Application of DNA Metabarcoding for Identifying the Diet of Asian Clam (Corbicula fluminea, Müller, 1774)
Round 1
Reviewer 1 Report
The authors investigated the applicability of DNA metabarcoding for the investigation of diet of freshwater macrobenthic bivalve Corbicula fluminea. The topic is highly relevant and interesting taking into account the rapidly increasing popularity of an idea to use DNA metabarcoding for monitoring, and since C. fluminea is known as “one of the more prolific freshwater invasive species in the world” (Weber et al., 2022 PLoS ONE 17(7): e0271402. doi.org/10.1371/journal.pone.0271402)
Several issues require more elaboration form the authors for the paper to become even more relevant and useful:
1. the current findings should be placed into the context of literature globally published on the topic,
2. a number of interesting technical and methodological details are missing and should be provided for readers to better understand what exactly was done and learn from this contribution,
3. and finally recommendations for the future studies could be refined.
L76-77 please briefly introduce the logic behind the idea to validated metabarcoding of potential Corbicula food using water-based metabarcoding data. Intuitively, this is not quite clear. Indeed, there is a growing need to validate the efficiency of DNA metabarcoding, but I would rather expect the validation of both potential food metabarcoding and water-based metabarcoding data using some more morphology-based data as reference, since such data is more straightforwardly interpretable.
Moreover, regarding samples for water-based metabarcoding - to study benthic species diet (main objective) obviously near-bottom sample of water should have being used, or, even better – both near-bottom and surface layer water samples – in comparison. This drawback is mentioned in the discussion shortly, but could receive some more attention: surface layer water masses may not even reach bottom layers if water column is strongly stratified.
Please specify water depth at each site.
Abstract:
L19-20: There are studies on diet of Corbicula (see and perhaps also use as reference Boltovskoy et al., 1995. Feeding selectivity of Corbicula fluminea (Bivalvia) on natural phytoplankton. Hydrobiologia 312 : 171-182). That is why additional aim or more detailed motivation/argumentation could also be provided here.
As main findings, specific dominant prey species or groups could be mentioned, instead of only just listing the numbers of OTUs detected. The overall role of those organisms in food web could further be highlighted.
L22: suggestion: “based on increasing salinity gradient”
L25: how is “total potential food” defined? An important concern I have is that if feeding is selective, and filtration is not, there can be non-digestible organisms identified, that are excreted/discharged b the clam afterwards without being consumed. Are they then not part of the diet/food? I think this require some clarification.
L27 Please describe in some details what could be suggested operationally as “methodological supplementation” (here and also in the discussion/conclusions). Those specific details and suggestions based on your experience with this investigation are extremely important for future studies and reuse.
Introduction: could provide little more details to the known diet specifications of Corbicula worldwide depending on the habitat and environmental settings.
L47 mentioning larval stage suggest the expectation that it was in focus here, which is a bit confusing. Weren’t only adult individuals of Corbicula sampled? Please specify.
L76: “sites exhibiting different salinity” could be more correct, since in this study only one snapshot is considered and no gradient/variability of salinity values at each site is documented.
Material and methods:
L80-81 remove “is one of the five major rivers in South Korea” – was said above
L88-89 is there vertical salinity gradient? Please describe: it could be more saline water masses at the bottom, as is common for the estuaries, due to compensating currents), or it it all fully mixed and no stratification is present? Please provide details on water depth/ level at each sampling site. What is the substrate type?
L91 NGS abbreviation – please give both the spelled-out version and the short form at the first use
L91 perhaps a naïve question, but can the filtration through a 0.45 μm influence the results of detected OTUs (i.e. remove some that were initially present in the sample)?
L96 suggestion: “… Vomit and Gut Contents…”
L97 how individuals were captured? What was the gear used?
L98 I am very curious how vomit content sample was received/extarcted, providing practical instructions or at least short details could help further studies. Since it is first mentioned in L75 before gut content, order could be consistent here.
L100 how exactly n=20 was distributed between two individuals from each site? By slicening/sectioning each gut?
L129 how the OTUs detected by not present in the NCBI reference database could influence the inferences of such study? Can it e.g. be issue with reference database, and not the reflection of natural diversity, that higher diversity was observed at lower salinity site?
Results:
L149-152 All abiotic parameters considered had higher values at site 2 than at site 1.
L156-160 what is the reason of higher paired-end reads in vomit content compared to gut content? Could be briefly mentioned in the discussion.
