only observed colonies of uniform shape on the PDA media and similar morphological patterns of hyphae in wet preparations that all are characteristic to F. solani.

L191-193: There is no difference between the 1.5 and 1.0 treatments. Please write clearly.

Thank you for this comment. Thanks for the grateful comment. At first glance, it appears that there are no significant differences between the two doses. However, upon examination, several tested parameters such as trypsin, amylase, SOD, lysozyme, THC, and post-challenge survival rate were significantly improved after the addition of 1.5 g/kg compared to others. Therefore, we recommend using this dosage for maximum benefits and high performance regardless of price value .

L191-194: The C4 group showed no significant difference from the C3 group in FW and WG.  Write correct sentences.

Thank you for this comment. Rephrasing all scientists using C1-C4 groups as a group definition was performed in the entire manuscript.

L192: weight gain (WG) → WG

Done as requested.

L193: final weight (FW) → final body weight (FBW)

Done as requested.

L195-196: feed conversion ratio (FCR) → FCR

Done

L197-198: Survival rate → SR

Done

L198 & 200: (P>.05) → (P≥0.05)

Done

L203: Shrimp → Pacific white shrimp

Done

L203: survival of Shrimp, → survival of Pacific white shrimp,

Done

L219 & 257: white shrimp → Pacific white shrimp

Done

L230: Total hemocyte counts → THC

Done

L251: survival rate → SR

Done

L261: at p ≤ 0.05 → p < 0.05

Done

L273: β glucan and fructooligosaccharide → β glucan and FOS

Done

Line 274: SGRs → SGR

Done

L279: β glucan and fructooligosaccharide (FOS) are → β glucan and FOS are

Done

L291: Litopenaeus vannamei → L. vannamei

Done

L294: fructooligosaccharide → FOS

Done

L318-320: Because it is the same result as other studies, you claimed that it improves antioxidant enzyme ability. However, it is not convincing enough to simply obtain the same results as studies with other raw materials and shrimp species. Please consider why β-1,3 GF enhances non-specific immunity.

Thank you for this comments. Shrimp have no immunological memory so that, β-1,3 GF  as a natural product could enhance non-specific immunity. A surge increase in THC along with exaggerated level of Lysozyme could be the possible cause. Lysozyme and prepheral hemocytes proved to have immunododulatory effect beside antimicrobial activity.

L326: What is LYZ?

Thank you for this comment, LYZ is the abbreviation of Lysozyme and we wrote lysozyme instead of LYZ.  

L327: Please write a review of your own study, not the results of other studies (how did lysozyme improve).

Indeed, lysozyme was expressed in most if not all hemocytes in circulation and in peripheral tissues. Hemocytes secrete lysozyme, and increases in hemolymph lysozyme activity have been associated with increases in their numbers. Thus, in the current study, the higher lysozyme activity may be congruent to the observed surge increases in hemocyte counts.

L348: total hemocyte counts → THC

Done as requested

L356: survival rate → SR

Done as requested

L362: Litopenaeus vannamei → L. vannamei

Done

L363-367: All the data shows linear trend to enhancing growth, immunity and Fusarium resistanve of shrimp in this study. However, we cannot identify the effect of β-1,3 glucan supplementation above 1.5 g/kg diet from the results in this study. Thus, we recommend to you to make a conclusion as 'further study is recommended under effect of dietary supplementation above 1.5 g/kg diet'.

Done as requested. The following statement was added to the conclusion part

In this study, our results showed an improvement in the growth, immunity, and Fusarium resistance of shrimp following supplementation with β-1,3 glucan at concentration of 1-1.5 g/kg diet. However, the impact of supplementing with β-1,3 glucan over 1.5 g/kg diet still not fully elucidated and required further investigations.

L365: [52] is a thesis not related to Pacific white shrimp. Need to be checked again.

It was removed.

Table 1: What is the ‘Aquastem’, ‘CMC’, ‘Vit.&Min.Mix’ and ‘min mix’? Abbreviations must be defined at their first mention in the text. Also, be clear about the name and components of ingredients in your formulation. Insufficient description of Ingredients. Fill in details about fish meal, fish oil, soybean meal, wheat flour, vitamin and mineral mix, etc..

Thank you for this comment. All was included as a table footnotes as mentioned above.

- You should mention the units of anlyzed data (Ex: %).  You have to check the total amount of ingredients (0.5 diet: total 997g / 1.0 diet: total 996g / 1.5 diet: total 997g). Why content of wheat flour is different in 0.5, 1.0, 1.5 g kg^-1 groups? Also, why does wheat flour increase in the 1.5 group?  Why was the min mix added again even though Vit&Min mix was added to the feed?

Thank you for this comment. We revised it carefully, addressed all mistyping, and added the full information about the ingredients.

- The crude protein of the experimental diets used in this study is significantly higher than that of other recent shrimp nutrition researches, especially in pond culture. Why did you set the crude protein in the experimental feed such high?  Change the experimental group in the dietary formulation (Ex; 0.0, 0.5, 1.0, 1.5 → C1, C2, C3, C4).

Thank you for valuable comment.  In general, this is the system on which this type of shrimp is raised in Egypt. However, we thank you for directing us to change the protein ratios later. Also, we changed all the groups to C1, C2, C3, C4 in all tables.

Table 2: You should change the significant figures of all parameter values correctly. Weight gain rate should be expressed as %, not g [Weight gain rate (g) → Weight gain rate (%)].

Thank you for this comment. The differences in the initial weight are quite small and were written in order to preserve the accuracy of the data. Based on statistical analysis, there was no any significant difference, as the standard deviation of C2 group is quite higher.

Also, there is no calculation of weight gain rate.  Why is there a significant difference in mean weight in the initial setting?

Thanks for the comment. We totally agree with comment. It was revised as % and its equation was mentioned in M&Ms section as follow: Weight gain rate (WGR %) = [final weight (g) – initial weight (g)] × 100 / initial weight (g). Following revision of the raw data, the initial weigh for all groups almost identical and showed no significant differences. The data are modified accordingly.

Table 3: You have to check the errors of array and significant digits of the table. The values of Con and 1.5 treatment are not sorted. Is it correct that there is no significant difference between the 0.5 test group and the other test groups in fat content?

 Thank you for this comment. Indeed, there is no statistically significant difference in body fat content.

Table 4:  Indicate the units for data.

Done as requested.

Table 5: You have to check the errors of array and significant digits of the table. Unify the format of the table. There is a lot of unnecessary spaces (Ex: Line 85, 485 and so on). SOD (U/L) → SOD (U/mg protein) or SOD (% inhibition). Remove the space in [MDA (nmol/ml ) → MDA (nmol/ml)] & [Respiaratory burstactivity ( mg/ml ) → Respiaratory burstactivity (mg/ml)]. It is better to unify the unit (ml → mL). Phenol oxidase → PO

Thank you for this comment. All the errors were corrected.vvv

Author Response File: Author Response.pdf

Round 2

Reviewer 1 Report

The author(s) have addressed the comments and suggestions and i recommend the acceptance of the manuscript.

Reviewer 2 Report

The manuscript is improved based on the suggested lines.