Cardiac Transcriptome and Histology of the Heart of the Male Chinese Mitten Crab (Eriocheir sinensis) under High-Temperature Stress
Round 1
Reviewer 1 Report
Comments and Suggestions for AuthorsChinese mitten crab is an important cultured species in China. With the advance of climate change, increasing temperature threatens culture activity. Improved understanding of the mechanism of high temperature stress might inform action to address that stress. Pan et al. conducted a study of the heart of male Chinese mitten crab, considering transcriptomic and histological responses. The science is straightforward and the results interesting. The presentation and the prose, though, will need considerable work before this manuscript can prove acceptable for publication. I have marked the manuscript to help the authors develop more readable prose. More context-specific comments follow.
Abstract. – At line 21 and throughout the manuscript, “heart slices” should be presented as “histological sections of the heart”.
Introduction. – At line 37 and throughout the manuscript, both common and Latin names should be presented for each species mentioned. I have marked the manuscript.
Methods. – At line 75, did the authors capture the crabs using nets or using cage traps? The English may or may not be correct.
At line 94, not “electricity”, but rather electrophoresis.
At line 97, we need manufacturers for the reverse transcriptase and random primers.
At line 127 and throughout the manuscript, it is “differentially expressed genes”.
At line 130, we need a citation or url for the omicsshare platform.
To Table 1, please add a column on the left and provide a more descriptive names for each gene, e.g.: heat shock protein 60A. The cryptic gene names are meaningless for most readers.
At lines 145-146, we need a manufacturer for the two reagents mentioned.
Results and Discussion. – At line 169, tell the reader what BUSCO stands for.
The first column of Table 2 should be labelled Treatment and the treatments should be told to the reader in the label or in a footnote to the table.
Figure 1 should be enlarged so it can be read.
The first column in Table 2 should be labeled Number of genes, and the second should be labeled BUSCO categorization.
At line 250, what kind of cell is an eIF5B knockdown cell? The reader wants to know what species and tissue it is from and the significance of the knockdown of that particular gene. There’s just too little here to understand the intent of the sentence.
In the caption for Figure 5, the second sentence is not needed. The reader should be told, however, what TA and TC mean.
Figure 6 is too small to read. I recommend that it be placed on a page oriented in landscape mode and then enlarged to fit that page.
Section 3.9 should be entitled Heart tissue histology.
At line 328, what agency supported the research? CARS is just not informative enough.
References. – The literature citations are not in journal stylistics. Article titles should not be enclosed in quotation marks. Species Latin names should be italicized. Other issues are also marked for the first 26 citations.
Comments for author File: Comments.zip
Comments on the Quality of English LanguageThe prose will need considerable work before this manuscript can prove acceptable for publication. I have marked the manuscript to help the authors develop more readable prose.
Author Response
Reviewer 1
Question 1: Abstract. – At line 21 and throughout the manuscript, “heart slices” should be presented as “histological sections of the heart”.
Answer: “Heart slice” has been replaced by “histological sections of the heart” throughout of the manuscript.
Question 2: Introduction. – At line 37 and throughout the manuscript, both common and Latin names should be presented for each species mentioned. I have marked the manuscript.
Answer: “sea bass” was added before “Dicentrarchus labrax”, “rainbow trout” has been replaced by “rainbow trout (Oncorhynchus mykiss)”, “Cancer magister” has been replaced by “Dungeness crab (Cancer magister)”, ” grass carps” has been replaced by ” grass carps (Ctenopharyngodon idellus)”, “Scophthalmus maximus” has been replaced by “turbot (Scophthalmus maximus)”, “Garra rufa” has been replaced by “doctor fish (Garra rufa)”, “Puntius sophore” has been replaced by “pool barb (Puntius sophore)”, “Oncorhynchus mykiss” has been replaced by “rainbow trout (Oncorhynchus mykiss)”, “zebra fish” has been replaced by “zebra fish (Danio rerio)”.
Question 3: Methods. – At line 75, did the authors capture the crabs using nets or using cage traps? The English may or may not be correct.
Answer: The crabs were captured by cage traps.
Question 4: At line 94, not “electricity”, but rather electrophoresis.
Answer: “electricity” was replaced by “electrophoresis”.
Question 5: At line 97, we need manufacturers for the reverse transcriptase and random primers.
Answer: Manufacturer of M-MuLV reverse transcriptase and random primers was Promega, USA.
Question 6: At line 127 and throughout the manuscript, it is “differentially expressed genes”.
Answer: “different expressed genes” has been replaced by “differentially expressed genes” in the manuscript.
Question 7: At line 130, we need a citation or url for the omicsshare platform.
Answer: url for the omicsshare platform was https://www.omicshare.com/.
Question 8: To Table 1, please add a column on the left and provide a more descriptive names for each gene, e.g.: heat shock protein 60A. The cryptic gene names are meaningless for most readers.
Answer: A column and full name were added on the left of Table 1
Question 9: At lines 145-146, we need a manufacturer for the two reagents mentioned.
Answer: Manufacturer of environmental Friendly Dewaxing Transparent Liquid I and II is Servicebio, China.
Question 10: Results and Discussion. – At line 169, tell the reader what BUSCO stands for.
Answer: BUSCO was the abbreviation of “Benchmarking Universal Single-Copy Orthologs”.
Question 11: The first column of Table 2 should be labeled Treatment and the treatments should be told to the reader in the label or in a footnote to the table.
Answer: Treatment was added in the first column. Treatment was listed in the table title (Table S1).
Question 12: Figure 1 should be enlarged so it can be read.
Answer: Figure 1 was enlarged in the manuscript.
Question 13: The first column in Table 2 should be labeled Number of genes, and the second should be labeled BUSCO categorization.
Answer: Number of genes and BUSCO categorization were listed in the first and second column, respectively.
Question 14: At line 250, what kind of cell is an eIF5B knockdown cell? The reader wants to know what species and tissue it is from and the significance of the knockdown of that particular gene. There’s just too little here to understand the intent of the sentence.