L160 Phred quality score – some reference/ detail on what that score is could be mentioned in the Methods
L164 Potential food were higher – reformulate – what is higher? Name a parameter/diversity/variable
L168 what could that mean? Is it larvae of this fish species that clams filter?
L180 there are no particular species data presented. Could perhaps species level be introduced to Fig 2? Figure2 label size could be increased. Nearly unreadable now.
L203 “that belong” – remove “was”
L193 and Caption Fig. 3 There seems to be some inconsistency: is it according to individuals or phyla? And what is actually the difference? What does is meant?
Fig. 3 Consider other scale for gut content or separate the figure e.g. to two plots above one other – now information for gut content is not really readable from this lower part of the plot (and according to Supplementary Materials there were many species and not only Chordata found in gut – how does it match?)
Table 2 shows higher values, i.e. higher diversity at site 2 – that contradicts to the sentence in the Abstract that site 1 is more diverse (L26). Please clarify
Discussion: the major point that require at least some attention in the discussion is how to combine morphological and DNA-based data to draw conclusions. Some practical advice and suggestions for monitoring, and clear documentation of gaps and shortcomings / challenges would be of great value!
L232 please mention and give some brief details on microscopy in the Methods section.
Again, isn’t it then vice versa that morphology/microscopy-based data could be used to evaluate applicability of NGS/DNA based methods – particularly when quantification is of importance.
Please highlight at least briefly an issue of how and if food and “bycatch” could be separated.
L251 suggestion “activity of these Bivalvia”
L253 What is length of variability ? variability of what?
One curiosity question – did water sample include Corbicula fluminea itself?
L280 “more complex“ than what? Perhaps add another sentence to clarify the point here.
Please refer to Supplementary material in the text
Is Table S1 based solely on two individuals?
To conclude: Conducting the study at two single sites does not allow much generality, and though I believe the data documented in the manuscript could be useful for larger audience of estuarine ecologists, the possible limitations for drawing out conclusions and formulating global inferences should be at least discussed. There is a needs of further development of knowledge about the use of DNA for quantification and assessment of biodiversity and its functions. Presented data is original and significant and should be published (after considering the points listed here).
Author Response
Response to Reviewer 1 Comments
The authors investigated the applicability of DNA metabarcoding for the investigation of diet of freshwater macrobenthic bivalve Corbicula fluminea. The topic is highly relevant and interesting taking into account the rapidly increasing popularity of an idea to use DNA metabarcoding for monitoring, and since C. fluminea is known as “one of the more prolific freshwater invasive species in the world” (Weber et al., 2022 PLoS ONE 17(7): e0271402. doi.org/10.1371/journal.pone.0271402). Several issues require more elaboration form the authors for the paper to become even more relevant and useful: 1. the current findings should be placed into the context of literature globally published on the topic, 2. a number of interesting technical and methodological details are missing and should be provided for readers to better understand what exactly was done and learn from this contribution, 3. and finally recommendations for the future studies could be refined.
Response: We thank you for your thoughtful suggestions and insights, which have enriched the manuscript and produced a more balanced account of the research.
L76-77 please briefly introduce the logic behind the idea to validated metabarcoding of potential Corbicula food using water-based metabarcoding data. Intuitively, this is not quite clear. Indeed, there is a growing need to validate the efficiency of DNA metabarcoding, but I would rather expect the validation of both potential food metabarcoding and water-based metabarcoding data using some more morphology-based data as reference, since such data is more straightforwardly interpretable. Moreover, regarding samples for water-based metabarcoding - to study benthic species diet (main objective) obviously near-bottom sample of water should have being used, or, even better – both near-bottom and surface layer water samples – in comparison. This drawback is mentioned in the discussion shortly, but could receive some more attention: surface layer water masses may not even reach bottom layers if water column is strongly stratified. Please specify water depth at each site.
Response: We have now explained why water-based metabarcoding was used to identify potential Corbicula fluminea prey species in this study by mentioning the importance of eDNA in species identification in an ecosystem (lines 91-96). We apologize for not including this information in the original manuscript. Water depth at each study site (line 106), as well as the depth from which water samples were collected (lines 108, 112), has been also specified.
Abstract:
L19-20: There are studies on diet of Corbicula (see and perhaps also use as reference Boltovskoy et al., 1995. Feeding selectivity of Corbicula fluminea (Bivalvia) on natural phytoplankton. Hydrobiologia 312 : 171-182). That is why additional aim or more detailed motivation/argumentation could also be provided here.
