Survival, Growth, and Development in the Early Stages of the Tropical Gar Atractosteus tropicus: Developmental Critical Windows and the Influence of Temperature, Salinity, and Oxygen Availability

Round 1

Reviewer 1 Report

GENERAL COMMENTS

The present manuscript by Martinez et al. describes the influence of three stressors – temperature, salinity and oxygen saturation – on the survival, growth and development in early life stages of A. tropicus. In general, the manuscript is well-written and easy to read, though I found quite long and descriptive in some sections, particularly from L144-306 and L322-380). I think it could be shortened in c. 20% without losing important info. However, there is an important issue that concerns me a lot, preventing me for moving this manuscript to a further step towards publication. This relates to what the author call acute and chronic in their study, for example when they say for temperature 33ºC are acute conditions and 36ºC are chronic conditions; or when they say 4 ppt of salinity are acute conditions and 6 ppt are chronic conditions. However, the definition of acute and chronic conditions does not have anything to do with stressor magnitudes referred by the authors, but instead how they happen in time: acute conditions are severe and SUDDEN in time, i.e. they appear and change or worsen rapidly; in contrast, chronic conditions develop and worsen over an extended period of time, they represent therefore a long-developing state. So, why you say 33ºC represents an acute stressor and 36ºC a chronic one (similar to salinity)??? This makes no sense, based on definitions of acute/chronic stressors. Indeed, 33ºC can be tested either as an acute stressor, as well as a chronic stressor (same for 36ºC). What you are testing is indeed a gradient of temperature and salinity (33 vs 36; 0 vs 4 vs 4), and that’s the way the manuscript should go. This is the major issue in the manuscript. Several details on fish capture and holding are needed. Figures should be re-drawn in a better quality, several graphics are hardly perceptible. A (short) conclusion section referring to the overall findings of your study as well as management implications and future lines of research is clearly needed (a background sentence is also missing in the abstract). Based on this (and particularly the acute/chronic conditions comment) my decision is to reject the manuscript, while encouraging the authors to re-submit a newer version based on the general, as well as the specific comment below.

SPECIFIC COMMENTS

L16 – First of all, you should start your abstract with a background sentence, stating why your study, the issue you are going to address, is important for science. In other words, what is the motivation of your study?

L82-84 – how did you come to such an assumption? In what basis? Any study to support? 

L84-85 – The same here. You should provide any background with valid references to support these statements.

L87 – “some development events”. Which events? Be specific.

L85-87 – But why this is important from the practical point of view? Why this can be of interest to science? This must also be referred in the abstract.

Material and methods – This section should follow the Introduction and should be moved accordingly.

L101 – These letters and the numbers are quite small and difficult to see. Suggest increasing font size.

Figure 1 – What are the units on the x- and y-axis? What do the dashed lines represent on Fig. 1c?

L105-106 – The sentence is unclear and should be re-written.

L146-147 – Please specify the values (e.g. a mean or median +- SD). The same applies to the lines below.

L149 – BM = body mass?

Figure 2 should be redrawn in a much better quality. The captions and letters on the graphics are very poor in quality and cannot be read (the same applies to the colour figures on the right). Please improve this and the other figures.

L178-180 – Why there are crosses (X) on the 2^nd and 3^rd development stages in fig. 2c? What do they mean?

L182-184 – The same comment about the quality of the figures. Salinity values and development stages are hardly distinguishable.

L187 – Lt= Total length?

L187-188 and throughout the paragraph (and also L145-155) - From the figure, it is not possible to see what is 33, 36 or 28ºC. Suggest including this on the caption. Once again, specify and provide – not only for Temp, but also for hypoxia and salinity- mean/median values between parentheses.

L193 – “Total length of P1-33°C and CE-36°C”. Total lengths are not of the experiments, but of the fish!

L193-194 – “increased significantly faster than the rest of the treatments”. How did you measure this variation? “significantly” – did you employ any statistical test? Provide statistics.

L195-197 and throughout the manuscript – Once again, please specify values.

L213-217 and throughout the manuscript – Which P1 and P2? By looking to the figures P1/P2 can be either acute or chronic stress, so please take this into consideration and specify accordingly. The same applies to the other paragraphs.