Answer: Eukaryotic translation initiation factor 5 (eIF5) is a GTPase for the initiation of protein translation, reduced eIF5B expression may disrupt proteostasis and trigger cellular processes associated with stress responses, eIF5 was up-regulated when eIF5B was knockdown in 293T and HepG2 cells by the CRISPR/cas9 system.
Question 15: In the caption for Figure 5, the second sentence is not needed. The reader should be told, however, what TA and TC mean.
Answer: The second sentence “The x-axis andy-axis represent the new data corresponding to the principal componetnt after dimensionality reduction.” was deleted. TA and TC were replaced by TSG and CG, respectively. TSG and CG indicate thermal stress group and the control group, respectively.
Question 16: Figure 6 is too small to read. I recommend that it be placed on a page oriented in landscape mode and then enlarged to fit that page.
Answer: The figure was oriental placed on a page in landscape (Figure 4).
Question 17: Section 3.9 should be entitled Heart tissue histology.
Answer: Section 3.9 has been changed as “Heart tissue histology”
Question 18: At line 328, what agency supported the research? CARS is just not informative enough.
Answer: This research was supported by the China's ministry of agriculture earmarked fund (grant number: CARS-48) and Anhui Province innovation platform of major science and technology projects (grant number: S202305a12020001).
Question 19: References. – The literature citations are not in journal stylistics. Article titles should not be enclosed in quotation marks. Species Latin names should be italicized. Other issues are also marked for the first 26 citations.
Answer: The references have been revised as pointed out.
Reviewer 2 Report
Comments and Suggestions for AuthorsTranscriptome and histology of male Chinese mitten crab (Eriocheir sinensis) under high temperature stress
This paper is describing a comparative evaluation of the heart transcriptome of male Chinese mitten crab (Eriocheirs sinensis) in response to temperature.
Abstract
Line 10. Delete “Temperature is an” and replace by “High temperatures are”.
Introduction
Line 39. Change “negative” by “negatively”.
Line 37 . Place “sea bass” before Dicentrarchus labrax
Line 44. Replace “getting higher and higher” by “increasing”.
Line 66. Change “on thermal stress is rare” by “ in response to thermal stress is still scanty”
scanty
M & M
Lines 81-83. Please edit the paragraph because in this current form it is hard to understand. “After acclimation for seven days, crabs were transferred into 185 L tanks, and divided into the thermal stress group and the control group (20 crabs each group) in triplicate. Water temperature in the thermal stress group and the control group maintained at 35 °C and 25 °C by aquarium heaters and air conditioner.
Please use the nomenclature “Thermal stress group (35 °C), and the control group (25 °C)”.
Line 117. Edit the sentence “ed by cor() in R environment”. In its current form it is uncompleted.
Results and Discussion
Line 156. Delete “Heart transcriptomes were sequenced at 24 h after the water temperature reached 25â—¦C and 35 â—¦C.”
Be concise with the nomenclature “Thermal stress group (35 °C), and the control group (25 °C)”.
Line 214 Replace “temperature increasing” by “the increase of temperature”.
Line 258. Comment on figure 5 and its legend. Change TA and TC by “TSG= Thermal stress group” and CG= Control group”.
Comments on the Quality of English Language
The information provided in this Ms is relevant, but relevant information is hindered by the low quality of the English. It is highly recommended to review the document by a Native English speaker.
Author Response
Reviewer 2
Question 1: Line 10. Delete “Temperature is an” and replace by “High temperatures are”.
Answer: “Temperature is an” replaced by “High temperatures are”.
Question 2: Line 39. Change “negative” by “negatively”.
Answer: “negative” replaced by “negatively”.
Question 3: Line 37 . Place “sea bass” before Dicentrarchus labrax
Answer: “sea bass” was added before “Dicentrarchus labrax”
Question 4: Line 44. Replace “getting higher and higher” by “increasing”.
Answer: “getting higher and higher” replaced by “increasing”.
Question 5: Line 66. Change “on thermal stress is rare” by “ in response to thermal stress is still scanty”
Answer: “on thermal stress is rare” replaced by “ in response to thermal stress is still scanty”
Question 6: Lines 81-83. Please edit the paragraph because in this current form it is hard to understand. “After acclimation for seven days, crabs were transferred into 185 L tanks, and divided into the thermal stress group and the control group (20 crabs each group) in triplicate. Water temperature in the thermal stress group and the control group maintained at 35 °C and 25 °C by aquarium heaters and air conditioner.
Answer: Section 2.2 has been rewritten as follows:
“After acclimation for seven days, the crabs were randomly divided into two groups that included the thermal stress group (TSG) and the control group (CG), each with three replicate tanks (185 L) of 20 individuals each. The water temperature in the thermal stress group and the control group was maintained at 35 °C and 25 °C, respectively, using aquarium heaters and air conditioners. Water temperature was elevated at a rate of 1 °C/24h. The control group was increased from 24 °C to 25 °C and maintained at 25 °C for 24 h. In the thermal stress group, water temperature was elevated from 24°C to 35 °C, and maintained at each integer temperature for 24 h. One-quarter of the water in the six experimental tanks was replaced with the same temperature water each day and aerated continuously. Food was not provided during the study period. The complete hearts of 5 individuals from each tank were collected from the control group and the thermal stress group at 24 h after water temperatures reached 25 °C and 35 °C, respectively. Five hearts from each group were separately placed in liquid nitrogen and then stored at -80 °C for transcriptome analysis. The other hearts were placed in 4% paraformaldehyde for histology.
Question 7: Please use the nomenclature “Thermal stress group (35 °C), and the control group (25 °C)”.
Answer: “Thermal stress group (35 °C), and the control group (25 °C)” were used in the manuscript.
Question 8: Line 117. Edit the sentence “ed by cor() in R environment”. In its current form it is uncompleted.
Answer: “analyzed by cor() in R environment” replaced by “analyzed by the cor() package in the R environment”.
Question 9: Line 156. Delete “Heart transcriptomes were sequenced at 24 h after the water temperature reached 25â—¦C and 35 â—¦C.”