As main findings, specific dominant prey species or groups could be mentioned, instead of only just listing the numbers of OTUs detected. The overall role of those organisms in food web could further be highlighted.
Response: We apologize for not including additional aims in the study. Since very little is known about the feeding selectivity of C. fluminea in natural conditions, we believe our study is unique in that the potential food sources of C. fluminea from the Seomjin River (a native habitat) have been identified using DNA metabarcoding (a highly efficient technique). Moreover, prey species were identified from the pseudofaeces and gut contents of C. fluminea and the water of its natural habitat, providing an ideal comparison for future research. Additionally, as per your suggestion, we have added the dominant prey species identified from the pseudofaeces and gut contents (lines 24-26).
L22: suggestion: “based on increasing salinity gradient”
Response: We have corrected this phrase as suggested (line 24).
L25: how is “total potential food” defined? An important concern I have is that if feeding is selective, and filtration is not, there can be non-digestible organisms identified, that are excreted/discharged b the clam afterwards without being consumed. Are they then not part of the diet/food? I think this require some clarification.
Response: We completely agree with this comment, and the phrase “total potential food” has been removed and the sentence has been modified.
L27 Please describe in some details what could be suggested operationally as “methodological supplementation” (here and also in the discussion/conclusions). Those specific details and suggestions based on your experience with this investigation are extremely important for future studies and reuse.
Response: We have added the required information as suggested (lines 359-361, 370-372).
Introduction: could provide little more details to the known diet specifications of Corbicula worldwide depending on the habitat and environmental settings.
L47 mentioning larval stage suggest the expectation that it was in focus here, which is a bit confusing. Weren’t only adult individuals of Corbicula sampled? Please specify.
Response: Yes, the Corbicula individuals sampled in this study were only adults. We have modified the sentence for better clarity (lines 59-61).
L76: “sites exhibiting different salinity” could be more correct, since in this study only one snapshot is considered and no gradient/variability of salinity values at each site is documented.
Response: This has been modified as suggested (line 90).
Material and methods:
L80-81 remove “is one of the five major rivers in South Korea” – was said above
Response: This has been corrected as suggested (line 99).
L88-89 is there vertical salinity gradient? Please describe: it could be more saline water masses at the bottom, as is common for the estuaries, due to compensating currents), or it it all fully mixed and no stratification is present? Please provide details on water depth/ level at each sampling site. What is the substrate type?
Response: According to a previous study (Lim, Y., & Baek, S. H. (2017). Assessment of Phytoplankton Viability Along the Salinity Gradient in Seomjin River Estuary, Korea. Journal of the Korean Society of Marine Environment & Safety, 23(5), 513-523), saltwater is completely mixed downstream vertically and the salinity gradient appears horizontally upstream of the freshwater zone. We have provided the details on the water depth at each sampling site (line 106). The substrate type was sandy (line 106).
L91 NGS abbreviation – please give both the spelled-out version and the short form at the first use
Response: We apologize for this oversight. This is has been corrected in the revised manuscript (Line 115).
L91 perhaps a naïve question, but can the filtration through a 0.45 μm influence the results of detected OTUs (i.e. remove some that were initially present in the sample)?
Response: The 0.45 µm membrane filter size is the preferred choice for microbial recovery and colony morphology, which was concluded after comparing the effects of a combination of pore size range on colony size and recovery. It is believed that the membrane filter pore size did not affect the results.
L96 suggestion: “… Vomit and Gut Contents…”
Response: We completely agreed with the comment, and modified as suggested.
L97 how individuals were captured? What was the gear used?
Response: We apologise for not adding this in our original manuscript. The method and gears used for the Corbicula collection have now been included in the Materials and Methods section (lines 121-123).
L98 I am very curious how vomit content sample was received/extarcted, providing practical instructions or at least short details could help further studies. Since it is first mentioned in L75 before gut content, order could be consistent here.
Response: We have now added the details of extracting the vomit/pseudofaeces content in the Materials and Methods section (lines 129-143).
L100 how exactly n=20 was distributed between two individuals from each site? By slicening/sectioning each gut?
Response: 10 Corbicula individuals were collected from each study site for the experiment. We have modified the sentence now.
L129 how the OTUs detected by not present in the NCBI reference database could influence the inferences of such study? Can it e.g. be issue with reference database, and not the reflection of natural diversity, that higher diversity was observed at lower salinity site?
