Gut microbiota of an Amazonian fish in a heterogeneous riverscape: integrating genotype, environment, and parasitic infections

ABSTRACT A number of key factors can structure the gut microbiota of fish such as environment, diet, health state, and genotype. Mesonauta festivus, an Amazonian cichlid, is a relevant model organism to study the relative contribution of these factors on the community structure of fish gut microbiota. M. festivus has well-studied genetic populations and thrives in rivers with drastically divergent physicochemical characteristics. Here, we collected 167 fish from 12 study sites and used 16S and 18S rRNA metabarcoding approaches to characterize the gut microbiome structure of M. festivus. These data sets were analyzed in light of the host fish genotypes (genotyping-by-sequencing) and an extensive characterization of environmental physico-chemical parameters. We explored the relative contribution of environmental dissimilarity, the presence of parasitic taxa, and phylogenetic relatedness on structuring the gut microbiota. We documented occurrences of Nyctotherus sp. infecting a fish and linked its presence to a dysbiosis of the host gut microbiota. Moreover, we detected the presence of helminths which had a minor impact on the gut microbiota of their host. In addition, our results support a higher impact of the phylogenetic relatedness between fish rather than environmental similarity between sites of study on structuring the gut microbiota for this Amazonian cichlid. Our study in a heterogeneous riverscape integrates a wide range of factors known to structure fish gut microbiomes. It significantly improves understanding of the complex relationship between fish, their parasites, their microbiota, and the environment. IMPORTANCE The gut microbiota is known to play important roles in its host immunity, metabolism, and comportment. Its taxonomic composition is modulated by a complex interplay of factors that are hard to study simultaneously in natural systems. Mesonauta festivus, an Amazonian cichlid, is an interesting model to simultaneously study the influence of multiple variables on the gut microbiota. In this study, we explored the relative contribution of the environmental conditions, the presence of parasitic infections, and the genotype of the host on structuring the gut microbiota of M. festivus in Amazonia. Our results highlighted infections by a parasitic ciliate that caused a disruption of the gut microbiota and by parasitic worms that had a low impact on the microbiota. Finally, our results support a higher impact of the genotype than the environment on structuring the microbiota for this fish. These findings significantly improve understanding of the complex relationship among fish, their parasites, their microbiota, and the environment.

Lines 487-501.Note.I very like that the authors discuss about the limitations of DNA-barcoding approach trying to explain observed results.For me, the most important limitation of this approach is impossibility to estimate the "size" of the factor "parasite infestation", I mean, such parameter as intensity level.Indeed, the lack of significant effect of parasite invasion in experiment where available information is categorized only as "yes" or "no" parasites in the fish gut is very vulnerable.
Lines 504-507 and 512-513.Note.I guess that the most important factor is the present or absent the first intermediated host for given cestode species rather than the type of water.Perhaps, in black water there are no specific zooplanktonic crustations that could be used as the first intermediate hosts by cestodes.
Lines 566-568.Similarly, we detected a higher abundance of Cyanobacteria in fish from white water, furthering the potential horizontal transfer of bacteria associated with the higher chlorophyll A concentration in white water sites.
Note.It is good example of horizontal transfer of bacterial taxa from aquatic compartments to fish gut.Similar observation was found by Parshukov et al. 2019.Probably this ms (Supporting Information File S4) will be useful: Variations of the intestinal gut microbiota of farmed rainbow trout, Oncorhynchus mykiss (Walbaum), depending on the infection status of the fish.2019.Journal of Applied Microbiology 127(2) DOI: 10.1111/jam.14302Lines 412 -414.However, as these taxa were not detected in the water microbiome sampled in each water type, it was not possible to link these discriminant features to a putative horizontal transfer from the water microbiome.Lines 544-546.Since the gut microbiota of fish is relatively isolated from the water, changes in physicochemical characteristics could have a limited influence on its communities.Lines 572-573.However, we cannot directly link these bacterial strains to the water bacterioplankton as we did not observe these in our water microbiome assessment.Lines 596-598.Overall, our results support a low but present effect of the environmental dissimilarity between black and white water environments on the taxonomic structure of the mid gut microbiota in M. festivus.
Note.Due to the author have not data associated with the bacterial community structure of water and fish gut estimated during several time points, low level of relationships between bacterial communities of water and fish gut is expected.Both type of bacterial communities (from water and fish gut) could be characterized by different time that is needed to change them.It could be expected that bacterial community in water is more sensitive and changed faster than the bacterial community in fish gut even if, as the authors pointed out, the gut bacterial community originated from the surrounding water.I understand that sample collection in nature is not the same and easy as in laboratory but the limited data from nature may led to erroneous conclusions.
Unfortunately, I did not find any speculations regarding to feeding features of M. festivus from black and white waters.The authors mentioned that this fish is detritivorous (line 157) and that host diet is very important factor (line 73) but there are no any analysis or speculations associated with potentially different microbiota of sediments itself in the bottom of white and black waters or differences in community of invertebrates inhabiting the bottom sediments of those places (both sediments and invertebrates may possess by specific microbiomes).That fact that the structure of community of invertebrates are different between black and white waters is partially supported by differences in the structure of fish parasites (as it was shown in the present ms) required invertebrates as the first intermediate hosts (for example, cestodes).Moreover, the authors may have such data because 18S rRNA analysis was conducted.It could be interesting to compare the gut content of fish from black and white water using 18 S data and, consequently, speculate about similarity of fish diets from studied different sites.
Probably these two papers about microbiota of fish parasites are also will be useful.
1) The effect of diet on the structure of gut bacterial community of sympatric pair of whitefishes (Coregonus lavaretus): One story more.2019PeerJ 7(9) DOI: 10.7717/peerj.80052) Composition of the microbial communities in the gastrointestinal tract of perch (Perca fluviatilis L. 1758) and cestodes parasitizing the perch digestive tract.2019.Journal of Fish Diseases 43(4).DOI:10.1111/jfd.13096Reviewer #2 (Comments for the Author): The authors present an interesting study of the gut microbiomes of M. festivus, an Amazonian wild fish, and the impact of genotype, water quality, and parasite incidence on the gut microbiome.The manuscript is well-written and then study and analyses are generally sound.That said, I do have several minor comments for the authors to consider: Line 98: Recommend replacing "horizontal transfer", which is a term that invoked genetic mechanisms, with "dispersal", which invokes the intended ecological mechanism.
Line 145: I have trouble following the defined scenario here, because it is presented in the context of strong versus weak environmental effects.However, the expecations outlined as a result of these effects includes genetic components.It thus rather seems that the context of the spectrum being defined in this scenario is actually about the strength of the environmental effects relative to the strength of the genetic effects.If that's an accurate interpretation of this scenario, it would help readers to be explicit.If I'm mistaken, clarification in this passage would help.
Line 198: Does the DNeasy kit include mechanical disruption of the samples (e.g., bead beating)?If not, it would be worth articulating whether this could be a caveat impacting the results, as many taxa may be recalcitrant to other forms of cellular lysis.
Line 222: Please specify the read length and whether the data produced by the MiSeq run was paired end.
Line 281: Phylogenetic diversity metrics are used, but it is not clear how the phylogeny was assembled.Please provide these details in the Methods.
Line 323: It isn't clear what is meant by "pooling does not influence the analysis of the results in the discussion" Line 390: The methods seem to indicate that PERMANOVA was used to evaluate the association between sample covariates and beta-diversity, yet here it seems that an ordination based correlation analysis was used.Can the authors clarify why this approach was used to address this analysis and not PERMANOVA, which tends to be more robust for these types of investigations since it is not dependent upon the oridation?Line 399: Adonis is often found to be sensitive to the order in which the regression covariates are listed.Have the authors validated that reordering genotype and water type in their PERMANOVA regression model produces consistent results to those reported here?Line 567: Are the authors confident in their Cyanobacteria annotations?Could these annotations alternatively result from the presence of chloroplast DNA in the fish diet?