L219 – “period 1,”, “first development period” (L211) – please be consistent in the use of nomenclature throughout the manuscript.

L220 – “and those hatched at 0.0 of salinity”. Aren’t these the control ones?

L223-224 – “hatched in either 4.0 and 6.0 presented significant LT differences compared to the control group and to those exposed during the third period” i) please provide names and units to the values, ii) presented significant? How significant? Higher? Lower? Test statistics?

L227 – How faster? Be specific.

L231 – SGR? Provide full name for an acronym when it is mentioned for the first time.

L255 – Statistics?

L258 – Figure 5e or 4e???

L260 – “a constant increase”. Does not sound well (either is constant or increases). Provide details.

L265 – “and a slightly increase…”. Be specific and provide details.

L268 – pdh or dph?

L274 – K?

Table 1 – What do the numbers represent? Mean? Medians? SD?

L323 – What you mean by “significantly enhanced”? The point is: increased or decreased?

L322-380 – In this section the authors compare how temperature, hypoxia and salinities affected the development stages. However by looking to Table 1, we don’t any temperature, hypoxia values or salinities, just P1, P2, P3, CE,…so there is no correspondence to what is written in the text and what is shown in the Table. This must be corrected – make the appropriate correspondence - so it is clear for the reader to confirm what is written in the text.

L339 – This is not a result and should be removed.

L342 – “the first and second developmental periods”. Again, what are in the Table the first or the second development periods?

L344-345 and throughout L322-380 – Where are these values on the table? Please make the appropriate correspondence.

L351, 353 and throughout this section- Please specify.

L389 – dot after “larvae”.

L482 – Avoid calling figures and tables in the Discussion.

L563 – A (short) conclusion section referring to the overall findings of your study as well as management implications and future lines of research is clearly needed!

L573 – What was the source of the animals? Were they captured in the wild?

L575-577 – What was the purpose of anesthesia and injection?

L578 – But how many fish in total you have? How many tanks? What was the mean and SD size for the fish?

L583 – Provide details of the tanks. For how long? Conditions? Did you monitor the water quality? What parameters were addressed?

L584-585 – So overall you had 9 (treatments) x 3 replicates x 50 fish = 1350 fish, is that so? Please clarify in the text.

L586 – “17 kPO2”. How was this value obtained?

L595-597 – As I said previously, the authors should address the reasons of selecting these periods. Why? Why are important?

L604 – What are the units for salinity?

L603- 605 – This needs to be better explained: i) how did you come to the values of Temp, salinities, and hypoxia? Why these in particular? Any reference to support them? Also it is difficult to understand the basis of these comparisons. For example, it would make sense comparing 33ºC vs 36ºC, but why 33ºC (acute) vs 36ºC (chronic)? What is the basis of the comparison? What do you want to show? The same goes for salinity. The definitions of acute and chronic as applied in the present study are highly questioned, see General comments.

L608 – What you mean by “Gradual switches between conditions occurred in short periods..”? Could you be more specific and give e.g. times in each condition? Were 3 hours the time of each experiment?

Figure 6 needs to be re-drawn in a better quality.

L620 – How many heaters were used? Provide also brand and model.

L631 – “bubbling nitrogen gas”. Concentration? Please provide further details.

L647 – What are the units for salinity? microS/cm? Other? Also “Water for the control population has a salinity of 0.0”. Even undisturbed river water (i.e with no pollution) has some salinity that results from natural factors (such as the geology of the catchment). This needs to be clear.

L657 – So again, how many fish did you have in total? 9 treatments x 150 fish = 1350 fish? Of the 150 fish for each treatment, you had 50 fish per each of the 3 development stages? Please clarify.

L662 - What third dimensional plots?

L672-673 – Any reference to support this formula? The same for the Fulton condition on L676.

L682 – What variables?

L698 – comparison instead of comparisons.

 

Author Response

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Author Response File: Author Response.docx

Reviewer 2 Report

The article Survival, growth, and development in the early stages of the tropical gar Atractosteus tropicus: Developmental critical windows and the influence of temperature, salinity and oxygen saturation is an interesting approach to study the main factors influencing the survival and growth of this species at different stages of development. I believe the experiments implied quite a lot of methodical work and I appreciate the efforts of the authors. However, in its present form, the article has some weakness and shortcomings that should be solved before publication.

