Biochemical Changes Occurring in Yellowfin Tuna Eggs during Embryonic Development

Round 1

Reviewer 1 Report

The manuscript describes the proximate composition of yellow fin tuna embryos during early development. The study design is very straight forward, and the manuscript is nicely written and easy to follow. My only real criticism would be that the data is relatively simple, and this limits its wider interest to those working outside of tuna broodstock and hatchery management/research.

A few minor comments

L115 – I find the sentence confusing, as the figure doesn’t show the time elapsed. It shows developmental stages, and the time is written in the sentence.

Discussion. In the first and second paragraph of the discussion it feels like the results are being repeated (e.g. L193-195 and L198-201). I also do not think the authors need to reference the figures in the discussion.

L222 – Could the authors include a reference for AST activity in the other organs?

Figures letters. For a few figures, the letters don’t make much sense to me. In Figure 1, the HC is designated bc, but what is the c for? No other group has a c. Same for Figure 3, all the groups with a b also have an a, so what are they different from?

Figure 2b – I would maybe split the axis so you can see the ammonia data better.

Figure 2d – The authors could use capital letters for one parameter, and lowercase for the other. At the LC stage, it is a bit difficult to know which parameter the letters are associated to.

Author Response

The manuscript describes the proximate composition of yellow fin tuna embryos during early development. The study design is very straight forward, and the manuscript is nicely written and easy to follow. My only real criticism would be that the data is relatively simple, and this limits its wider interest to those working outside of tuna broodstock and hatchery management/research.

 Thank you for your critical comments and suggestions, which have helped us improve our manuscript. As indicated in the following responses, we have taken consideration of all these comments and suggestions and hope that the revisions are satisfactory.

• We agree that the data in this study may limit the wider interest of those working outside of the tuna broodstock and hatchery management/research. However, we believe that the results of this study may also be applied to the hatchery management of other fish species similar to tuna for which artificial production of seedlings is difficult.
• Moreover, the global demand for tuna species has been increasing daily because of its high meat quality and worldwide popularity of sashimi and sushi cuisine. To meet the increasing global demand, the tuna aquaculture industry is expanding rapidly using natural juveniles, which rapidly diminishes the natural stock and disturbs the natural ecosystems. Only approximately five species of tuna are widely used for human consumption. Even if the information from this study is applied only to the management of these tuna species, it will have a great impact on reducing the high dependency on natural stocks and contribute to the development of a sustainable aquaculture system.

A few minor comments

L115 – I find the sentence confusing, as the figure doesn’t show the time elapsed. It shows developmental stages, and the time is written in the sentence.

 As Reviewer 1 has pointed out, we have added the elapsed time in the figure legend of Figure 1.

Discussion. In the first and second paragraph of the discussion it feels like the results are being repeated (e.g. L193-195 and L198-201). I also do not think the authors need to reference the figures in the discussion.

 The first and second paragraph of the discussion was compactly summarized.

• The reference to the figures in the discussion has been deleted.

L222 – Could the authors include a reference for AST activity in the other organs?

• We cited the following two studies on the activity of AST in other tissues:

33. Horio Y, Tanaka T, Taketoshi M, Uno T, Wada H. Rat Cytosolic Aspartate Aminotransferase: Regulation of its mRNA and Contribution to Gluconeogenesis. Biochem. 1988, 103, 805-808. DOI: 10.1093/oxfordjournals.jbchem.a122350.
34. Bennett, M.D., Woolford, L., O'Hara, A.J., Nicholls, P.K., Warren, KS. Clinical Chemistry Values and Tissue Enzyme Activities in Western Barred Bandicoots (Perameles bougainville). Clin. Pathol. 2008, 37, 221-224. DOI: 10.1111/j.1939-165X.2008.00040.x.

 Figures letters. For a few figures, the letters don’t make much sense to me. In Figure 1, the HC is designated bc, but what is the c for? No other group has a c. Same for Figure 3, all the groups with a b also have an a, so what are they different from?

 This means that groups with different letter show a significant difference, whereas those with the same letter do not.

Figure 2b – I would maybe split the axis so you can see the ammonia data better.