Answer: “Heart transcriptomes were sequenced at 24 h after the water temperature reached 25â—¦C and 35 â—¦C.”was deleted.
Question 10: Be concise with the nomenclature “Thermal stress group (35 °C), and the control group (25 °C)”.
Answer: “Thermal stress group (35 °C), and the control group (25 °C)” concised to “TSG” and “CG”, respectively, in the main document.
Question 11: Line 214 Replace “temperature increasing” by “the increase of temperature”
Answer: “temperature increasing” replaced by “the increase of temperature”
Question 12: Line 258. Comment on figure 5 and its legend. Change TA and TC by “TSG= Thermal stress group” and CG= Control group”.
Answer: TA and TC were replaced by TSG and CG, respectively. TSG and CG indicate thermal stress group and the control group, respectively.
Question 13: Comments on the Quality of English Language. The information provided in this Ms is relevant, but relevant information is hindered by the low quality of the English. It is highly recommended to review the document by a Native English speaker.
Answer: The document has been revised by a Native English speaker.
Reviewer 3 Report
Comments and Suggestions for AuthorsTingshuang Pan, Tong Li, Min Yang, He Jiang, Jun Ling, and Qian Gao
Transcriptome and histology of male Chinese mitten crab (Eriocheir sinensis) under high temperature stress
fishes-2856077
This work analyzes the effects of a heat stress experiment (experimental temperature of 35 ºC versus control temperature of 25 ºC) on the transcriptome and cardiac histology of male Chinese mitten crab.
De entre los genes que mostraron expresión diferencial destacan 59 familias de factores de transcripción que incluyen 851 factores de transcripción.
Among the genes that showed differential expression, 50 transcription factor families including 851 transcription factors stand out.
The presence of short simple repeats was also analyzed, with the AC/GT motif being the most abundant.
Analysis of heart samples showed significant alterations in myocardial fibers, number of nuclei and connective tissue.
The work presented uses methods that are adequate, although not very novel, to the objective presented and is potentially useful for the study of thermal stress in this species.
My recommendation is that the manuscript should be reconsider after major revision.
MAJOR COMMENTS
- In my opinion, an important aspect to improve is the justification of the experimental temperature (35 ºC). Why is this temperature used and not another one? The introduction is not clear enough about the range of temperatures that these animals suffer. I think it is important to describe in more detail the natural conditions in which this species lives and the aquaculture conditions.
- Another important aspect to justify is the number of individuals finally analyzed. The experimental and control groups consisted of 20 individuals each and were performed in triplicate. However, when presenting the results, there is no mention of these replicates or of possible variation between and within replicates. For example, in the principal component analysis it is mentioned that the samples within the groups were clearly grouped and that the groups were clearly separated. However in Figure 5 one of the TC samples is clearly separated from the rest of the samples in that group.
- The Discussion and Conclusions are rather descriptive. The authors should make an effort to present more clearly what can be concluded from the results regarding the response of this species to elevated temperatures.
- Some figures and tables could be placed as Supplementary Material (e.g. table 2 and figure 1).
- Although I am not a native English speaker, I think the text needs revision to correct typos, some confusing phrases and verb tenses.
- Manuscript title. Since the study of the heart is a fundamental part of the manuscript, this should be reflected in the title of the manuscript.
- Section 2.1. and 2.2. It is necessary to justify certain aspects of the experimental design and sampling.
- P2 L75-6, "Healthy male E. sinensis (60. 1 ± 2.6 g) were caught by cages from our experiment base ponds and acclimated in 20000 L plastic tanks"
What is the reason that only males were used, what is their original origin (wild or captive-bred), and what is the approximate or total number of animals acclimatized in these tanks?
- P2 L81-2, "After acclimation for seven days, crabs were transferred into 185 L tanks, and divided into the thermal stress group and the control group (20 crabs each group) in triplicate"
Please describe how individuals were assigned to each tank and how each tank was assigned to the stress or control group.
- References. It is necessary to revise the format of the species names in many references, some of them do not have the genus with the first letter in capital letters and others have not been italicized.
- P2 L95-7, "The total mRNA was fragmented by ultrasonic, and the fragmented mRNA was reverse transcribed into the first strand cDNA by M-MuLV reverse transcriptase and random primers"
Please detail sonication details and add information about the supplier of M-MuLV reverse transcriptase.
- P3 L132-3, "In our study, eight DEGs related immune or apoptosis were selected to verify RNA- Seq result by qRT-PCR. RNA for heart qRT-PCR was the same as transcriptome"
Please provide the rationale to select these particular DEGs.
- Figura 4. This figure is redundant, the information is presented in the main text (P6 L194-5).
- P7 L202-4, "In this study, 4007 DEGs (Table S1) were identified between the two groups, containing 2660 up-regulated and 1347 down- regulated DEGs, numbers of up-regulated DEGs were more than the down-regulated DEGs"
The last part of the sentence does not provide additional information about what is numerically stated.
- Figure 8. What statistical values doe the bars in Figure 8B represent?
Why was a break added to the y axis in Figure 8B?
- Abbreviations "TA" and "TC" are not defined in the text or figure / table legends.
- How many transcriptomes / individuals were sequenced?
- How many samples in the qPCR?
MINOR COMMENTS
- P1 L25-6, "About 4,007 DEGs and protein processing in endoplasmic reticulum pathway were mostly modulated at high temperature in Chinese mitten crab, indicating their importance in crab adaption to high temperature stress"
The use of "about" suggests that the exact number is not given, but here it is being given. I suggest changing the phrase to "About 4,000 SDRs..." or "Four thousand seven SDRs".
- P1 L31-2, "With the global warming, extreme high temperature occurred in China and other regions in summer [2]."
There are probably better references to support the occurrence of extreme temperature events than a review of statistical methods for analyzing extreme precipitation and temperature events.
- P1 L32-3, "Aquatic organisms are ectothermic animals, whose temperate fluctuates with water temperature [3]." The reference ("Ecophysiological biomarkers defining the thermal biology of the caribbean lobster panulirus argus.")