In the general view I think the objectives of the investigation are not clearly exposed, the methods used are quite confusing and difficult to understand and reproduce. The terminology used to describe different treatments changes along the text. The authors denominate ‘’chronic’’ and ‘’acute’’ to describe the different conditions and it is not clear, sometimes, if the several stressors were applied independently or at the same time to each treatment group.  The results section and plots should be reorganized to present the information in a more consistent and clearer way. The discussion section is well exposed, but I think the conclusions must be improved and concisely summarize the authors’ main contributions to the present knowledge on this field.

Specific comments:   

L18. Add units to salinity

L34-47. References should be related to fish studies, or at least a general reference that explains the terms you are defining in the text. It seems that these references were randomly selected.   

L43-45. But the authors did not perform multidimensional experiments.

L48. I do not agree with this statement. There are plenty of works on survival and growth of several fish species, mainly those intended for aquaculture production or pet-trade. You could specify tropical fish or the herein studied species.

L76 and throughout the text. Once you have mention Atractosteus tropicus the first time you may use the abbreviate form A. tropicus afterwards.

L83-85. I think these hypotheses should be reformulated and the objectives of the study properly exposed. If you want to demonstrate the first periods of development are the most sensitive to stressors, then you should compare them to other stages (adult period, for example). Also, if you state the stressors will have their greatest effects at the same time is because you intend to test them at the same time and independently, and if I understood properly, you tested the stressors one by one. By the way, this is something that is not always clear along the text since you usually refer to Control conditions: 28 °C, 0 ppt of salinity, normoxia, Acute conditions: 33ºC, salinity 4, hypoxia and Chronic conditions:  36ºC, salinity 6 and hyperoxia. And still CE acute and chronic. It is really difficult to understand the experiment in the way it is presented (see other comments bellow).

L91. It seems to me that the information exposed in this section is basically the same than in section 2.2. In addition, with some contradictory statements.

L94-95. ‘’Higher percentages of survival were observed in the control group (84%), followed by treatments hatched in temperature of 28°C (76-81%)’’. Control group and temperature 28ºC are not the same??

L99. The figures are difficult to understand due to the low quality. Please, provide figures in a format with higher quality. Survival curves? They are not curves. Also, please, be consistent with the nomenclature you use for the different treatments. Here they are P1-33ºC or -36ºC, later you name them ‘’acute’’ or ‘’chronic’’. I strongly suggest adopting the nomenclature that indicates the period and variable.

L114. I suggest organizing the different sub-sections in the same way. In this case you divided the main section in the different periods (i.e. 2.2.1. Period 1 (P1)- Fertilization to hatch), and then the stressors (- Temperature,-Hypoxia/hyperoxia, -Salinity). Present the results in the same way for the next sections to make easier the reading and understanding.

L143. Body Morphology and Growth ….as a function of….? Be consistent in the way you present your results.

125-126. This is in contradiction with L110-111.

L158. Figure 2. I do not understand why the authors present the data in this way: ‘’Temperature experiment’’, ‘’hypoxia/ hyperoxina’’ experiment and ‘’salinity experiment’’ were different in each treatment but then there are Control: 28 °C, 0 ppt of salinity, normoxia, Acute: 33 °C, 4 ppt of salinity and hypoxia, Chronic: 36 °C, 6 ppt of salinity, hyperoxia) which seems that these conditions are applied together to the same group of individuals. Also I do not see why you named acute and chronic to these two treatments. They were applied exactly for the same time of exposure. There is no acute or chronic treatment here, only two different conditions. Maybe you could denominate the ‘’continuous exposure (CE)’’ as ‘’chronic’’ but you still differentiate between CE chronic and acute. This is very confusing for the reader. The plots d-f are also quite confusing. The x and z axes refers both to the time of exposure, so I do not see the point in presenting the same information in two different axes. Period 1 comprises from fertilization to hatch but the z axis ‘’development’’ starts in hatching. Furthermore the P1,P2,P3 are repeated in the x axes and then, the temperature, oxygen and salinity conditions are presented at the top of each graph and because of the small size of the figure is quite hard to read. I suggest a more intuitive way to present your data and resize the plot or provide a figure with better quality.  