 The changes in the amounts of ammonia are mentioned in the results section. The amounts of ammonia at the 2C, LC, EG, E, K, H, BH, 50H, and HC stages were 0.003±0.002, 0.005±0.003, 0.006±0.003, 0.007±0.001, 0.007±0.001, 0.013±0.005, 0.013±0.003, 0.009±0.001, and 0.010±0.001 nmol/egg and larva, respectively.

Figure 2d – The authors could use capital letters for one parameter, and lowercase for the other. At the LC stage, it is a bit difficult to know which parameter the letters are associated to.

 Thank you for your comment. Significant differences are shown using the letters “wxyz.”

Reviewer 2 Report

Generally, the paper is an interesting study on biochemical changes in yellowfin tuna eggs during development.

The manuscript needs improvement and I have listed several points in the following:

Major concerns:

In material and methods description of sample preparation and processing for biochemical analysis is missing but necessary. Appropriate extraction procedures are essential for correct analysis procedure and reproducibility of the data. The chorion of embryos is a protein structure. It is unclear if it was included in analysis.  Also sample composition (numbers of eggs and larvae per assay) should be described).

At the time point 50% hatch: do you analyze non-hatched embryos or hatched?

Discussion contains unclear passages and vague conclusion

Specific points:

Line 24, 25, abstract: increase in protein concentration is due to organ differentiation: could it also be related to larvae growth?

Line 45: The sentence is unclear in this context

Line 46/47: This sentence is incomplete – and: how can biochemical changes in embryos contribute to enhance egg quality

In the text (e.g. line 49/50, 224/225, 246/247) there are descriptions that in tuna larvae organ differentiation occurs after hatching. This indicates that tuna larvae hatch in a less developed stage than other species. This point needs additional information and discussion.

Line 72: derive eggs from one or more females/spawning events?

line 86 how were eggs digested?

line 107: Please indicate egg/larvae numbers per analysis

Statistics: individual eggs and larvae and those from different spawns may differ in weight – It might be more precise to report in units/mg egg or larvae

Line 119-121: the last 2 sentences could be deleted

Line 118: how can you explain that dry weight of eggs decreases:  is this a loss of organic material?

Legend to Fig. 1: Better to say: Changes in dry weight of eggs and larvae of yellowfin tuna

Line 145: Better say: embryonic development instead of morphological changes

Fig. 2: Ammonia are not visible in the diagram. I suggest to use second y-axis.

Discussion:

line 189: dry weight of eggs decreased: this needs discussion

First paragraph of discussion are mainly results and should be shortened

line 202 say: similar pattern of temporal changes

Discussion lines 198 – 208: I think the conclusion of the study is as follows: TG and FAA are both energy resources, TG the most important because of the higher energy content

Lines 209-218: this part of discussion is unclear – it is stated that the used type of energy resource depends on egg size. I see no indication from discussion.  

Lines 220-222: Enzymes were investigated as markers for organ development. However, enzymes were high during first cleavage, decreased and re-decreased in advanced development stages. This is an indication that they are not only markers of organ differentiation but also involved in other functions.

Lines 225/226: why are analytes values high in first cleavage stages

Lines 224/225: do tuna larvae hatch in a less developed differentiation stage than the other species

Line 227: Enzyme activities cannot be consumed

Lines 233/234 the quantity of TG was sufficient to accelerate embryonic development – this is an unclear statement – can energy resources or metabolites influence the development time of embryos?

Lines 246/247: the delayed organ function is due to the low amount of available FAA: how is this related? How can you conclude that organ function correlates with FAA concentration

Author Response

Reviewer 2

Generally, the paper is an interesting study on biochemical changes in yellowfin tuna eggs during development.The manuscript needs improvement and I have listed several points in the following:

 Thank you for your critical comments and suggestions, which have helped us improve our manuscript. As indicated in the following responses, we have taken consideration of all these comments and suggestions and hope that the revisions are satisfactory.

Major concerns:

In material and methods description of sample preparation and processing for biochemical analysis is missing but necessary. Appropriate extraction procedures are essential for correct analysis procedure and reproducibility of the data. The chorion of embryos is a protein structure. It is unclear if it was included in analysis.  Also sample composition (numbers of eggs and larvae per assay) should be described).