- P2 L47-8, "According to Li [13] report, immune related enzyme peaked at 12h or 24h, and then decreased by the thermal stress"
Correct "According to Li [13] report..." to "According to Li et al. [13] report...".
- P3 L112-4, "Simple sequence repeats (SSR) in transcriptome was predicted by the *MIcroSAtellite* (http://pgrc.ipk-gatersleben.de/misa/)"
Please indicate the program version.
- P3 L116-7, "Pearson’s correlation coefficient and principal component analysis (PCA) for sample replicates and stability of samples were analyzed by cor() in R environment"
Add and apropriate citation for the R language.
- P3 L129-30, "The significant enriched GO terms for DEGs were analyzed on omicsshare platform"
If possible provide additional details / information.
- P4 L144-7, "Cut sections (5 μm thick) were immersed in sequence in Environmental Friendly Dewaxing Transparent Liquid I for 20min, Environmental Friendly Dewaxing Transparent Liquid II for 20min, Anhydrous ethanol I for 5min, Anhydrous ethanol II for 5min, 75% Ethyl alcohol for 5min, and then rinsed with tap water".
Please provide manufacturer details for these reagents.
- P4 L156-63, "Heart transcriptomes were sequenced at 24 h after the water temperature reached 25 â—¦C and 35 â—¦C. In this study, when low quality sequences were removed from the thermal stress group and the control group libraries, the raw reads number was from 37.73 M to 55.73 M, the clean reads number was from 37.33 M to 55.48 M, and clean bases were from 5.57 G to 8.28 G. Values for Q20 and Q30 of the sequenced libraries were from 96.82 % to 98.14 %, and from 91.67 % to 94.31 % individually, and GC content was from 38.54 % to 43.95 % (Table 2), indicating that the sequence data were high quality and reliable for further study"
It would probably not be necessary to give so many details.
In "Values for Q20 and Q30 of the sequenced libraries were from 96.82 % to 98.14 %, and from 91.67 % to 94.31 % individually, ..." do you mean "respectively" instead of "individually"?
- P4 L166-7, "Max length, min length, and average length of unigenes was 36,785 bp, 201 bp, and 1,190 bp individually"
Do you mean "respectively" instead of "individually".
- Table 3. The legend of the table is too short, please expand it.
- P6 L194-5, "In this study, 59 TF families including 851 TFs were detected from all Unigenes of Eriocheir sinensis transcripts"
The species names should be in italics.
- P7 L206-8, "DEGS mainly focused in protein processing in endoplasmic reticulum, ribosome biogenesis in eukaryotes, glycine, serine and threonine metabolism, protein export, *insect hormone biosynthesis pathway*"
Any explanation for the insect hormone biosynthesis pathway?
- P7 P224-6 "According to Oksala [36] study, HSPs (HSP60, HSP70, HSP90, HSC70) from Carra rufa muscle were more elevated in high temperature water (34.4 °C) than those in normal temperature water (25.4 °C)"
Correct "According to Oksala [36] study..." to "According to Oksala et al. [36] study..."
- P7-8 L234-6, "HSPe1 was a member of HSP10 family, and could be expressed more in liver, brain, head kidney than that of gill, heart, and spleen when stressed by heat shock [42]"
I am not sure what are you trying to said in this sentence. Please reformulate it.
- P8 L238-9, "HSPbp1 could be significant up- regulated when zebrafish treated by high temperature (35 °C ) [21]"
Reference 21 is not a paper on zebrafish, is it the reference you wanted to mention in this line?
- Figure 5. It contains redundant information (labels above the figure and axis).
- P8 L264-6, "The most three enriched cellular component (CC) items included “cellular anatomical entity”, “protein-containing complex”, “virion component”"
The authors should try to explain why the “virion component” is one of the most enriched component.
- P11 L302-3, "When male E. sinensis aquacultured at 25 °C for 24 h, horixontal stripes of myocardium fibres and nucleus are clear (Figure 10A)"
Typo in "horixontal"
- P12 L317-20, "Heart DEGs in the high temperature stressed group were significantly enriched in 1377 GO terms, and the top 20 KEGG pathways were protein processing in endoplasmic reticulum et al."
Why is "et al" at the end of the sentence?
- P12 L321-4, "This research domenstrated the transcriptome changes in male E. sinensis at high temperature, and 317 will be helpful for better understanding the adaption of Chinese mitten crab to high temperature challenge"
Typo in "domenstrated".
Although I am not a native English speaker, I believe the text needs revision to correct typos, some confusing phrases and verb tenses.
Author Response
Reviewer 3
Question 1:. - In my opinion, an important aspect to improve is the justification of the experimental temperature (35 ºC). Why is this temperature used and not another one? The introduction is not clear enough about the range of temperatures that these animals suffer. I think it is important to describe in more detail the natural conditions in which this species lives and the aquaculture conditions.
Answer: In China, most E. sinensis are cultured in earth ponds filled with submerged macrophytes (Hydrilla verticillate) and Nuttall’s waterweed (Elodea nuttallii), and the depth of the water is approximately 1.5 m. Chinese mitten crabs rest at the bottom of the pond and are surrounded by water weeds. Although water temperature on the surface of the pond can reach 37 °C or even higher in summer, the water temperature at the bottom of the pond surrounded water weeds can only reach 35 °C or even lower. Previous study demonstrated that E. sinensis could molt normally at 35 °C, and the changes in diel water temperature from 28 °C to 35 °C did not affect molting [12]. Thus, 35 °C can be used as the thermal stress temperature.
Question 2:. - Another important aspect to justify is the number of individuals finally analyzed. The experimental and control groups consisted of 20 individuals each and were performed in triplicate. However, when presenting the results, there is no mention of these replicates or of possible variation between and within replicates. For example, in the principal component analysis it is mentioned that the samples within the groups were clearly grouped and that the groups were clearly separated. However in Figure 5 one of the TC samples is clearly separated from the rest of the samples in that group.