The same for Figure 3, Figure 4 and Figure 5.

L264. What do you mean by ‘’treatments without exposure’’?

L583. ‘’For each experiment (…) eight experimental treatments’’ or eight experimental treatments in total?

L663. Indicate the figure you are referring to.

 

Author Response

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Author Response File: Author Response.docx

Reviewer 3 Report

This paper presented the detailed data on complicated effects of environmental factors as stressors on survival, growth and development in the 3 early life stages of the tropical gar. Results are valuable to researchers in the related fields and will be expected to contribute to progress in ecological science of larval fishes.

 

However, I could not understand and evaluate the contents of this paper sufficiently, because descriptions of the experimental methods were incomplete and the resolution of figures (Fig.1-Fig. 5) were so poor that I could not read letters and numbers in the figures. This paper needs to be thoroughly revised to improve intelligibility.

 

Material and Methods

In the methods, I could not find important information on rearing experiments and sampling protocols.

L584-585; “50 larvae per triplicate, per treatment” Do you mean 50 larvae per tank or 16.6 larvae (50/3) per tank for a treatment?　Size of the experimental tank?

L624-627, 641-644, 647; It seems there are 3 different control groups corresponding to 3 different stressors. Please state that 3 control groups had the same external conditions as 28℃, 17 kPO2, 0 salinity. Does 1 control group have 1 tank or 3 tanks?

L656-659 Survival; did you use only one tank for one treatment (a total of 9 tanks) in the survival experiments?

L662-664; Data for morphological variables were taken every day. How many fish were sampled from each treatment?

L667; “n = 20” If one tank started with 50 eggs, all fish will disappear within 3 days after fertilization.

L679-687; If you sample 30 fish per treatment to analyze developmental events every day after hatching, a total of 360 fish are needed for one treatment. You wrote 50 larvae per triplicate at L584.

L690; The formula to estimate Q10 is not correct, should be Q10=(R1/R2^{)(10/(T1-T2))}

I also do not understand how to estimate Q10 values using the data, because R values decreases with increase of temperature T.

L702; “(n = 20 from every treatment)” Do you mean you sampled 20 fish at each end of period (3 periods)?

L705; “(n = 30 from every treatment)” Now I do not understand the sampling protocol at all.

L706; All statistical differences were showed with P<0.001 in figures. I am wondering why there were no cases at other statistical significant levels such as P<0.05 and <0.01.

Table 1; I do not understand the method to obtain data shown in Table 1. When and how many were sampled at each treatment (15 control groups, 52 acute treatment groups, 52 chronic groups)?

 

Results

L165; “N=20 for each treatment” Same as above, I do not understand the meaning of this value “20”. In addition, “N” should be “n”. Did you distinguish n and N? Originally n and N have different meanings in statistics.

L106; “normoxia and were” should be “normoxia were”

L216　“form” should be “from”

L210-218 and Fig. 2; P1 hyperoxia (chronic) showed the highest TL in Fig. 2 b (period 3), but P2 hypoxia (acute) seemed highest at the end of the experimental period in Fig. 2 e. Fig. 2b and Fig. 2e do not match each other.

L244; SRG should be SGR

L262; “CE-6.0 and P1-6.0” should be “P1-6.0 and CE-6.0”. Follow the order.

Fig. 5; Factor K decreased with developmental time in Fig. 5 a,b,c, but K increased in Fig. 5 d,e,f. Why Figs. a,b,c and Figs. d,e,f are different?

 

Tables 1 and 2

L314; “Significant differences within treatments for every experiment” And also L701-702 said “within time for each treatment”. Actually I do not understand how to read the results of statistical tests in Tables 1 and 2. As mentioned below, tables include *, **, ***, s, and b.

Tables 1 and 2; What are “b” at the value of 94±4.3^*b in Table 1 and ^“a” in Table 2?

Caption of Table 1 and 2; Do *, **, *** indicate the significance levels at P<0.05, P<0.01, and P<0.001, respectively?

Table 1; You should show data in the order of Temperature, Oxygen and Salinity under Treatment by following those in Figs. 2-5.

Table 2; Why Q10 decreased in the case of 28-36 (<1)? while Q10 increased in the cases of 28-33 and 33-36 (>1).