Response: Many OTU clustering methods have been proposed, most of which use a threshold of 97% sequence identity. Typically, this threshold is considered as a given rather than as a tunable parameter, following the conventional wisdom that 97% corresponds approximately to species. Accordingly, we analyzed the OTUs in this study.
Stackebrandt, E., & Goebel, B. M. (1994). Taxonomic note: a place for DNA-DNA reassociation and 16S rRNA sequence analysis in the present species definition in bacteriology. International journal of systematic and evolutionary microbiology, 44(4), 846-849.
Results:
L149-152 All abiotic parameters considered had higher values at site 2 than at site 1.
Response: We have now included this sentence “All abiotic parameters had higher values at site 2 than at site 1” in the results section (lines 209-210).
L156-160 what is the reason of higher paired-end reads in vomit content compared to gut content? Could be briefly mentioned in the discussion.
Response: This is probably because of more undigested prey in the vomit content than in the gut content. We have now included this information in the discussion part.
L160 Phred quality score – some reference/ detail on what that score is could be mentioned in the Methods
Response: Base calling accuracy, measured by the Phred quality score (Q score), is the most common metric used to assess the accuracy of a sequencing platform. For example, if Phred assigns a Q score of 30 (Q30) to a base, this is equivalent to the probability of one incorrect base call in 1000 times. Low Q scores can increase false-positive variant calls, which can result in inaccurate conclusions and higher costs for validation experiments. We have now included this information in Results (lines 218-223).
L164 Potential food were higher – reformulate – what is higher? Name a parameter/diversity/variable
Response: We have modified the sentence.
L168 what could that mean? Is it larvae of this fish species that clams filter?
Response: Interestingly, we found OTUs from O. mykiss, which is a fish, in C. fluminea individuals from the two sites even though C. fluminea cannot consume O. mykiss directly. Although we could identified the fish species, it was difficult to distinguish between adult and larval stages of the fish through DNA barcode analysis. Therefore, we are not sure of if C. fluminea feeds on the fish larvae specifically. We and have now included this information in the discussion part (Line 315-330).
L180 there are no particular species data presented. Could perhaps species level be introduced to Fig 2? Figure2 label size could be increased. Nearly unreadable now.
Response: We completely agree with the reviewer’s comment and have increased of Figure 2 label size and added a table with species-level information Table 2
L203 “that belong” – remove “was”
Response: We have accepted and corrected this as suggested.
L193 and Caption Fig. 3 There seems to be some inconsistency: is it according to individuals or phyla? And what is actually the difference? What does is meant?
Response: We mean according to phyla.
Fig. 3 Consider other scale for gut content or separate the figure e.g. to two plots above one other – now information for gut content is not really readable from this lower part of the plot (and according to Supplementary Materials there were many species and not only Chordata found in gut – how does it match?)
Response: We completely agree with this comment and modified the sentence as suggested.
Table 2 shows higher values, i.e. higher diversity at site 2 – that contradicts to the sentence in the Abstract that site 1 is more diverse (L26). Please clarify
Response: This has been corrected.
Discussion: the major point that require at least some attention in the discussion is how to combine morphological and DNA-based data to draw conclusions. Some practical advice and suggestions for monitoring, and clear documentation of gaps and shortcomings / challenges would be of great value!
L232 please mention and give some brief details on microscopy in the Methods section. Again, isn’t it then vice versa that morphology/microscopy-based data could be used to evaluate applicability of NGS/DNA based methods – particularly when quantification is of importance. Please highlight at least briefly an issue of how and if food and “bycatch” could be separated.
Response: The information related to microscopy was included in the method. The usefulness of microscopic examination in quantification was described in the discussion.
L251 suggestion “activity of these Bivalvia”
Response: We completely agreed with the comment, and modified as suggested (line 329).
L253 What is length of variability ? variability of what?
One curiosity question – did water sample include Corbicula fluminea itself?
Response: The water sample contained Corbicula fluminea individuals.
L280 “more complex“ than what? Perhaps add another sentence to clarify the point here. Please refer to Supplementary material in the text
Response: Food web may be more complex. We completely agree with this comment and modified the sentence accordingly (line 370).
Is Table S1 based solely on two individuals?
Response: Ten individuals were examined at each study site. Microscope and NGS analysis were conducted for the same individual. The content has been revised.