Answer: Ten individuals were used for heart transcriptomes sequencing（Table S1）. Five individuals in the thermal stress group (35 °C), and another five in the control group (25 °C). The aquaculture experiment was performed in three tanks each group. To improve the transcriptomic data reproducibility, five individuals of each group were used for transcriptome sequencing.
Although one of the TSG samples is separated from the rest of the samples in thermal stress group, the five TSG samples clustered at the same side, and clearly separated from the control group (CG).
Question 3:. - The Discussion and Conclusions are rather descriptive. The authors should make an effort to present more clearly what can be concluded from the results regarding the response of this species to elevated temperatures.
Answer: In conclusion part, “This research demonstrated DEGs of heat shock proteins, transcription factors, and pathways of protein processing in the endoplasmic reticulum were sensitive in male E. sinensis at high temperature” was added.
Question 4:. - Some figures and tables could be placed as Supplementary Material (e.g. table 2 and figure 1).
Answer: Table 2 and figure 1 were placed as Supplementary Material.
Question 5:. - Although I am not a native English speaker, I think the text needs revision to correct typos, some confusing phrases and verb tenses.
Answer: The manuscript has been revised by a Native English speaker.
Question 6:. - Manuscript title. Since the study of the heart is a fundamental part of the manuscript, this should be reflected in the title of the manuscript.
Answer: heart was added in the title as “Transcriptome and histology of male Chinese mitten crab (Eriocheir sinensis) heart under high temperature stress”
Question 7:. - Section 2.1. and 2.2. It is necessary to justify certain aspects of the experimental design and sampling.
Answer: Section 2.2 has been rewritten as follows:
After acclimation for seven days, the crabs were randomly divided into two groups that included the thermal stress group (TSG) and the control group (CG), each with three replicate tanks (185 L) of 20 individuals each. The water temperature in the thermal stress group and the control group was maintained at 35 °C and 25 °C, respectively, using aquarium heaters and air conditioners. Water temperature was elevated at a rate of 1 °C/24h. The control group was increased from 24 °C to 25 °C and maintained at 25 °C for 24 h. In the thermal stress group, water temperature was elevated from 24°C to 35 °C, and maintained at each integer temperature for 24 h. One-quarter of the water in the six experimental tanks was replaced with the same temperature water each day and aerated continuously. Food was not provided during the study period. The complete hearts of 5 individuals from each tank were collected from the control group and the thermal stress group at 24 h after water temperatures reached 25 °C and 35 °C, respectively. Five hearts from each group were separately placed in liquid nitrogen and then stored at -80 °C for transcriptome analysis. The other hearts were placed in 4% paraformaldehyde for histology.
Question 8:. - P2 L75-6, "Healthy male E. sinensis (60. 1 ± 2.6 g) were caught by cages from our experiment base ponds and acclimated in 20000 L plastic tanks". What is the reason that only males were used, what is their original origin (wild or captive-bred), and what is the approximate or total number of animals acclimatized in these tanks?
Answer: In summer, male crabs are larger and easier stressed by high temperature than female crabs in pond, when water temperature reached 35 °C or even higher for long time. Male crabs were selected as experimental sample precede to female.
All the experiment crabs are captive-bred in the pond.
300 male crabs were acclimated in 20000 L plastic tanks.
Question 9:. - P2 L81-2, "After acclimation for seven days, crabs were transferred into 185 L tanks, and divided into the thermal stress group and the control group (20 crabs each group) in triplicate. Please describe how individuals were assigned to each tank and how each tank was assigned to the stress or control group.
Answer: After acclimation for seven days, crabs were randomly divided into two groups with the thermal stress group (TSG) and the control group (CG), each group had three replicate tanks (185 L) of 20 individuals each. The control group was elevated from 24 °C to 25 °C, and maintained at 25 °C for 24 h. In the thermal stress group, Water temperature was elevated from 24°C to 35 °C, and maintained at each integer temperature for 24 h.
Question 10:. - References. It is necessary to revise the format of the species names in many references, some of them do not have the genus with the first letter in capital letters and others have not been italicized.
Answer: The species names in references have been revised to italic, and first letter of species name is in capital letters now.
Question 11:. - P2 L95-7, "The total mRNA was fragmented by ultrasonic, and the fragmented mRNA was reverse transcribed into the first strand cDNA by M-MuLV reverse transcriptase and random primers". Please detail sonication details and add information about the supplier of M-MuLV reverse transcriptase.
Answer: Manufacturer of M-MuLV reverse transcriptase was Promega, USA.
Question 12:. - P3 L132-3, "In our study, eight DEGs related immune or apoptosis were selected to verify RNA- Seq result by qRT-PCR. RNA for heart qRT-PCR was the same as transcriptome"
Please provide the rationale to select these particular DEGs.
Answer: The standard to select particular DEGs for qRT-PCR as below:
- Valified DEGs in one set of samples had FPKM values at least> 10.
- DEGs with high group stability and greater fold difference between groups were preferred.
Question 13:. - Figure 4. This figure is redundant, the information is presented in the main text (P6 L194-5).
Answer: Figure 4 was deleted.
Question 14:. - P7 L202-4, "In this study, 4007 DEGs (Table S1) were identified between the two groups, containing 2660 up-regulated and 1347 down- regulated DEGs, numbers of up-regulated DEGs were more than the down-regulated DEGs"
The last part of the sentence does not provide additional information about what is numerically stated.
Answer: “numbers of up-regulated DEGs were more than the down-regulated DEGs.” was deleted in the manuscript.
Question 15:. - Figure 8. What statistical values doe the bars in Figure 8B represent?
Why was a break added to the y axis in Figure 8B?
Answer: bars in Figure 8B represent P < 0.05. Because the control group is too short to demonstrate the column, it is easy to see the control group column by adding a break to the y axis.
Question 16:. - Abbreviations "TA" and "TC" are not defined in the text or figure / table legends.
Answer: In Table 2, Figure 5 and Figure 8B, "TA" and "TC" were replaced by “CG” and“TSG”, respectively.
Question 17:. - How many transcriptomes / individuals were sequenced?
