Reduced Hypoxia Tolerance and Altered Gill Morphology at Elevated Temperatures May Limit the Survival of Tilapia (GIFT, Oreochromis niloticus) under Global Warming

Round 1

Reviewer 1 Report (Previous Reviewer 3)

The authors of the manuscript made the revisions requested by the referees. The paper is therefore well usable and easy to understand.

 There are still some points to be corrected:

- in the text, in line 328 the authors of the citation are incorrect; you have to change with Jung et al.

 In the references there are 4 errors to be corrected:

- reference 8 should be modified as follows: Beuvard C, Imsland AKD, Thorarensen H

- reference 20 should be modified as follows: Jung EH, Brix KV, Richards JG, Val AL, Brauner CJ

- reference 21 should be modified as follows: Eliason EJ, Anttila K: Temperature and the Cardiovascular System. Fish Physiol., 36 (part B): 235-297.

- reference 33 should be amended as follows: Román-Palacios C, Wiens JJ by adding the file in brackets (8).

For these reasons, a second round of review is needed.

Author Response

Dear reviewer:

Thank you very much for your comments. The references have been revised according to your suggestions. Thank you very much.

Best wishes

Yours sincerely Dr. Zhou

Reviewer 2 Report (Previous Reviewer 1)

statistics not reported

Author Response

Dear reviewer:

Thank you very much for your comments. Previously I did not have a well understanding of pseudoreplication, there are some bias in my statistical analysis. After reading a research paper on experimental design (https://bmcneurosci.biomedcentral.com/articles/10.1186/1471-2202-11-5), I profoundly understand the mean of pseudoreplication.

Since the three temperature treatment are created within each of 3 recirculation systems, one for each temperature, i.e., they are not independent observations. Real replicates would require an independent recirculation system for each. According to this reason, I have taken two major revisions in the new manuscript:

1) Reanalyzed the data by using mixed effects models in ANOVA and include tank as a random effect. In addition to p-values, F values are shown to interpret the ANOVA results.

2) Statistics were reported in the Results.

For more details, please see the update manuscript and the attachment file. Thank you very much for your patience.

Best wishes.

Yours sincerely Dr. Zhou

Author Response File: Author Response.pdf

Reviewer 3 Report (New Reviewer)

Dear Authors,

You wrote very novelty and important during thise time article about temperature tolerence. I would like to ask you are the conclusions drawn from a homogeneous cohort somewhat exaggerated? Are further studies planned on more genetically diverse populations?

Detailed comments and comments are provided below:

M&M line 132 and 133 - repeating

Line 166 - please check the speces in this line.

2.5 Paraffin sectioning - please change to: histological analysis.

In this paragraph please change: wax to paraffin; wax tape to paraffin sections; sentence: Embedded wax blocks were used to produce sagittal 169 sections (8 µm thickness) to Embedded paraffin blocks were sectioned sagittally (8 µm thickness). (or were cut sagittaly into 8um).

Axio Imager2 device - what is it? Microscope? Please, precise.

Line 327-328 please use another word for reduce (synonim).

Sincerely,

R

Author Response

Dear reviewer:

Thank you very much for your comments. The suggestions are very good. Since the conclusions were drawn from a homogeneous cohort and we planned to use more genetically diverse populations in the further studies.

I have revised one by one according to your suggestion. Thank you very much. However, there are still two comments which I'm not sure.

1) M&M line 132 and 133 - repeating.

For line 132 and 133 ".........The mean body weights of fish in which ventilatory frequency was tested were 100.8 ± 5.6, 101.7 ± 6.0, and 95.9 ± 4.1 g in the 28, 32, and 36 °C treatments, respectively.........."I'm sorry for I didn't find the repeated words or sentences. Could you please point out it in more detail?

2) Line 166 - please check the speces in this line.

For line 166 “........using 0.05% 3-aminobenzoic acid ethyl ester methanesulfonate (MS-222).The first gill containing arch was cut into small pieces and ........”I'm sorry for I didn't get the point. Could you please point out it in more detail?

Thank you for your patience. Best wishes.

Yours sincerely Dr. Zhou

This manuscript is a resubmission of an earlier submission. The following is a list of the peer review reports and author responses from that submission.

