Morphology and Genetic Structure Profile of Farmed Snails Cornu aspersum aspersum and Cornu aspersum maximum in Greece
Round 1
Reviewer 1 Report
The manuscript "Morphology and genetic structure profile of farmed snails Cornu aspersum aspersum and Cornu aspersum maximum in Greece" enters the topics of the journal. In this study, the utility is verified.
The study deepens the knowledge of two species and 8 subpopulations of snails distributed in different ecosystems of Greece. In this research, the knowledge of the polymorphism of snails of the two subspecies using morphological and molecular markers was deepened. Snails were categorized according to the shell colour and bands. three clusters among snail populations were obtained and six loci (Ha5, Ha6, Ha8, Ha9, Ha10 and Ha11) were tested for their diversity. The results showed a common gene pool brood stock in Greece.
It is recommended to clearly indicate the objective of the research and the justification for the study. The title of the paper should be included in the objective.
Currently two species appear in the title, then it seems that they analyze 8 subpopulations, which also come from 7 farms with different systems.
The axis of the work should be the two species that are characterized by quantitative morphometric characters and other qualitative meristic characters. Later the polymorphisms are used to see its genetic structure.
Comments:
The manuscript says:
According to the sampling procedure, a total of 160 snail specimens of the two infraspecific taxa of the species C.aspersum [7]. 4 Cornu aspersum aspersum and 120 snails Cornu aspersum maximum were collected from 7 snail farms located in Greece (Figure 1).
I think it refers to 40 specimens of Cornu aspersum maximun
I recommend that with the morphometric and meristic measurements of all the snails (the 160). An ACP and cluster could be done so that we can see how they are grouped.
Another different analysis would be to see the variables with the greatest power to discriminate between species. To do this, with all the morphometric and meristic measurements, make a discriminant, seeing which variables show the greatest discriminant value and serve to differentiate the species.
If we want to work with populations (8) we have few data, but it also escapes the objective of the work
The relevant factor is the species and that is how the work should be conducted. The separation by farms (populations) Figure 2, 3 and 4 is not very relevant and could be eliminated from the work.
The sample size to measure morphometric and meristic variables is very low. In methodology we talk about color, etc. and in no case are the patterns shown. The existence of band patterns on the shell is indicated, which are also not present. Snails have different ages, breeding systems, etc. That may explain the morphometric differentiation.
In Figure 5, I would like to see in each of the components which variables are the ones that affect it.3
Delete Figure 6, Table 2, and Figure 7. Irrelevant. One comment is enough.
In the discussion, point out what is relevant, what is related to the objective of the work. For example, when it says that if we start from wild populations, the successive generations decrease in size, it is not correct. we and other authors have obtained the opposite whenever it is a snail in suitable breeding conditions
It is a good job and I encourage its correction. Differentiating species and analyzing genetic variability is of great importance. But you have to focus the work according to the objective. I think that this paper is very promising.
Author Response
We would like thank the Reviewer for his/her comments. The responses are included in the file.
Author Response File: 
Author Response.pdf
Reviewer 2 Report
REFEREE´S COMMENTS ON
Manuscript I.D.: sustainability-1981208
Manuscript Title: Morphology and genetic structure profile of farmed snails Cornu aspersum aspersum and Cornu aspersum maximum in Greece.
Corresponding author: Efkarpia Kougiagka.
Review Assigned Date: October 7, 2022
Review Due Date: October 20, 2022
GENERAL COMMENTS
This manuscript (ms) aims to investigate genetic and morphological profiles of two snails of commercial interest in Greece. Despite I am not expert in snails, being my area of expertise related to fish, a number of questions came to my mind, e.g. I could not infer what is the actual contribution of this ms to the current state of knowledge; I also wondered what aspects do this research improve in relation to the current farming procedures of these snails species; what aspects are novel compared to what has been done to date; in summary, why is this study worth. None of these questions are clearly answered, neither in the abstract nor in the introduction.
However, what really concerns me about this ms the poor English writing used throughout the different sections, and the lack of consistency in the terminology used. For instance, the first sentence in the Abstract is grammatically unaccepted. I will not go any further with more examples but I strongly encourage the authors to deeply review the English grammar before any further submission, and before entering into any scientific aspects related to the novelty of their study. Probably, they should forward the document to any trustworthy proofreading service before any resubmission. It is up to the authors to amend the entire manuscript for a second opinion.
In its current form, I could not read the introduction section with fluidity and it was so difficult to me to grab clear ideas. This section is extremely longwinded and imprecise, so many paragraphs seem to fall away from the specific topics that are supposed to be tackled. The different paragraphs are unconnected and in some cases repetitive lacking of clarity about what is the main objective of the work. No matter if the goal of this study is presented in the discussion section, it should also be posted clearly in the introduction. This reader simply gets tired, bored and finally lost after reaching the fourth paragraph of the introduction. I strongly suggest focusing on the specific theme to be tackled without moving forward into any irrelevant topics such as those related to fish, sheep, etc. This is not an introductory section of a book but it should be the introduction of a scientific paper. Please summarize the current state of the situation and state the intent of your study outlining the key characteristics and what is supposed to be the actual contribution/originality of this study. Do not go beyond these aspects.
In its actual state I cannot go any forward before it is amended so that it conforms to correct grammar and proper structure. Hence, I will not deep into minor aspects as I consider the aforementioned major aspects fatal. The authors are encouraged to amend those major comments before going into any other issues.
Author Response
We would like to thank the Reviewer for his/her comments. The responses are included in the file.
Author Response File: Author Response.pdf
Reviewer 3 Report
Please consider the improvement of the language quality in a few instances and the clarity of the statements in the materials and methods section. Please improve the quality of the figures as mentioned in the reviewed manuscript.