 Thank you for your suggestion. All eggs and larvae used in this study were analyzed, including the chorion of the embryos. All experiments were performed using 0.1-1.0 g eggs or larvae. After weighing, the number of eggs or larvae was counted to calculate the measurements per egg or larva.

 At the time point 50% hatch: do you analyze non-hatched embryos or hatched?

 At the 50% hatching time point, both non-hatched and hatched embryos were analyzed.

 Line 24, 25, abstract: increase in protein concentration is due to organ differentiation: could it also be related to larvae growth?

 As the larva grows, the organs are thought to differentiate.

Line 45: The sentence is unclear in this context

 We meant to convey that it is necessary to explore the related aspects that can lead to the technical development of the mass production of yellowfin tuna seedlings.

 Line 46/47: This sentence is incomplete – and: how can biochemical changes in embryos contribute to enhance egg quality

 If we can obtain information on which nutrients have been utilized during the embryonic developmental stages, it may provide the opportunity to improve the egg quality by enriching these nutrients in the eggs through broodstock diet manipulation.

 In the text (e.g. line 49/50, 224/225, 246/247) there are descriptions that in tuna larvae organ differentiation occurs after hatching. This indicates that tuna larvae hatch in a less developed stage than other species. This point needs additional information and discussion.

 As reported by Tanaka et al., the digestive system of yellowfin tuna immediately after hatching was similar to that of most marine fish that hatched from small pelagic eggs. By contrast, as with freshwater fish and marine fish that take a long time to hatch, organogenesis is considerably progressed at the time of hatching.

• We have added the following sentence to the Discussion: Tanaka et al. reported that the digestive system of yellowfin tuna immediately after hatching was similar to that of most marine fish that hatched from small pelagic eggs. We further measured enzyme activity to determine at what stage of egg development tissue differentiation progressed.

32. Tanaka, M. Stusies on structure and function of the digestive system in teleost larvae I: Development of the digestive system during prelarval stage. Japanese Journal of Ichthyology. 1969, 16 (1).

Line 72: derive eggs from one or more females/spawning events?

 The eggs used in this study were collected from one female fish that spawned in a single day.

  line 86 how were eggs digested?

 We apologize for this oversight. It should be “homogenized” and not "digested."

line 107: Please indicate egg/larvae numbers per analysis

 All experiments were performed using 0.1-1.0 g eggs or larvae. After weighing, the number of eggs or larvae was counted to calculate the measurements per egg or larva.

Statistics: individual eggs and larvae and those from different spawns may differ in weight – It might be more precise to report in units/mg egg or larvae

 We agree with the observation of Reviewer 2 that individual eggs and larvae and those from different spawns may differ in weight. We accept that it might be more precise to report the data in units/mg of eggs or larvae.

• However, in this study, we examined the endogenous energy of one egg or larva. Therefore, the changes in the amount of enzyme in one egg or larva were measured and compared with the data of other nutrients analyzed.

Line 119-121: the last 2 sentences could be deleted 

• As Reviewer 2 suggested, we have removed these two sentences.

Line 118: how can you explain that dry weight of eggs decreases:  is this a loss of organic material? 

• The decrease in the dry weight of eggs is thought to be due to the utilization of the nutrients in the eggs for tissue development.

Legend to Fig. 1: Better to say: Changes in dry weight of eggs and larvae of yellowfin tuna 

• Thank you for your suggestion. We have rewritten “Change in dry egg and larval weights with development of yellowfin tuna eggs” as “Changes in the dry weights of eggs and larvae of yellowfin tuna.”

Line 145 (Line 135?): Better say: embryonic development instead of morphological changes 

• As suggested, we have rewritten “morphological changes” as “embryonic development.”

Fig. 2: Ammonia are not visible in the diagram. I suggest to use second y-axis. 

• The changes in the amounts of ammonia are mentioned in the results section. The amounts of ammonia at the 2C, LC, EG, E, K, H, BH, 50H, and HC stages were 0.003±0.002, 0.005±0.003, 0.006±0.003, 0.007±0.001, 0.007±0.001, 0.013±0.005, 0.013±0.003, 0.009±0.001, and 0.010±0.001, respectively.