Round 1

Reviewer 1 Report

General comments:

This manuscript investigated the effects of long term (147 days) thermal acclimation to three temperatures (28-36 C), as well as acute hypoxia on the survival, ventilation frequency, resting metabolic rate, red blood cell concentration, hemoglobin content, gill morphology and gene expression from whole transcriptome. While I appreciate the immense effort that went into this experiment, there are some confounding factors and missing information that need to be addressed. Namely, total ammonia was above acceptable levels (0.1 mg/L) and dissolved oxygen were below normoxic levels (6mg/L). The levels of ammonia and oxygen shown in table 1 are widely considered stressful for fish. Important details are missing from the methods sections, such as the rate of oxygen draw down during the hypoxia trial and the duration fish were in hypoxic conditions. Statistics are widely missing from the results section. The discussion could improve by focussing on the significant results observed and linking them better. The manuscript would highly benefit from English language editing. Line numbers would have been helpful for review.

Specific comments:

“When temperature in the water rising, due to the limitation of oxygen supply, after meeting the requirement of routine metabolic, the excess energy used for other aerobic activities is limited. This will lead to the decline of biological adaptability, such as reduce disease resistance [10], stimulate intestinal inflammatory response, reduce growth performance [11] and cause germ cell abnormalities, resulting in infertility [12].” Awkward sentences and links unclear, please rephrase.

“After a period of high temperature adaptation, the metabolic rate of fish will be lower than the original level under the same temperature conditions.” – what is this supposed to mean, what period and why? Your results show the opposite – higher resting metabolic rate under higher temperature, as expected.

“The insufficient supply of oxygen at high temperature leads to the decline of animal aerobic capacity, which in turn results in the declining of locomotion, growth, reproduction and etc.” Remove “etc.” throughout and be specific. References are needed here.

“This is like the barrel effect in which the shortest piece of wood determines the water level of the barrel.” I don’t understand this comparison.

“This shows that gill tissue is the most sensitive tissue to high or low temperature.” No, this shows that gills are sensitive to low temperature in some zebrafish and tilapia, but not grass carp.

Methods:

Table 1: Problematic water parameters:

1.)    Why are the pH levels so different between temperature treatments?

2.)    Oxygen levels below 5mg/L are stressful for fish, including tilapia (normoxia=6mg/L). The high temperature treatment had oxygen levels below 4, which can confound result for metabolic rate, survival, gill morphology, blood physiology.

3.)    Ammonia levels are very high. The only safe concentrations of ammonia for fish are below 0.1 mg/L. Lethal concentrations depend on fish species but range from 0.2 to 2.0 mg/L. Ammonia levels listed in table 1 are stressful for fish and can severely skew results, especially in combination with stressful levels of oxygen, temperature and pH.

Figure 1 is not necessary, it adds no information we cannot gleam from the text.

2.4 Acute hypoxia trial – what was the rate of O2 drawdown? How long were the fish in hypoxic conditions?

2.6 Transcriptome analysis – what three fish? Put together? Methods unclear here.

How many fish from each replicate were sampled for each parameter? Were statistics done on each fish, or each replicate? How did tank effect factor into analyses?

3.1 Ventilatory frequency, routine metabolism rate and survival rate – I’m not convinced fish in 36°C were in normoxia. Normoxia=6mg/L and above. The 36°C group had a higher ventilation frequency but a lower metabolic rate? Please explain. Statistics completely missing from this section. Stating a few p-values is not enough.

Figure 2 & Fig 3: n=5? per replicate or per treatment? I was under the impression that the temperature treatment was done in triplicate (n=3). If multiple fish were used from one replicate, it needs to be stated that tank effect was checked and fish were pooled. In that case n should be some multiple of 3.

Fig. 2C: are any of these significant?

Figure 3: is this from the acute hypoxia challenge? Hypoerplasia, hypertrophy, lamellar clubbing and epithelial lifting are common effects of acute hypoxia challenge, but I’d be surprised to see lamellar thickness per se change so quickly. The curling of the lamellae is interesting. Was this observed in all sectioned fish, or only in some? It would be good to see some data and variation of these effects to compliment the histology pictures.

3.3 Gill transcriptome analysis: I’m not familiar with the Rich Factor. Log-fold changes seem more straight forward to me.

Fig 5: again it would be imperative to know how long these fish were in acute hypoxia and what the rate of oxygen draw down was to be able to interpret this data.

4.3 Elevated temperature decreased the routine metabolic rate – it doesn’t look like RMR was significantly different between treatments in this study? Statistic values are missing. Also, MRM was not measured, so this discussion section doesn’t seem relevant. I don’t see an “The inhibition of aerobic metabolism under high temperature” in the data.

Author Response

Dear reviewer:

Thank your very much for your comprehensive comments and a lots of good suggestions, which help me largely to improve my manuscript. Specifically, there are four major improvements have been made:

• Statistic have been added into the results section.
• Discussion about the RMR has been largely improved.
• The experimental method chapter revised in more detail.
• English has been polished.

In addition, questions about the total ammonia and dissolved oxygen were fully discussed in the response letter. For more details, Please see the attachment.