Answer: Ten heart transcriptomes were sequenced（Table S1）. Five transcriptomes in the thermal stress group (35 °C), and five in the control group (25 °C)
Question 18:. - How many samples in the qPCR?
Answer: In order to verify the transcriptome results, six samples were used for qPCR. Three samples from the thermal stress group (35 °C), and another three samples from the control group (25 °C).
Question 19:. - P1 L25-6, "About 4,007 DEGs and protein processing in endoplasmic reticulum pathway were mostly modulated at high temperature in Chinese mitten crab, indicating their importance in crab adaption to high temperature stress"
The use of "about" suggests that the exact number is not given, but here it is being given. I suggest changing the phrase to "About 4,000 SDRs..." or "Four thousand seven SDRs".
Answer: "About 4,007 DEGs” has been changed as “A total of 4,007 DEGs”
Question 20:. - P1 L31-2, "With the global warming, extreme high temperature occurred in China and other regions in summer [2]."
There are probably better references to support the occurrence of extreme temperature events than a review of statistical methods for analyzing extreme precipitation and temperature events.
Answer: The reference has been changed as below:
Zhang, G., G. Zeng, X. Yang and Z. Jiang. Future changes in extreme high temperature over china at 1.5 c–5 c global warming based on cmip6 simulations. Advances in Atmospheric Sciences 38 (2021): 253-67.
Question 21:. - P1 L32-3, "Aquatic organisms are ectothermic animals, whose temperate fluctuates with water temperature [3]." The reference ("Ecophysiological biomarkers defining the thermal biology of the caribbean lobster panulirus argus.")
Answer: “The reference has been changed as “Gotthard, K. Growth strategies of ectothermic animals in temperate environments. Environment and animal development (2001): 287-304.”
Question 22:. - P2 L47-8, "According to Li [13] report, immune related enzyme peaked at 12h or 24h, and then decreased by the thermal stress"
Correct "According to Li [13] report..." to "According to Li et al. [13] report...".
Answer: "According to Li [13] report..." has been corrected as "According to Li et al. [13] report..."
Question 23:. - P3 L112-4, "Simple sequence repeats (SSR) in transcriptome was predicted by the *MIcroSAtellite* (http://pgrc.ipk-gatersleben.de/misa/)"
Please indicate the program version.
Answer: The program MIcroSAtellite version is v2.1 was added.
Question 24:. - P3 L116-7, "Pearson’s correlation coefficient and principal component analysis (PCA) for sample replicates and stability of samples were analyzed by cor() in R environment"
Add and apropriate citation for the R language.
Answer: A reference has been added.
Wei, T., V. Simko, M. Levy, Y. Xie, Y. Jin and J. Zemla. Package ‘corrplot’. Statistician 56 (2017): e24.
Question 25:. - P3 L129-30, "The significant enriched GO terms for DEGs were analyzed on omicsshare platform"
If possible provide additional details / information.
Answer: url for the omicsshare platform “https://www.omicshare.com/”was added in the manuscript.
Question 26:. - P4 L144-7, "Cut sections (5 μm thick) were immersed in sequence in Environmental Friendly Dewaxing Transparent Liquid I for 20min, Environmental Friendly Dewaxing Transparent Liquid II for 20min, Anhydrous ethanol I for 5min, Anhydrous ethanol II for 5min, 75% Ethyl alcohol for 5min, and then rinsed with tap water".
Please provide manufacturer details for these reagents.
Answer: Manufacturer of environmental Friendly Dewaxing Transparent Liquid I and II is Servicebio, China.
Question 27:. - P4 L156-63, "Heart transcriptomes were sequenced at 24 h after the water temperature reached 25 â—¦C and 35 â—¦C. In this study, when low quality sequences were removed from the thermal stress group and the control group libraries, the raw reads number was from 37.73 M to 55.73 M, the clean reads number was from 37.33 M to 55.48 M, and clean bases were from 5.57 G to 8.28 G. Values for Q20 and Q30 of the sequenced libraries were from 96.82 % to 98.14 %, and from 91.67 % to 94.31 % individually, and GC content was from 38.54 % to 43.95 % (Table 2), indicating that the sequence data were high quality and reliable for further study"
It would probably not be necessary to give so many details.
In "Values for Q20 and Q30 of the sequenced libraries were from 96.82 % to 98.14 %, and from 91.67 % to 94.31 % individually, ..." do you mean "respectively" instead of "individually"?
Answer: "individually" has been replaced by "respectively".
Question 28:. - P4 L166-7, "Max length, min length, and average length of unigenes was 36,785 bp, 201 bp, and 1,190 bp individually"
Do you mean "respectively" instead of "individually".
Answer: "individually" has been replaced by "respectively".
Question 29:. - Table 3. The legend of the table is too short, please expand it.
Answer: The legend of Table 3 was expanded as “BUSCO analysis for the Eriocheir sinensis transcriptome.”
Question 30:. - P6 L194-5, "In this study, 59 TF families including 851 TFs were detected from all Unigenes of Eriocheir sinensis transcripts"
The species names should be in italics
Answer: “Eriocheir sinensis” is in italics now.
.
Question 31:. - P7 L206-8, "DEGS mainly focused in protein processing in endoplasmic reticulum, ribosome biogenesis in eukaryotes, glycine, serine and threonine metabolism, protein export, *insect hormone biosynthesis pathway*"
Any explanation for the insect hormone biosynthesis pathway?
Answer: Molting and metamorphosis are essential for Chinese mitten crab growth and development. In insects molting and metamorphosis are primarily under the control of two insect hormones, ecdysone and juvenile hormone. In this study, ecdysone and juvenile hormone in insect hormone biosynthesis pathway were down-regulated under the thermal stress, this may be caused by more energy were used for survival not for growth.
Question 32:. - P7 P224-6 "According to Oksala [36] study, HSPs (HSP60, HSP70, HSP90, HSC70) from Carra rufa muscle were more elevated in high temperature water (34.4 °C) than those in normal temperature water (25.4 °C)"
Correct "According to Oksala [36] study..." to "According to Oksala et al. [36] study..."