Discussion:

line 189: dry weight of eggs decreased: this needs discussion 

• The decrease in the dry weights of eggs is thought to be due to the utilization of the nutrients in the eggs for tissue development.
  

First paragraph of discussion are mainly results and should be shortened 

• As recommended, we have shortened the first paragraph of the discussion.
  

line 202 say: similar pattern of temporal changes 

• We have rewritten “similar pattern” as “similar pattern of temporal changes.”
  

Discussion lines 198 – 208: I think the conclusion of the study is as follows: TG and FAA are both energy resources, TG the most important because of the higher energy content 

• Thank you for your suggestion. The following sentence has been added: “Although TG and FAA are energy resources, TG is the more important because of its higher energy content.”
  

Lines 209-218: this part of discussion is unclear – it is stated that the used type of energy resource depends on egg size. I see no indication from discussion.   

• We have mentioned “the endogenous energy source in the eggs is not defined by the egg size alone.”
  

Lines 220-222: Enzymes were investigated as markers for organ development. However, enzymes were high during first cleavage, decreased and re-decreased in advanced development stages. This is an indication that they are not only markers of organ differentiation but also involved in other functions. 

• There are studies that describe the synthesis of protein precursors in the egg envelope in some fish species in the mother fish. As Reviewer 2 pointed out, enzyme levels were high during the first cleavage and decreased during the advanced development stages. This indicates that enzymes are not only markers of organ differentiation but are also dependent on the health and nutritional status of the mother fish.

35.    Yamagami, K. Studies on the Hatching Enzyme (Choriolysin) and its Substrate, Egg Envelope, Constructed of the Precursors (Choriogenins) in Oryzias latipes: A Sequel to the Information in 1991/1992. Zoolog. Sci. 1996, 13, 331-340. DOI: 10.2108/zsj.13.331. 

Lines 225/226: why are analytes values high in first cleavage stages

• Studies have described the synthesis of protein precursors in the egg envelope in some fish species in the mother fish. Therefore, the high levels in the first cleavage stages are considered to be because the amount synthesized in the mother fish was provided to the eggs.

35.    Yamagami, K. Studies on the Hatching Enzyme (Choriolysin) and its Substrate, Egg Envelope, Constructed of the Precursors (Choriogenins) in Oryzias latipes: A Sequel to the Information in 1991/1992. Zoolog. Sci. 1996, 13, 331-340. DOI: 10.2108/zsj.13.331.

Lines 224/225: do tuna larvae hatch in a less developed differentiation stage than the other species 

• As reported by Tanaka, the digestive system of yellowfin tuna immediately after hatching was similar to that of most marine fish that hatched from small pelagic eggs.
• We have added the following sentence to the Discussion: Tanaka et al. reported that the digestive system of yellowfin tuna immediately after hatching was similar to that of most marine fish that hatched from small pelagic eggs. We further measured enzyme activity to determine at what stage of egg development tissue differentiation progressed.
  

32. Tanaka, M. Stusies on structure and function of the digestive system in teleost larvae I: Development of the digestive system during prelarval stage. Japanese Journal of Ichthyology. 1969, 16 (1).

 Line 227: Enzyme activities cannot be consumed

• We apologize for this error. It should be “detected and not “consumed.”

Lines 233/234 the quantity of TG was sufficient to accelerate embryonic development – this is an unclear statement – can energy resources or metabolites influence the development time of embryos?

• Thank you for your suggestion.
• The possibility that TG was a key factor was ruled out; the related sentence has been revised as “Accordingly, although the quantity of TG was sufficient to accelerate embryonic development, there are important factors other than TG.”
• We believe that FAA influences the development time of embryos.

Lines 246/247: the delayed organ function is due to the low amount of available FAA: how is this related? How can you conclude that organ function correlates with FAA concentration

• Since amino acids are the source of proteins, we suggest that low amounts of amino acids contribute to delayed organ function.

Round 2

Reviewer 2 Report

The manuscript was properly revised and I have no further comments.

Author Response

Thank you for your review.