See the marked pdf copy.
Comments for author File: Comments.pdf
Author Response
We would like to thank the reviewer for his/her comments. The responses are inclunded in the file.
Author Response File: Author Response.pdf
Reviewer 4 Report
1. The Introduction has low coherence between paragraphs. Authors should make subtitles or link the paragraphs in some way. Especially the last one.
2. Line 126 “4 Cornu aspersum aspersum and 120 snails Cornu…”. Apparently it means 40 snails Cornu aspersum aspersum.
3. The name of the species in full form should be indicated at the first mention in the text. In the following, the abbreviated form is used (C.a.aspersum, C.a.maximum).
4. Authors should add photos of different morphs of C. aspersum to illustrate them.
5. The sequences of the microsatellite markers used in the analysis should be given.
6. "Post-hoc comparisons were performed using the Tukey test." Before that, it was written that the authors used "t-test and Bonferroni corrections". This test with Bonferroni correctionsis also used specifically for post-hoc comparisons. It is not clear why use two tests to solve one problem. We need clarification here.
7. In the “Results” section, data about generators of snail farms is given - this information should be in the Materials and Methods section, because these are not research results.
Also authors must explain how these data were used in the analysis (it seems to me that in no way).
8. In M&M, authors must indicate the use of PCA to solve certain goals.
9. The caption to Table 1 says “For C.a.aspersum data t-test and for C.a.maximum one-way Anova followed by Tukey test and Kruskal-Wallis test were performed.”
It is not clear which version of the analysis of variance was used: parametric or nonparametric (Kruskal-Wallis test)? If both options, in which cases which one?
If only the Student's test was used for the subspecies C.a.aspersum, does this mean that the Bonferroni correction was applied here, as indicated in the M&M? If so, why? because there are only two samples and correction for multiple comparisons is not needed here. If not, why is it listed in M&M?
10. In table 1 there are indices "a", "b", "c", but there is no decoding - what do they mean?
11. The same applies to the row "Statistics" in table 1 - the values of which of all the criteria used by the authors are given here? Apparently, this is only the result of ANOVA or Kruskal-Wallis test. Then where are the results of pairwise comparisons (Tukey's test) for С.a.maximum?
12. Table 2 is not very informative. Such information is best presented graphically. However, in Figure 6, as far as I can see, this information is actually presented. Not as a percentage, but apparently in absolute terms and in shares of the total number of snails. But in this figure it is not clear where which subspecies of snails is. The explanation says that this is indicated by asterisks. But only two populations have asterisks. It is better to depict subspecies of snails in the form of two different shapes (for example, a circle and a square or something else).
13. The title of Figure 6 needs to be changed. "Map of Greece showing the shell pattern..."is not appropriate. This is not a map of Greece. “Distribution of shell patterns…” or “Map of pattern distribution… among studied populations” would be more correct.
14. Describing Table 5, the authors write “C.a.aspersum the farmed populations OMR snails showed the higher HOBS value for all loci while FIS for all loci of KNT1 snails was higher than OMR snails. Locus Ha6 presented the higher number of alleles (23) and the higher HOBS (0.650)". Are the differences between populations statistically significant?
15. Also from table 5: it is important to compare HEXP HOBS. First, whether they differ statistically significant. Secondly, it is quite possible that some populations have HOBS values closest to HEXP.
16. What does "observed heterozygosity of locus Ha5 had a strong relationship with biotic load" mean in line 143? What is "biotic load"? How is it measured?
17. Lines 210-211 "darker shells are observed in habitats with colder climates, while the lighter shells are observed in habitats with warmer climates". In this research, the relationship between temperature and shell color was not studied, so this statement is speculative. Moreover, on line 224 the authors write “As the environment of all farms did not present differences…”
18. Lines 256-257 "In our data, for both farmed subspecies the observed heterozygosity of all populations of both species was lower than the expected" - where were these differences shown to be statistically significant?
19. Lines 274-275 "In our study, all populations of C.a.maximum snails were larger in size and weight than the C.a.aspersum populations." Have populations of different subspecies been compared? Populations within subspecies were compared.
20. Lines 275-276 "Farmed and wild C.a.aspersum snails had no significant differences in weight and shell morphometric characteristics". Where is the data on wild populations of C.a.aspersum? They are not included in the study at all.
21. If the conclusions refer only to genetic variability, then why was the study of morphological variability and variability of shell color carried out?
22.Also in the conclusions “This fact was reinforced by the obtained haplotype network” - which haplotype network is meant? The study did not construct any haplotype network.
Author Response
We would like to thank the Reviewer for his/her comments. The responses are included in the file.
Author Response File: Author Response.pdf
Round 2
Reviewer 1 Report
1) In your answer the objective of the paper has been clearly described. I ask that this clarity and conciseness of the language be incorporated into the work (before the material and methods)
Our goal was to investigate the morphological polymorphism and describe for the first time the genetic structure of farmed snails which are a (NO. famous) food product consumed worldwide (22, 53). Th1e results obtained in this research is a part of a research project focused on snail farming in Greece. Our goal is to investigate the snail farms and the factors affecting snail quality. The snails Cornu aspersum aspersum and C.a.maximum used for polymorphism study are the main farmed subspecies in Greece (22,53).
2) I suggest that you justify in the article that the sample size is sufficient for the eight populations. I suggest you include a quote that reinforces it
I think that the authors have made a great effort to improve the article, answering the main questions.
Congratulations, now I think it is suitable for publication
Author Response
We would like to thank the reviewer. Our answer is in the file.
Author Response File: Author Response.pdf
Reviewer 4 Report
The authors improved the manuscript in accordance with the comments in the previous review.
Author Response
We would like to thank the reviewer. Our response is in the file.
Author Response File: Author Response.pdf