Answer: "According to Oksala [36] study..." has been replaced by "According to Oksala et al. [36] study...".
Question 33:. - P7-8 L234-6, "HSPe1 was a member of HSP10 family, and could be expressed more in liver, brain, head kidney than that of gill, heart, and spleen when stressed by heat shock [42]"
I am not sure what are you trying to said in this sentence. Please reformulate it.
Answer: The sentence has been changed as “HSPe1 is a member of HSP10 family, and can be more highly expressed in liver, brain, and head kidney than in gill, heart, and spleen when stressed by heat shock[42]”
Question 34:. - P8 L238-9, "HSPbp1 could be significant up- regulated when zebrafish treated by high temperature (35 °C ) [21]"
Reference 21 is not a paper on zebrafish, is it the reference you wanted to mention in this line?
Answer: Sorry, the reference was wrong cited. The reference has been revised as "Comparative transcriptome analysis of heat-induced domesticated zebrafish during gonadal differentiation." BMC Genomic Data 23 (2022): 39.
HSPbp1 could be significantly up-regulated in zebra fish (Danio rerio) during gonadal differentiation when treated by high temperature (35 °C) [44].
Question 35:. - Figure 5. It contains redundant information (labels above the figure and axis).
Answer: The second sentence “The x-axis andy-axis represent the new data corresponding to the principal componetnt after dimensionality reduction.” was deleted. TA and TC were replaced by TSG and CG, respectively. TSG and CG indicate thermal stress group and the control group, respectively.
Question 36:. - P8 L264-6, "The most three enriched cellular component (CC) items included “cellular anatomical entity”, “protein-containing complex”, “virion component”"
The authors should try to explain why the “virion component” is one of the most enriched component.
Answer: A comprehensive sequence and structure analysis of major virion proteins indicates that they evolved on about 20 independent occasions, and in some of these cases likely ancestors are identifiable among the proteins of cellular organisms. Although the replication modules of at least some classes of viruses might descend from primordial selfish genetic elements, bona fide viruses evolved on multiple, independent occasions throughout the course of evolution by the recruitment of diverse host proteins that became major virion components [51].
- Krupovic, M. and E. V. Koonin. Multiple origins of viral capsid proteins from cellular ancestors. Proceedings of the National Academy of Sciences 114 (2017): E2401-E10.
Question 37:. - P11 L302-3, "When male E. sinensis aquacultured at 25 °C for 24 h, horixontal stripes of myocardium fibres and nucleus are clear (Figure 10A)"
Typo in "horixontal"
Answer: "horixontal" has been replaced by "horizontal"
Question 38. - P12 L317-20, "Heart DEGs in the high temperature stressed group were significantly enriched in 1377 GO terms, and the top 20 KEGG pathways were protein processing in endoplasmic reticulum et al."
Why is "et al" at the end of the sentence?
Answer: "et al" was deleted.
Question 39:. - P12 L321-4, "This research domenstrated the transcriptome changes in male E. sinensis at high temperature, and 317 will be helpful for better understanding the adaption of Chinese mitten crab to high temperature challenge"
Typo in "domenstrated"
Answer: "domenstrated" has been replaced by "demonstrated"
Question 40:. Comments on the Quality of English Language
Although I am not a native English speaker, I believe the text needs revision to correct typos, some confusing phrases and verb tenses.
Answer: “The document has been revised by a Native English speaker.
Round 2
Reviewer 1 Report
Comments and Suggestions for AuthorsChinese mitten crab is an important cultured species in China. With the advance of climate change, increasing temperature threatens culture activity. Improved understanding of the mechanism of high temperature stress might inform action to address that stress. Pan et al. conducted a study of the heart of male Chinese mitten crab, considering transcriptomic and histological responses. The science is straightforward and the results interesting. Thi is a revised manuscript and reads rather well now. There remain a few, minor matters needing attention. I have marked the manuscript to guide that minor revision. More context-specific comments follow.
Methods. – At line 116, can the authors provide the manufacturer for the DNA polymerase I?
At line 130, we need a citation or url for the BLASTp software.
Results and Discussion. – At lines 179 and 180, what does “G” mean?
At line 253, the Latin name for rainbow trout is Oncorhynchus mykiss. Then it need not be given at lines 259.
References. – There remain a few stylistic issues with the literature citations. I’ve marked them.
Comments for author File: 
Comments.pdf
There remain a few, minor matters needing attention. I have marked the manuscript to guide that minor revision.
Author Response
Reviewer 1
Question 1: Methods. – At line 116, can the authors provide the manufacturer for the DNA polymerase I?
Answer: The manufacturer for the DNA polymerase I is Promega, USA.
Question 2: At line 130, we need a citation or url for the BLASTp software.
Answer: url for the BLASTp is added in the paper.
Question 3: Results and Discussion. – At lines 179 and 180, what does “G” mean?
Answer: “G” is the nucleic acid length unit. 1 Gb=1,000,000,000 bp. 1000 bp=1kb, 1000kb=1Mb, 1000Mb=1Gb.
Question 4: At line 253, the Latin name for rainbow trout is Oncorhynchus mykiss. Then it need not be given at lines 259.
Answer: Oncorhynchus mykiss removed from lines 259 to lines 253.
Question 5: References. – There remain a few stylistic issues with the literature citations. I’ve marked them.
Answer: I have revised the references as marked.
Reviewer 3 Report
Comments and Suggestions for AuthorsTingshuang Pan, Tong Li, Min Yang, He Jiang, Jun Ling, and Qian Gao
Transcriptome and histology of male Chinese mitten crab (Eriocheir sinensis) under high temperature stress
fishes-2856077
The issues and suggestions I made in my previous review have been satisfactorily addressed by the authors. I have a number of additional comments, but my opinion is that the manuscript could be accepted after a minor revision.
MINOR COMMENTS
- P2 L50-2, "Although water temperature on the surface of the pond can reach 37 °C or even higher in summer, the water temperature at the bottom of the pond surrounded water weeds can only reach 35 °C or even lower."
