Evaluation of the Viral Diversity of Artemia Cysts from Saline Lakes in Kazakhstan Using Viral Metagenomics Analysis
, Kobey Karamendin 1, Zhanara Mazhibayeva 3, Yermukhammet Kassymbekov 1, Temirlan Sabyrzhan 1,2, Kuanysh Isbekov 3, Saule Assylbekova 3 and Aidyn Kydyrmanov 1,*Round 1
Reviewer 1 Report
In this study, the authors conducted a metagenomic analysis of Artemia (brine shrimp) cysts collected in salt lakes in various regions of Kazakhstan. The data obtained undoubtedly expand our knowledge of the diversity of viruses in various aquatic organisms. On the whole, the manuscript makes a good impression. However, there are questions that I would like to clarify. In addition, I have a number of comments that I hope will help the authors improve the text and the general understanding of their results.
L. 66-68, 401 – How do your results reveal the viral dynamics in salt lakes?
Figure 1 – Sign the map of Kazakhstan (center). Complete the figure (right) with a list of sampling sites identified as numerals.
Section 2.1 – Include the details of sampling and pre-treatment of the samples prior to nucleic acid extraction.
L. 97 – When homogenizing, you used lysis buffer. How sure are you that the virus particles were not damaged by this?
L. 132-139 – What do you mean by "the reads" here - original reads or assembled sequences (contigs)?
Table S2 - What do the numbers in the tables mean?
Table 1 – I am confused by the low values of the minimum and average length of the contigs. It seems that many reads remained unassembled. Specify what was the length of the raw reads, paired or unpaired?
L. 186-193 – Unnecessary repetition of tabular data. Present summarized data.
L. 199 - Do you mean "reads" or "assembled sequences"? Check the text carefully to make sure there are no inaccuracies.
L. 204-206 – Rewrite this sentence. The meaning is not clear.
L. 227 – Specify that “one of these families” was unclassified Caudovirales (Table S3).
L. 234-236 – Specify here which family.
L. 242 – It would be better to replace "viruses" with "viral contigs" since you are dealing with incomplete genomes, right?
Figure 2 – Move Figure 2b to Figure 4a and merge as one Figure 2(a,b). Do the same with figure 4. In the caption to the Figure 2(b) decipher the abbreviation “UN”.
L. 303 – Rewrite “Artemia reo-like virus belonged to the unclassified Reoviridae family” for “Artemia reo-like virus belonged to the unclassified Reoviridae”.
L. 331 – Also rewrite “clustering with other viruses belonging to the CMNV”.
L. 353, 359 – Figure 5a?
Figure 5 – Generate a single tree for dicistro-like viruses, to show relationships of Artemia dicistro-like virus 1 and Artemia dicistro-like virus 2.
Table S4 – Add a column with numbering/designation of contigs.
Discussion – Here it is better to remove the subheadings-families (Reoviridae and others).
L. 402 – “The seven viral families” Here it is appropriate to clarify which virus families.
Have other virome studies (of water samples or aquatic organisms) been carried out in the salt lakes of Kazakhstan or other countries? In any case, this should be mentioned in the text of the manuscript.
Add a graphic representation of the contigs (Artemia viruses) you described, indicating the identified ORFs; possibly along with closely related viruses.
The English translation is quite competent and understandable.
Author Response
Please see the attachment.
Author Response File: 
Author Response.pdf
Reviewer 2 Report
Firstly, I have doubts whether the article falls under the scope of Special issue Infections of Aquatic Animals. Especially since it was determined that this Special Issue will promote original knowledge about marine biology, while also relating to marine ecology and public health issues. However, the decision to accept the article to SI belongs to the editor Dr. Danny Morick.
I have a few comments that may help improve the manuscript:
The Introduction lacks broader information about what pathogens Artemia carries, and what other species living in these lakes may be threatened by them. Considering the Special Issue scope, have there been confirmed cases of viral infections in these lakes in other aquatic animals? Please describe it in more detail. In several places, Artemia should be capitalized by analogy with the rest of manuscript eg. in lines 57,59.
In Materials and Methods, please describe the methods used to collect Artemia samples. How were the cysts separated from the lake water, in what conditions were they transported to the laboratory? How long did it take to start the analysis? In the case of metagenomic analyses, proper conservation of the research material is crucial, there is no information to determine whether the procedure was appropriate. The Supplementary Table S1 lacks information about the salinity and temperature of the water during sampling. This is crucial information due to the shortened survival of viruses in salt water and high temperature. Why August was chosen as the sampling month, please explain in the manuscript.
I have a few comments for the Discussion and Conclusions also.
In the Discussion, issues related to environmental conditions (temperature and salinity) and ecological conditions, e.g. the presence of other species in the studied reservoirs, should be supplemented, due to the fact that, for example, the Yellow Head Virus mentioned in the line was not found, but is highly species-specific. Are there Penaeus monodon shrimp farms nearby? is it possible to transfer the virus, e.g. by birds to these lakes, that the authors are surprised that there is no this virus in Artemia samples? Please take into account the ecological aspect of the food chain relationship between the species existing in the lakes and Artemia and discuss the results obtained in this respect.
In lines 499-501 the authors mention the differentiation of results due to various factors, e.g. salinity, temperature and sample size. Since nothing is known about temperature and salinity, it is difficult to say whether this fact applies to the results obtained.
And what effect does sample size have on which viruses?
In Conclusion: Artemia - capital letter (line 513) or Artemia cysts?
Also, there are no specific conclusions in Conculsions. This should be corrected by referring to the hypothesis, the purpose of the research, which should be clearly specified in the Introduction and should be proven by the Results and Disscusion.
Author Response
Please see the attachment.
Author Response File: Author Response.pdf
Round 2
Reviewer 1 Report
The authors took into account my comments and recommendations and made appropriate additions and clarifications. However, a few minor comments still remain.
L 131-133: Indicate the length of the reads (or the kit used, 2x250 or 2x300).
L 245-246: Remove "one of these families remains", or replace "families" with "group".
Figure 1: Enlarge the block with the names the sampling sites. Enlarge also the font in Figure 2. This will make the images easier to read.
Present the reference genomes In Figures 3-6(b) more fully, supplementing them with gene/domain designations. Also increase their resolution.
The English translation is quite competent and understandable.
Author Response
Please see the attachment.
Author Response File: Author Response.pdf
Reviewer 2 Report
The authors have satisfactorily addressed most of my concerns. In particular, the authors have improved and supplemented the Discussion and Materials and methods. The only remaining concerns I have with the abstract are seemingly minor. In lines 20-21 authors wrote: "It has also furnished crucial insights for the identification, surveillance, and management of viral infections that impact brine shrimp and fish species populations."
In my opinion, the results in the manuscript say nothing about the surveillance and management of viral infections affecting populations of brine shrimp and fish species, since no impact of the viruses detected on the Artemia population was demonstrated. There are no results from, for example, of different seasons and no evidence that the occurrence of the identified viruses may affect population dynamics.
I would suggest correcting this expression in Abstract.
Author Response
Please see the attachment.
Author Response File: Author Response.pdf