Best wishes

Dr. Yan Zhou

Author Response File: Author Response.pdf

Reviewer 2 Report

The article entitled "Reduced hypoxia tolerance and altered gill morphology at elevated temperatures may limit the survival of GIFT tilapia (Oreochromis niloticus) during the global warming" is certainly very interesting and of scientific relevance. However, the authors base their morphological results exclusively on one stain, H/E. This appears to be a limited result, if taken as the only morphological result. The authors should evaluate the morphological alterations using also other stains, to also highlight any quantitative and qualitative changes of specific cells, such as rodlet cells and mast cells. I suggest the authors to use different stains, including trichrome like Mallory and Masson, or histochemical stains like AB/PAS. It could be interesting to stain the sections with GIEMSA, for a general evaluation of the immune cells. The gills, being organs in direct contact with the outside, offer a first physical but also cellular barrier, hosting a wide range of highly specialized cells. Overall, the article can be appreciated and considered for publication after a major revision, with the addition of other histological data.

Author Response

Dear reviewer:

Thank your very much for your comprehensive comments and a lots of good suggestions, which help me largely to improve my manuscript. For more details, please see the attachment.

Best wishes

Dr. Yan Zhou

Author Response File: Author Response.pdf

Reviewer 3 Report

The paper fishes_1743930 examines the tolerance to hypoxia and temperature rise phenomena in farmed tilapia. The authors conducted a well-developed and articulated experiment, using an adequate number of subjects to demonstrate the effects described in 3 different temperature conditions to mimic a state of temperature-dependent stress and hypoxia in a species that tolerates high temperatures well.

I want to congratulate the authors for the experimental design and the scientific approach used: it could be useful to verify with the same methods the response of tilapia to the temperature rise, but with different oxygen content conditions, higher, to observe the response both at the branchial level and at the level of aerobic metabolism, precisely to have a broader and more complete panorama of the responses to these stress conditions, mimicking possible treatment scenarios of the problem in breeding conditions.

The discourse becomes extremely interesting precisely because of the concept of global warming, which at all latitudes presents itself with important aspects both on wild and farmed fish fauna.  The study of possible effects in numerous fish species with different optimal temperature ranges could give many answers to the problems that have arisen in the last decade.

Returning to the article, the authors clearly described the status of knowledge on the subject in the introduction.

In the materials & methods all the parts covered are well written and clear;  in sub-chapter 2.2 perhaps it could be useful to also insert the average initial and final weight of the fishes in the 3 different treatments, to better understand the effects in the three different conditions.

The results are also clear and well written; also in this case, for easier use of the text, it could be useful to expressly insert the survival rate of the various tanks treated in numerical form, to give greater prominence to the results themselves.

Finally, the discussion is well argued and clear.

For all these reasons, in my opinion, this manuscript is ready for publication after a simple minor revision.

Author Response

Dear reviewer:

Thank your very much for your comprehensive comments and a lots of good suggestions, which help me largely to improve my manuscript. For more details, Please see the attachment.

Best wishes

Dr. Yan Zhou

Author Response File: Author Response.pdf

Round 2

Reviewer 1 Report

Review 2 of “Reduced hypoxia tolerance and altered gill morphology at elevated temperatures may limit the survival of GIFT tilapia (Oreochromis niloticus) during the global warming”

Statistics:

There is still insufficient detail on the statistical tests to understand exactly what was done. The problem is, that multiple animals were sampled per tank which raises the issue of pseudoreplication. It is necessary to include tank as a random effect in the analysis to address this problem, for example by used mixed effects models, which is a standard procedure in experiments where multiple samples are taken from one replicate. In addition to p-values, F values are needed to interpret the ANOVA results.

It is unclear what the %s refer to. % deformities, such as curling of lamellae, have no S.D. Were all fish from one treatment lumped into one without controlling for replicate tanks? Percentage data is not well suited to analysis with ANOVA, a more appropriate test would be a GLMM.

Specific comments:

“When temperature in the water rising, due to the limitation of oxygen supply, after meeting the requirement of routine metabolic, the excess energy used for other aerobic activities, such as disease resistance [10], growth [11] and reproduction [12] is limited.” This sentence needs to be revised.

Author Response

Dear reviewer:

Thank you very much for your patience. I have revised the manuscript according to your suggestion. The major responses in the attachment including:

• Why use the pooling strategies in RNA-seq studies?
• How to analysis of different expressed genes within groups?
• Change the two-way ANOVA to Chi-square test to compare the curling rate.
• Revised the obscure sentence.

Best wishes

Your sincerely Yan Zhou

Author Response File: Author Response.pdf