I understand that you mean that the water temperature does not usually exceed 35°C, not that there is anything to prevent it from exceeding that temperature.
- P2 L134-6 "Pearson’s correlation coefficient and principal component analysis (PCA) for sample replicates and sample stability were performed using the cor() package in the R environment [27]"
I suppose you mean the cor() function.
- P3 L105-6, "Five hearts from each group were separately placed in liquid nitrogen and then stored at -80 °C for transcriptome analysis."
In response to a question I raised in the first review, the authors indicate that 5 samples per group (control and heat stress) were used to improve reproducibility. This is reasonable, but it is not clear to me how the selection was made - did they come from all three replicates or just one of them? Was it random?
- P5 L152-5, "In our study, eight DEGs related to immunity or apoptosis were selected to verify the RNA-seq results using qRT-PCR. RNA used for heart qRT-PCR was the same as that used for transcriptome analysis. qRT-PCR was performed using a SYBR Premix Ex Taq kit (Invitrogen) according to the manufacturer’s snstructions."
Typo in "snstructions".
- P6 L200-1, "Figure 1. Distribution of 43,606 E. sinensis unigenes in four protein databases, including SwissProt, KEGG, KOG and Nr."
The legend of Figure 1 is placed on a different page than the figure itself.
- P6 L225-30, "Molting and metamorphosis are essential process for the growth and development of Chinese mitten crabs. In insects, molting and metamorphosis are primarily controlled by two insect hormones that include ecdysone and juvenile hormones. In this study, ecdysone and juvenile hormones in the insect hormone biosynthesis pathway were down-regulated under thermal stress, possibly due to the observation that more energy was used for survival and not growth."
In my previous review I asked for an explanation for the importance of this pathway in a crustacean. Are these hormones conserved between crustaceans and insects (which is probably logical)?
- P7 L237-9, "After E. sinensis was exposed to hot (32 °C) or cold water (4 °C) for 0 h, 2 h, 6 h, 12 h, or 24 h, EsTreh expression was gradually downregulated [23]".
Suggestion. Change "... for 0 h, 2 h, 6 h, 12 h, or 24 h..." to "... for 0, 2, 6, 12, or 24 h..."
Author Response
Reviewer 3
Question1 : - P2 L50-2, "Although water temperature on the surface of the pond can reach 37 °C or even higher in summer, the water temperature at the bottom of the pond surrounded water weeds can only reach 35 °C or even lower."
I understand that you mean that the water temperature does not usually exceed 35°C, not that there is anything to prevent it from exceeding that temperature.
Answer: Aquatic weeds can block sunshine to the earthen pond bottom directly, so the water temperature at the bottom is lower than on the surface.
Question 2: - P2 L134-6 "Pearson’s correlation coefficient and principal component analysis (PCA) for sample replicates and sample stability were performed using the cor() package in the R environment [27]", I suppose you mean the cor() function.
Answer: Yes.
Question 3: - P3 L105-6, "Five hearts from each group were separately placed in liquid nitrogen and then stored at -80 °C for transcriptome analysis."
In response to a question I raised in the first review, the authors indicate that 5 samples per group (control and heat stress) were used to improve reproducibility. This is reasonable, but it is not clear to me how the selection was made - did they come from all three replicates or just one of them? Was it random?
Answer: Two, two, and one sample was random selected from the first, second, and third tank in each group, respectively. Thus, five samples can be got from each group.
Question 4: - P5 L152-5, "In our study, eight DEGs related to immunity or apoptosis were selected to verify the RNA-seq results using qRT-PCR. RNA used for heart qRT-PCR was the same as that used for transcriptome analysis. qRT-PCR was performed using a SYBR Premix Ex Taq kit (Invitrogen) according to the manufacturer’s snstructions."
Typo in "snstructions".
Answer: “snstructions” has been revised as “instruction”
Question 5: - P6 L200-1, "Figure 1. Distribution of 43,606 E. sinensis unigenes in four protein databases, including SwissProt, KEGG, KOG and Nr."
The legend of Figure 1 is placed on a different page than the figure itself.
Answer: Figure 1 and the legend have been placed on the same page now.
Question 6: - P6 L225-30, "Molting and metamorphosis are essential process for the growth and development of Chinese mitten crabs. In insects, molting and metamorphosis are primarily controlled by two insect hormones that include ecdysone and juvenile hormones. In this study, ecdysone and juvenile hormones in the insect hormone biosynthesis pathway were down-regulated under thermal stress, possibly due to the observation that more energy was used for survival and not growth."
In my previous review I asked for an explanation for the importance of this pathway in a crustacean. Are these hormones conserved between crustaceans and insects (which is probably logical)?
Answer: Ecdysone and juvenile hormones are conserved between crustaceans and insects [32, 33].
- Xu, Y., M. Zhao, Y. Deng, Y. Yang, X. Li, Q. Lu, J. Ge, J. Pan and Z. Xu. Molecular cloning, characterization and expression analysis of two juvenile hormone esterase-like carboxylesterase cDNAs in Chinese mitten crab, Eriocheir sinensis. Comparative Biochemistry and Physiology Part B: Biochemistry and Molecular Biology 205 (2017): 46-53.
- Shen, H., Y. Ma, Y. Hu and X. Zhou. Cloning of the ecdysone receptor gene from the Chinese mitten crab, Eriocheir sinensis, and sexually dimorphic expression of two splice variants. Journal of the World Aquaculture Society 46 (2015): 421-33.
Question 7: - P7 L237-9, "After E. sinensis was exposed to hot (32 °C) or cold water (4 °C) for 0 h, 2 h, 6 h, 12 h, or 24 h, EsTreh expression was gradually downregulated [23]".
Suggestion. Change "... for 0 h, 2 h, 6 h, 12 h, or 24 h..." to "... for 0, 2, 6, 12, or 24 h..."
Answer: "... for 0 h, 2 h, 6 h, 12 h, or 24 h..." to " has been revised as "... for 0, 2, 6, 12, or 24 h...".
