Movement Behavior of the Dusky Grouper Epinephelus marginatus (Lowe, 1834) in Early Life Stages

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

This work is very interesting and well presented. My minor suggestions are as follow:

·      I urge authors to clearly establish the objective of the work. This work aimed to describe the swimming behavior of larvae 3 to 10 days post hatched to identify body contraction, fin movements and responses to prey under laboratory conditions.

·      So, the title has to be modified to something like: Behavioral patterns of 3 to 10 days post hatched larvae of the Dusky grouper Epinephelus marginatus (Lowe, 1834)

·      How many larve were used for how many experiments? This information must be included in the abstract.

·      The experimental design must be more specific in the statistical tests used to compare experiments. In Materials and methods section is mentioned that “Statistical analysis was conducted using non-parametric methods, specifically the Kruskal-Wallis test (P < 0.05) and Dunn's post hoc test, to assess differences between treatments (presence and absence of prey) and with respect to the ontogenetic development of the larvae”. However, it is not specifically mentioned which variables were tested with which tests?

Author Response

This work is very interesting and well presented. My minor suggestions are as follow:

• I urge authors to clearly establish the objective of the work. This work aimed to describe the swimming behavior of larvae 3 to 10 days post hatched to identify body contraction, fin movements and responses to prey under laboratory conditions.

Yes, we agree. This objective was described on the original manuscript, lines 54-56, using basically the same words as suggested by the reviewer, and we kept as it is.

 

• So, the title has to be modified to something like: Behavioral patterns of 3 to 10 days post hatched larvae of the Dusky grouper Epinephelus marginatus (Lowe, 1834)

We thank the reviewer for the suggestion. We have added the common name of the species first, but we prefer to maintain the notion of “movement behavior” instead of the general statement of “behavioral patterns” because we would like to emphasize the main topic of this study, which relates to movement in particular, and not behavior in general. Finally, we believe that “early life stages” depict with reasonable accuracy the intended notion that we are dealing with recently hatched larvae.

 

• How many larve were used for how many experiments? This information must be included in the abstract.

The information was included.

 

• The experimental design must be more specific in the statistical tests used to compare experiments. In Materials and methods section is mentioned that “Statistical analysis was conducted using non-parametric methods, specifically the Kruskal-Wallis test (P < 0.05) and Dunn's post hoc test, to assess differences between treatments (presence and absence of prey) and with respect to the ontogenetic development of the larvae”. However, it is not specifically mentioned which variables were tested with which tests?

Correct. We changed the sentence accordingly: the new version states that the tested variables were routine speed, burst speed, caudal beat frequency, C-start duration, and rest duration.

Reviewer 2 Report

Comments and Suggestions for Authors

This manuscript provides a detailed analysis of the early life history behavior of Epinephelus marginatus larvae and proposes a new perspective contrary to past beliefs. I strongly recommend the author to enhance the discussion on the relationship between larval behavior and aquaculture. Additionally, the author should create detailed comparison tables listing the various behaviors of different fish species at different stages, and different live feed organisms were used in these studies. This would be beneficial. These efforts can enhance the value of this paper.

The following are specific suggestions:

L47 The author should provide a detailed explanation of the significance of larval fish feeding behavior in aquaculture to enhance comprehension of the connection between studying feeding behavior and enhancing larval survival rates. For example, the changes in larval feeding behavior under different water quality conditions (such as temperature, turbidity, light intensity, stocking density, etc.) and their impact on aquaculture performance. References should be included.

L79 Why not transport fertilized eggs? Generally, transporting larvae seems to result in a higher mortality rate.

L81 What impact does handling stress have on the feeding rate of fish larvae?

L87 Why use Temora turbinate and Acartia tonsa in this study?

L125 The author should explain which statistical software and plotting software were used.

L144 It is inappropriate to compare behaviors of different ages (dph).

L335 How do the behaviors of Epinephelus marginatus larvae differ in their preference for preying on rotifers or copepods? What are the differences between different age (dph)? It is suggested that the author can enhance the discussion in this aspect.

 

L365 How can studying larval behavior enhance aquaculture production? Could you please provide more specific details?

 

Comments for author File: Comments.pdf

Author Response

• This manuscript provides a detailed analysis of the early life history behavior of Epinephelus marginatus larvae and proposes a new perspective contrary to past beliefs. I strongly recommend the author to enhance the discussion on the relationship between larval behavior and aquaculture. Additionally, the author should create detailed comparison tables listing the various behaviors of different fish species at different stages, and different live feed organisms were used in these studies. This would be beneficial. These efforts can enhance the value of this paper.

The connectivity between larval fish behavior and aquaculture has been explored in more detail on a new paragraph before the end of the Introduction. The reviewer’s suggestion regarding the preparation of a table with a list of behaviors is indeed very interesting but we believe it would pertain to a review article dealing with fish behavior in general. It goes beyond the scope of this manuscript.

 

The following are specific suggestions:

• L47 The author should provide a detailed explanation of the significance of larval fish feeding behavior in aquaculture to enhance comprehension of the connection between studying feeding behavior and enhancing larval survival rates. For example, the changes in larval feeding behavior under different water quality conditions (such as temperature, turbidity, light intensity, stocking density, etc.) and their impact on aquaculture performance. References should be included.