To conclude: Conducting the study at two single sites does not allow much generality, and though I believe the data documented in the manuscript could be useful for larger audience of estuarine ecologists, the possible limitations for drawing out conclusions and formulating global inferences should be at least discussed. There is a needs of further development of knowledge about the use of DNA for quantification and assessment of biodiversity and its functions. Presented data is original and significant and should be published (after considering the points listed here).
Reviewer 2 Report
General Comments
This paper reports on an interesting proof-of-concept study evaluating the potential for DNA metabarcoding to aid in diet analysis of bivalves. Replication was low, but the study still provides useful information especially given that the technique is novel. I found the Introduction to be well written aside from some minor wording issues. (These wording issues appear throughout the text, so some careful editing is in order.) The Methods are sound, but they currently lack important detail required to replicate the study. I provide suggestions for addressing this below. I found the Results section to be hard to follow. It would benefit from a careful reorganization and more precise wording. Perhaps reporting the results in named sections that match sections in the Methods would help. I also feel that the microscopy efforts reported in the Discussion belong in the Results. I provide some more detailed suggestions below. Overall, this is an interesting paper that will be useful and of interest to many in the field.
Detailed Comments
L81: Please reference Fig. 1 here.
L90: Spell out NGS at first mention.
L85-101: Please describe how you collected and transported the water samples, how much water was filtered, how you collected the animals, how you collected the vomit, and how many animals were collected.
L97: “preserved in” isn’t the correct word here as water does not preserve DNA. “Placed into” would be more appropriate.
L98: Stored for how long?
L98: I’m not a bivalve expert but I believe the “vomit” is more appropriately termed “pseudofeces”.
L100: I don’t follow how the sample size is n=20 if there were “2 individuals from each study site” and there were only 2 study sites.
L104: Should you spell out the “g” in gDNA?
L104: You say you used 2 regions but then only describe the use of only one region (v9)
L111: I think “taxonomic diversity” would be a more appropriate phrase here.
L106-110: I don’t think you need to justify why you didn’t use COI. But a citation supporting the validity of v9 (i.e., the “broad range…” claim) would be helpful.
L123-124: Citations are needed for software listed here
L126: What software was used to cluster? Be sure it is clear which software was used to perform each task (with citation) and which functions where appropriate.
L131: Is “uncultured” the correct term here?
L135: Please clarify that “abundance data” refers to read counts.
L137-142: Please clarify these descriptions of the 2 diversity indices. As written, they are not very informative or precise.
L145: I recommend the term “water chemistry” instead of “water quality”
Table 1: Again, I recommend the term "water chemistry" in the caption.
L164: I recommend deleting the first sentence here. It isn’t supported.
L167: Interesting! Are these mites known to be aquatic?
L169: does this mean the proportion of reads? Please clarify.
L170: What is a similarity cutoff? I was confused by this mentioning of “species” before taxonomy assignment was discussed. Can this be clarified?
L172: Isn’t this already stated in the Methods?
L162-204: I think this entire section needs to be reorganized so that it is clear what results are being reported in each section.
L212: The main difference seems to be between sites seems to be in the vomit. The gut contents have similar diversity between locations. Also, with so little replication, you cannot really say definitely that the diversity differs. Please be careful how you word this and what conclusions you draw from this, both in the Results and the Discussion.
L220: More diverse than what? Please clarify.
L230-238: I think these microscopy results should be reported in the Results section.
Author Response
Response to Reviewer 2 Comments
This paper reports on an interesting proof-of-concept study evaluating the potential for DNA metabarcoding to aid in diet analysis of bivalves. Replication was low, but the study still provides useful information especially given that the technique is novel. I found the Introduction to be well written aside from some minor wording issues. (These wording issues appear throughout the text, so some careful editing is in order.) The Methods are sound, but they currently lack important detail required to replicate the study. I provide suggestions for addressing this below. I found the Results section to be hard to follow. It would benefit from a careful reorganization and more precise wording. Perhaps reporting the results in named sections that match sections in the Methods would help. I also feel that the microscopy efforts reported in the Discussion belong in the Results. I provide some more detailed suggestions below. Overall, this is an interesting paper that will be useful and of interest to many in the field.
Response: We thank you for your thoughtful suggestions and insights, which have enriched the manuscript and produced a more balanced account of the research.
Detailed Comments
L81: Please reference Fig. 1 here.