 

L341, 353; “The larvae exposed to temperature” What do you mean “temperature”? You mean “larvae of the temperature-treatment group”?

L321-380; In Q10 sections, you mentioned about the significant temperature sensitivities (Q10>2.5). I do not understand the statistical method to test the temperature sensitivity. In addition, the results of statistical test for the temperature sensitivity were not shown in Table 2.

L387-388; “temperature and dissolved oxygen decreased survival during incubation,” is strange as English. You mean “high temperatures and extreme oxygen conditions decreased survival during incubation”?

L389 “killed all larvae” should be “killed all larvae.” Add “.”

L394 “decrease” should be “affect”

 

Discussion Critical Windows for Survival；

Discussion in this section is shallow. How did temperature and DO affect survival of embryo and larvae, and their mechanisms?

 

L411-414; “These findings suggest that the period of transition from lecithotrophy to exotrophy could represent a critical window not just for growth but also for survival, especially when the young larvae must cope with the changing environment and the development of feeding strategies.” has been generally accepted since Hjort (1914, Fluctuations in the great fisheries of northern Europe viewed in the light of biological research. Rapp. P.-v. Reun. Cons. Perm. Int. Explor. Mer. 20:1–228.)

L439, 560; “metamorphosis” is usually used to indicate the transition from larval stage to juvenile stage. I am not sure the onset of exogenous feeding can be called “metamorphosis”.

L543 “which ca occur” → “which can occur”

L559 “that” → “than”

L723 6. Patents ?

 

Concerning discussion, I would like to recommend authors to read Fry (1971, The effect of environmental factors on the physiology of fish. In Hoar, W.S., Randall, D.J. eds. Fish Physiology 5. Academic Press, New York, 1-98.) which is classic but basic.

Author Response

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Author Response File: Author Response.docx

Reviewer 4 Report

General comments.

As the background of the reviewer is different from this study, following comments are mostly related to editorial issues. Please follow other reviewer's comments for scientific matters.

Pages are incorrectly numbered. Pages 10 and 11 are not numbered. From page 12, the number starts from "2 of 26."

"Materials and methods" comes after "Discussion". It looks for me unusual. In most of journals, I believe, "Materials and methods" comes after "Introduction".

Specific comments.

L. 97. The title "2.1.1 Temperature" should be moved to below the legend of Figure 1.

L. 389: "... killed all larvae" => Add a period at the end of this sentence.

L. 419-420: no relationship between => no significant relationship between

L. 430: Gymnpcephalus => Gymnocephalus  (Replace "p" with "o".)

L. 431: humbpldtianum => humboldtianum (Replace "p" with "o".)

L. 462-463: The preposition "in" is duplicated.

L. 543: which ca occur => which can occur (OK?)

L. 690:  "is the" is duplicated.

L. 691: R1 => R₁

L. 723: "6. Patents" =>  It looks like a title of an independent section. If not applicable, it may be deleted.

Figures. Many of characters and numbers on the graphs are too small, not clear and not easy to read. The size should be enlarged.

Figure 1. Characters and numbers below "Survival (%) are too small to read.

Figures 2-5. Explanations of symbols, and characters beside the scales of 3D graphs are too small to read.

 

Author Response

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Round 2

Reviewer 1 Report

I'm overall satisfied with the changes and comments provided by the authors, in particular on how they dealt with my major concern, removing the terms "accute" and "chronic" from the manuscript which were previously (and erroneously) allocated to specific values of temperature and salinity. It now makes much more sense focusing on a set of different treatments of increasing values for temperature (28,33,36) and salinity (0,4,6,) and air saturation  (30,95,117). Well done.

Author Response

Thank you very much for your time and the useful comments which really improved our manuscript. Thank you!

Reviewer 2 Report

The manuscript has significantly improved and is in the present form much clearer and understandable, allowing for a better appreciation of the interest of the study. I acknowledge authors’ efforts in answering reviewer’s questions as well as correcting and editing the text and figures. I believe the manuscript is now ready for publication.

Author Response

We appreciate your time and all the useful comments that improved our manuscript. Thank you!

Reviewer 3 Report

 

 

Comments for author File: Comments.docx

Author Response

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