Thank you for suggesting this. We believe the new paragraph starting on line 54 satisfies the request.

• L79 Why not transport fertilized eggs? Generally, transporting larvae seems to result in a higher mortality rate.

The reviewer is correct about the effectiveness of transporting larvae compared to fertilized eggs. It is an excellent suggestion, but we did not have access to the eggs. However, in this study, the commercially available larvae corresponded to the first feeding phase: at 1-3 dph, the larvae still have the enclosing membrane and the yolk reserve, making transport and survival less problematic. As such, we believe the original sentence concerning this topic does not need further changes.

• L81 What impact does handling stress have on the feeding rate of fish larvae?

Detailed explanations about the care taken to minimize stress prior to experimental observations were added to the text (Lines 101-110).

• L87 Why use Temora turbinate and Acartia tonsa in this study?

Those were the copepod species available in the natural environment during the experiments; they are common species in tropical and subtropical coastal environments, and easy to cultivate, making them good candidates for live feed. We apologize for the mistake in the name of the Acartia species in the original version of the manuscript. The correct is Acartia (Odontacartia) lilljeborgii.

• L125 The author should explain which statistical software and plotting software were used.

This information is now available on lines 147-148.

• L144 It is inappropriate to compare behaviors of different ages (dph).

The reviewer is correct, but in this case the comparison is between diets, and not between ages. The larvae utilized in this comparison had different ages but the number of individuals of each age was balanced, meaning that age did not interfere in the analysis.

• L335 How do the behaviors of Epinephelus marginatus larvae differ in their preference for preying on rotifers or copepods? What are the differences between different age (dph)? It is suggested that the author can enhance the discussion in this aspect.

This is reported in the first four paragraphs of the Results section, and now summarized on the first paragraph of the Conclusions:

• L365 How can studying larval behavior enhance aquaculture production? Could you please provide more specific details?

The connectivity between larval fish behavior and aquaculture has been explored in more detail in a new paragraph before the end of the Introduction.

 

Reviewer 3 Report

Comments and Suggestions for Authors

The purpose of the study is unclear in that authors did not describe clearly what they want to obtain through the study. There are various factors that influence the preying behaviors of larvae: the age (development stage), types of live food, or something else. This must be considered and clarified what factor you want to apply to studying the prey behavior. Accordingly, the experimental design and pertinent approach should be described in the methods section.

line 87-88: How to separate the two adult copepods Temora turbinate and Acartia tonsa?

A schematic diagram of the experimental device is suggested to be drawn in order to further understand how the experiment was conducted.

line 96-99: larvae aged at 3-5 dph were divided into three sessions in the study. What is the purpose of doing so? The procedure of feeding live food in larvae cultivation at early stage should be provided in detail. What procedure is performed about the experiments with larvae aged at 6-10 dph?

Routine and burst swimming speed, and relative and absolute swimming speed were used in the study, what is their role? what is the difference of routine and burst swimming speed, and between relative and absolute swimming speed? How to calculate these parameters?

Conclusions are not informative. Authors excessively presented the results of others without summarizing their own findings.

Author Response

• The purpose of the study is unclear in that authors did not describe clearly what they want to obtain through the study. There are various factors that influence the preying behaviors of larvae: the age (development stage), types of live food, or something else. This must be considered and clarified what factor you want to apply to studying the prey behavior. Accordingly, the experimental design and pertinent approach should be described in the methods section.

Thank you for the comment, we agree and have added more details on the purposes of the study on the final paragraph of the Introduction.

• line 87-88: How to separate the two adult copepods Temora turbinate and Acartia tonsa?

The senior author is an experienced copepodologist, and both species are among the most common coastal copepods in the study area, easily recognizable from other calanoid copepods. Adult individuals are distinguished from juveniles by several morphological characters, including the number of somites in the urosome, presence of a mature genital somite in females, presence of visible oocytes etc.

• A schematic diagram of the experimental device is suggested to be drawn in order to further understand how the experiment was conducted.

The diagram is now available (Figure 1).

• line 96-99: larvae aged at 3-5 dph were divided into three sessions in the study. What is the purpose of doing so? The procedure of feeding live food in larvae cultivation at early stage should be provided in detail. What procedure is performed about the experiments with larvae aged at 6-10 dph?

The use of copepod nauplii in our experiments was restricted to the early larval fish stages (3-5 dph) because of difficulties in offering the same naupliar developmental stage to grouper larvae beyond a 5-day time interval. This is now stated in lines 123-127.

• Routine and burst swimming speed, and relative and absolute swimming speed were used in the study, what is their role? what is the difference of routine and burst swimming speed, and between relative and absolute swimming speed? How to calculate these parameters?

The difference between these swimming modes relies on the instantaneous speeds, which are calculated from the distance traveled by the fish larvae at a certain time interval. This information if important to characterize the movement behavior.

• Conclusions are not informative. Authors excessively presented the results of others without summarizing their own findings.

We believe the Discussion section is focused on discussing our own results in comparison to the relevant literature. We agree with the reviewer that a  summary of the major results was absent from the Conclusions section, which is now corrected, with new text inserted at the beginning of the section.

 

Round 2

Reviewer 3 Report

Comments and Suggestions for Authors

no comments