Identification and Characterization of Heme Oxygenase-1 from Litopenaeus vannamei Involved in Antioxidant and Anti-Apoptosis under Ammonia Stress

Round 1

Reviewer 1 Report

In the present manuscript Huang et al worked-on identification and characterization of Heme Oxygenase-1 from the Litopenaeus vannamei, a shrimp verity which is economically important. The experiment was designed to test the hypothesis that HO-1 can be highly induced by a variety stress causing substances such as ammonia. HO-1 has also been studied to link to cell protection by reducing inflammation, apoptosis, and regulating cell proliferation. This was attained through a knockdown test. The experiment was well-executed and explained. Although the experimental hypothesis of this study contributes to a better theoretical framework, some minor issues must be addressed before publication.

 

Minor Corrections:

 

1.     Line 52: “7°C fish” ? paraphrase the sentence.

2.     Line 62: “Lv-HO-1” must be italics

3.     Line 77/118/182/Figure 7: Consistency needed in writing acronym; h/hours

4.     Line 85: Primers used were not quoted.

5.     Line 95: Reference could be mentioned to the software used.

6.     Line 105: It's critical to specify which side of the shrimp was injected intramuscularly.

7.     Line 106: “was injected in”, paraphrase the sentence.

8.     Line 70/108/269: Consistency needed in writing acronym; d/day

9.     Line 118: HP tissue is mentioned twice. Grammatical error.

10.  Line 151-154: Paraphrase in the sentence for better clarity.

11.  Line 158: The spelling for BLAST should be corrected throughout.

12.  Line 169: The word "relatively heighted" is rarely used.

13.  Figure 3: use of phrases like “at various time points/ other time points” is confusing the reader.

14.  Figure 4: dsRNA-HO-1? Or dsRNA-Lv-HO-1?

15.  Section 3.1.: Issues in writing italics.

16.  Line 212: acronym “SOD” - abbreviation needed.

17.  Line 213: dsRNA-HO-1? Or dsRNA-Lv-HO-1?

18.  Line 213 Figure 5: Would be better if the explanation was referred to 5A and 5B separately.

19.  Line 215: dsRNA-HO-1? Or dsRNA-Lv-HO-1?

20.  Figure 4 and Figure 5: The label has dsRNA-HO-1 instead of dsRNA-Lv-HO-1? hope I didn’t get it wrong through the flow.

21.  Figure 7: dsRNA-EGFP+NH4CL, Check for subscript and superscripts throughout.

22.  Line 231 & 232: Mentioned the time point to explain the significance between groups.

Author Response

Thank you very much for your letter and the comments about our paper submitted to Diagnosis and Treatment of Disease in Fish. These comments are all valuable and helpful for improving our article. The manuscript was carefully revised by using  blue text according to the comments. We responded point by point to each comment as listed below, along with a clear indication of the location of the revision.

Changes for review issues are shown in blue.

 Hope these will make it more acceptable for publication.

Thank you very much again.

Author Response File: Author Response.pdf

Reviewer 2 Report

This paper reports on the identification and characterization of Heme Oxygenase-1 (HO1) from the whiteleg shrimp (Litopenaeus vannamei), a stress-responsive enzyme that counteract signals such as reactive oxygen species and inflammation. A variety of molecular techniques were used to characterize transcript levels. In addition, a positive control (ammonia exposure) was used to test its involvement in stress response and its knockdown caused a significant decrease in antioxidant capacity. The authors conclude that HO-1 can be induced by oxidative stress therefore being important in the regulation of the antioxidant system and reducing apoptosis in this model.

In general, this is a well-written manuscript with an interesting approach and design with results showing potential scientific implications for the field and expanding our knowledge on the characterization of HO1 system and the involved response. Yet, some revisions must be taken in consideration and some issues need to be addressed in order to improve the quality of this manuscript:

-          L44, in vitro and in vivo should be in italics.

-          L69, please include SD for temperature and salinity.

-          L79, further description of RNA isolation and cDNA synthesis is required.

-          L113, what were the water characteristics?

-          L113, why was a PBS group used? Was it expected to cause changes in HO-1?

-           L118, was solution changed to maintain ammonia levels? In addition, why so high concentration of ammonia was used?

-          L119, why were 30 shrimps exposed if only 3 were used? Besides, how many replicates were used?

-          L120, how were these parallel experiments run? Were them exposed in the same tank? Were these 30 shrimps collected in triplicate? Review the experimental unit.

-          L121, more info is required for the qRT-PCR.

-          L130, how were tissues fixed?

-          L134, a quantification of histopathologic lesions should be included.

-          L135, further details are required for TUNNEL assay.

-          L146, which test was used to verify homogeneity of data?

-          L204, an image of the control group is missing. Also, a scale should be included in the figures.

-          L221, when there are no statistical differences between groups, there is no need to include letters (e.g. Fig 5A).

-          L240, quantification of apoptosis signals is required.

-          L267, L289, how do these results of chronic exposures (42 and 72 days) relate with the results obtained here with an acute exposure (3 days)?

Author Response

Thank you very much for your letter and the comments about our paper submitted to Diagnosis and Treatment of Disease in Fish. These comments are all valuable and helpful for improving our article. The manuscript was carefully revised by using red text according to the comments. We responded point by point to each comment as listed below, along with a clear indication of the location of the revision.

Changes for review issues are shown in red.

 Hope these will make it more acceptable for publication.

Thank you very much again.

Best wishes

Author Response File: Author Response.pdf

Round 2

Reviewer 1 Report

The authors have revised all the comments. 

Author Response

Thank you very much for your advice. 

Author Response File: Author Response.docx

Reviewer 2 Report

The authors have improved the manuscript. However, some details are still missing:

-       Regarding RNA isolation and cDNA synthesis, the information inserted is not sufficient to replicate the study. As such, further details need to be included or a reference for the method included.

Author Response

Thank you very much for your advice. We have revised it, and we have referred to the material method of RNA extraction and cDNA synthesis in the previously published articles “LECT2 Protects Nile Tilapia (Oreochromis niloticus) Against Streptococcus agalatiae Infection”, “SP protects Nile tilapia (Oreochromis niloticus) against acute Streptococcus agalatiae infection”. RNAiso Plus and PrimeScript™ RT reagent kit with gDNA Eraser (TaKaRa, Dalian, China) were used for the experimental operation. And all procedures are carried out in strict accordance with the kit instructions.

Author Response File: Author Response.docx