Aurantiochytrium sp. Meal as Feed Additive for Pacific White Shrimp Reared under Low Temperature and Challenged by WSSV in Association with Thermal Stress

Round 1

Reviewer 1 Report

In general, I consider that the Introduction section has sufficient information and relevance to the topic covered.

On the other hand, I suggest the following modifications to Materials and Methods for better understanding: 

The information provided in section 2.4 is adequate and important within the content of the article, however, I consider that this section could be relocated within or  right after section 2.1 since both mention proximal analyzes of the ingredients of the experimental diets.

In section 2.6, I suggest including a brief explanation of how the viral inoculum was prepared and more detail on the oral inoculation method used (forced-feeding, ad libitum or per os). 

I deeply appreciate a brief explanation on how the number of viral particles was calculated. Although the authors mention how they detected the presence of the virus in the WSSV inoculum, and in the organisms (Hi-PCR® WSSV Semi-Q PCR Kit) this methodology does not refer to viral quantification, only to detection. Also, I would appreciate more information about the kit.

Regarding the results, Table 5 and Figure 2 are a bit difficult to understand. Inevitably, looking at the values in the table and the very low deviation values, would make me assume that all the treatments present significant differences between them. I would appreciate it if the low standard deviation values with the non-significant differences presented were explained somewhere in the text.

At the end of the WSSV challenge, was a PCR test performed to verify that the shrimp were infected with WSSV? If so, I suggest including it in the results.

Finally, the experiment concludes that the 4% dose treatment showed the lowest mortality in shrimp after viral challenge with WSSV, however, the result of the 3% dose treatment requires further explanation. Assessing the concentration of viral copies in dead, dying and surviving organisms would be suitable to gather information and help explain the results of all the different treatments. Therefore I suggest that you consider it for future work.

Author Response

Dear reviewer,

We are grateful for your suggestions and have included the requested information in the text. We thank you for your careful review and suggestions, which have significantly improved our manuscript. We hope that we have met all the requirements and are open to new corrections if needed. Below you will find brief comments on your questions and information added to the manuscript.

- Section 2.4 was moved to 2.2, bringing it closer to the section that mentions -the analyzes of Aurantiochytrium meal, as suggested.

- In section 2.6, we added more information about the viral inoculum: a summary of its preparation and how the material was quantified.

- Table 5 had incorrect standard deviation values. The deviations had not been transformed into percentages. The correction has been made. We apologize for the error. We have included an illustration (Figure 1) to facilitate understanding of the WSSV challenge in our study.

- At the end of the WSSV challenge, dead animals were tested by PCR kit. The information was included in the text. The quantification with WSSV dosis in dead animals has already been performed in our laboratory in other studies. The pattern of results confirmed the doses used in the infection. Unfortunately, in the present studies we did not quantify the virus in dead animals, but only detected it with PCR kit in the post-infection and post-mortality. We appreciate the suggestion for future studies with the WSSV challenge.

Reviewer 2 Report

The present study aimed to evaluate how the shrimp P. vannamei juveniles receiving five dietary concentrations of Aurantiochytrium sp. in a clean water system, at 22 °C are affected in growth and in some immunological,  and microbiological parameters. The effect of  WSSV challenge to aniamls receiving differnet doses of this feed additive was alos tested.This is a very straight forward study which is of minor interest as a scientific report but is of interest for those engaged in farming shrimp and trying to get higher survival of shrimp to challenge with the deleterious WSSV. Thus this manuscript can be considered but this reviewer recommends it to be shortened primarily for this reason.

Lines 239-242  Please specify the details of the parameters tested and how many individual aniamls were used in each of the test.

The results reported give no or extremely small evidence that this feed additive has any effect of growth or bacterial load (Table 4 and Figuer 1) as well as on other parameters.

The experiemnt with WSSV challenge give some better effect . Noteworthy is that 1% is quite good compared to the higher concentrations used albeit 4% gave a higher survival rate.Is this a valid observation or is this probably because of few individuals used?

OK

Author Response

Dear reviewer,

We are grateful for your suggestions and have included the requested information in the text, which have significantly improved our manuscript. We hope that we have met all the requirements and are open to new corrections if needed. Below you will find brief comments on your questions and information added to the manuscript.

- Agree. We have, accordingly, included a detailed description about the animals and parameters (lines253-257)

- We agree with your comment that the effects of Aurantiochytrium on growth performance and bacterial counts are not statistically significant. We had a long discussion about the feed used and the possible factors that led to these results. And indeed, we were able to establish that the effects of the additive were only statistically noticeable when we exposed the animals to a viral challenge with WSSV and the associated thermal stress. However, the breeding conditions were 22ºC (atypical temperature for shrimp). For this reason, the authors believe it is important to maintain data on growth performance and gut bacterial counts, as the growth pattern of the shrimp and bacteria differs from that reported by other authors at ideal temperatures. For future studies at suboptimal temperatures, these data may be helpful in experimental design as they provide information on the growth of the species as well as the bacteria at 22 ºC.

- Regarding the 1% treatment having good results in immune parameters, it is a valid observation, we even mentioned it in Line 320 and Line 485-489. We understand that 1% was enough to cause some improvement in the shrimp's immune system, especially after the first phase of cultivation (9 weeks in 22 degrees, at clear water system). However, when we subjected the animals to challenges (WSSV and thermal stress) these results were not reflected in better survival and did not demonstrate statistically better results in any isolated phase or complete challenge. It may be that 1% of the inclusion of Aurantiochytrium meal is sufficient only for cultivation at low temperatures, without the occasion of WSSV challenge. Therefore, we chose to keep mentioning its immune performance, but without classifying this treatment prominently, considering the full scope of the study (with WSSV post-infection results).