Response: We have referenced “Figure 1” for the sentence “the Seomjin River is located in the southwest of the Korean Peninsula” (lines 99).
L90: Spell out NGS at first mention.
Response: This has been corrected (line 116).
L85-101: Please describe how you collected and transported the water samples, how much water was filtered, how you collected the animals, how you collected the vomit, and how many animals were collected (Line 112-117, 122-130, 132-147).
Response: We have described the methods for collecting and transporting the water samples (lines 105-113), the quantity of water that was filtered (line 114), and the methods of animal collection (lines 121-127) as well as pseudofaeces extraction (lines 129-131). We didn't focus on the number of animals we collected, because the study did not aim for quantitative collection. However, we selected 20 individuals from the collected specimens (10 from each study site) for our experiment.
L97: “preserved in” isn’t the correct word here as water does not preserve DNA. “Placed into” would be more appropriate.
Response: We completely agree with this comment and have made the required changes (line 129).
L98: Stored for how long?
Response: The individuals were stored at 4 °C for 48 h (line 130).
L98: I’m not a bivalve expert but I believe the “vomit” is more appropriately termed “pseudofeces”.
Response: Thank you making this suggestion. We have modified the terminology.
L100: I don’t follow how the sample size is n=20 if there were “2 individuals from each study site” and there were only 2 study sites.
Response: We apologize for this confusion. 10 individuals from each study site were used for the experiment. Thus, there were 20 individuals in total. We have modified the sentence accordingly.
L104: Should you spell out the “g” in gDNA?
Response: We completely agree with this comment; however, we believe “DNA” is more appropriate here than “genomic DNA” (line 151).
L104: You say you used 2 regions but then only describe the use of only one region (v9)
Response: The two regions refer to the reverse and forward primers of 18S v9. We have modified the sentence accordingly.
L111: I think “taxonomic diversity” would be a more appropriate phrase here.
Response: We completely agree with this comment and have made the required change (line 162).
L106-110: I don’t think you need to justify why you didn’t use COI. But a citation supporting the validity of v9 (i.e., the “broad range…” claim) would be helpful.
Response: We have now included the references of previous studies supporting the use of v9 (Line 153-156).
L123-124: Citations are needed for software listed here
Response: We have added the citations for the software listed in the study (lines 174-185).
L126: What software was used to cluster? Be sure it is clear which software was used to perform each task (with citation) and which functions where appropriate.
Response: UCLUST was used for clustering. We have added the names of software used for performing each task (lines 174-181).
L131: Is “uncultured” the correct term here?
Response: We have changed “uncultured” to “Non -assigned” (line 183).
L135: Please clarify that “abundance data” refers to read counts.
Response: We have clarified this (line 190).
L137-142: Please clarify these descriptions of the 2 diversity indices. As written, they are not very informative or precise.
Response: “Among the most popular metrics used to quantify diversity are Shannon index, believed to emphasize the richness component of diversity, and Simpson’s index, emphasizing the evenness component [54]. Simpson’s index, which considers the number and abundance of species, was used to quantify habitat biodiversity. A greater value of Simpson’s index indicates higher potential food diversity. Shannon index considers the number of individuals as well as the number of taxa. It varies from 0 for communities with only a single taxon to high values for communities with many taxa, each with few individuals.” We have added these descriptions for the diversity indices.
L145: I recommend the term “water chemistry” instead of “water quality”
Table 1: Again, I recommend the term "water chemistry" in the caption.
Response: We have changed “water quality” to “water chemistry” (lines 201, 211).
L164: I recommend deleting the first sentence here. It isn’t supported.
Response: We have removed this sentence.
L167: Interesting! Are these mites known to be aquatic?
Response: The species of mites was collected from a moss (Sphagnum sp.) in a seepage area We have now included this in the discussion part.
L169: does this mean the proportion of reads? Please clarify.
Response: We have reorganized this portion for better clarity.
L170: What is a similarity cutoff? I was confused by this mentioning of “species” before taxonomy assignment was discussed. Can this be clarified?
Response: We have removed “species” from this part.
L172: Isn’t this already stated in the Methods?
Response: We have removed this portion from the Results.
L162-204: I think this entire section needs to be reorganized so that it is clear what results are being reported in each section.
Response: We have reorganized this portion for better clarity.
L212: The main difference seems to be between sites seems to be in the vomit. The gut contents have similar diversity between locations. Also, with so little replication, you cannot really say definitely that the diversity differs. Please be careful how you word this and what conclusions you draw from this, both in the Results and the Discussion.
Response: We have revised the phrasing in the discussion part (lines 365-373).
L220: More diverse than what? Please clarify.
Response: We meant that metabarcoding allows the identification of diverse potential food sources (lines 291-2912).
L230-238: I think these microscopy results should be reported in the Results section.
Response: We have now included the microscopy data in the Methods as well as Result part.
Round 2
Reviewer 2 Report
This manuscript is much improved over the initial submission I reviewed. However, I still think it needs a careful read-through to improve the flow of the language. I also noticed some issues with two of the figures. Overall, I’m very happy with the reorganization and added detail. The changes have markedly improved the quality of this useful paper.
L28-29: I’m still not sure what “methodological supplementation” is and why it is a logical future direction for the research.
L34: replace “highest productive” with “among the most productive”
L59: replace “invertebrate species” with “invasive bivalves” as it seems strange to switch from talking about invasive bivalves to all invertebrates
L187: change “an important tool” to “a tool”
L246: I think some Phyla are missing from the legend of Figures 2 and 3. For example, I see no label for “Arthropoda” even though it is the main taxa in the clam samples.
Table 3: Please indicate in the legend what units these numbers are in. Also, I don’t think they need decimals if they all end in “.00”.
L302-303: This sentence needs to be reworded. Please do a careful grammar check throughout before resubmitting.
L346-347: “…and both the genus and species were first recorded from 346 South Africa, where it was collected from a moss (Sphagnum sp.) in a seepage area…”: The location of collection of the type specimen for this mite does not seem relevant to mention here.
L364: change “difference in salinity gradient” to “difference in salinity”
L366-367: I’m not sure what this means: “…our data lacked any of the high-throughput sequence analysis available currently.” Please clarify.
Author Response
Response to Reviewer 2 Comments
This manuscript is much improved over the initial submission I reviewed. However, I still think it needs a careful read-through to improve the flow of the language. I also noticed some issues with two of the figures. Overall, I’m very happy with the reorganization and added detail. The changes have markedly improved the quality of this useful paper.
Response:
Thank you for giving me the opportunity to work on your response letter. We appreciate your positive consideration of this paper. We have done our best to revise this paper according to the valuable comments from reviewer.
L28-29: I’m still not sure what “methodological supplementation” is and why it is a logical future direction for the research.
Response:
We completely agree with this comment. We changed it. Please see the line 27-29.
L34: replace “highest productive” with “among the most productive”
Response:
Thank you making this suggestion. We have corrected this phrase as suggested.
L59: replace “invertebrate species” with “invasive bivalves” as it seems strange to switch from talking about invasive bivalves to all invertebrates
Response:
Thank you making this suggestion. We have corrected this phrase as suggested.
L187: change “an important tool” to “a tool”
Response:
Thank you making this suggestion. We have corrected this phrase as suggested.
L246: I think some Phyla are missing from the legend of Figures 2 and 3. For example, I see no label for “Arthropoda” even though it is the main taxa in the clam samples.
Response:
In the analysis result, only clam samples (C. fluminea) belonged to Arthropoda. This is judged as individual information and the OTU of C. fluminea (clam samples) was removed and analyzed.
Table 3: Please indicate in the legend what units these numbers are in. Also, I don’t think they need decimals if they all end in “.00”.
Response:
We have added the required information as suggested and have modified the Table.
L302-303: This sentence needs to be reworded. Please do a careful grammar check throughout before resubmitting.
Response:
English proof-reading has been done before re-submission of the manuscript.
“We performed microscopy in parallel to assess the applicability of morphological methods in analysing the PF and gut contents.”
L346-347: “…and both the genus and species were first recorded from 346 South Africa, where it was collected from a moss (Sphagnum sp.) in a seepage area…”: The location of collection of the type specimen for this mite does not seem relevant to mention here.
Response:
We completely agree with this comment, and the phrase has been removed and the sentence has been modified.
L364: change “difference in salinity gradient” to “difference in salinity”
Response:
Thank you making this suggestion. We have corrected this phrase as suggested.
L366-367: I’m not sure what this means: “…our data lacked any of the high-throughput sequence analysis available currently.” Please clarify.
Response:
We would like to convey the implication that there were fewer paired-end reads identified in gut contents compared to the PF content of corbicula. We completely agree with this comment, and have modified the sentence for better clarity