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The paper entitled "Gut microbiota of an Amazonian fish in a heterogeneous riverscape: Integrating Genotype, Environment and Parasitic infections" focused on estimation of different factors that potentially affect on fish gut bacterial community structure.I very like that more and more studies started to include parasites as a potential factor that may modulate gut bacterial community.The present study is very well written, logically presented and statistical analyses were professionally conducted.In the same time, there are several main queries (from my point of view) that have to be addressed.Please see them below.
Lines 72-73.From these, four usually stand out as the most important: environmental conditions, host diet, genotype, and physiological condition Note.In the introduction part the authors described environmental conditions, genotype, and physiological condition but nothing was mentioned about the effect of host diet on the bacterial community structure.I guess that such important factor as diet has to be mentioned as well.
Lines 175-176.Field trips were conducted from September to December 2018-2019 during the dry season.
Note.How did the authors estimate the potential effect of "year"?The microbiota of aquatic ecosystem can be altered from year to year.The mentioned period (dry season) included four months (September-December). Please clarify the potential effect of different months on the gut bacterial structure and support it by statistical analyses.For example, both March and May belong to "spring" season but various water physico-chemical characteristics could be significantly different between them.
Lines 195-197.A total of 167 midgut samples were collected and processed.DNA was extracted from 20 mg of a midgut segment for each fish.We specifically selected the middle section of the intestine to be constant in the tissues analysed.
Note.Please clarify the potential effect of content in midgut of fish on general bacterial structure of this gut segment.Did the authors estimate the midgut fullness?It is known that the diversity estimates in gut mucosa and gut content could be different.I guess that among 167 specimens there were fish with different levels of gut fullness.
Lines 458-462.For instance, we detected the presence of 135 nonhost-related ASVs from 53 different genera across our 167 Mesonauta festivus samples.In comparison, Minich et al. (2018) detected less than 50 Eukaryotic operational taxonomic units (OTUs) in their faeces and gut samples of aquaculture fish.
Note.Sorry, I do not understand why the authors try to find relations between the level of parasite infestation in fish populations from nature and fish cultivated under aquaculture conditions?It is expected that these communities will be very different.
Lines 487-501.Note.I very like that the authors discuss about the limitations of DNA-barcoding approach trying to explain observed results.For me, the most important limitation of this approach is impossibility to estimate the "size" of the factor "parasite infestation", I mean, such parameter as intensity level.Indeed, the lack of significant effect of parasite invasion in experiment where available information is categorized only as "yes" or "no" parasites in the fish gut is very vulnerable.
Lines 504-507 and 512-513.Note.I guess that the most important factor is the present or absent the first intermediated host for given cestode species rather than the type of water.Perhaps, in black water there are no specific zooplanktonic crustations that could be used as the first intermediate hosts by cestodes.
Lines 566-568.Similarly, we detected a higher abundance of Cyanobacteria in fish from white water, furthering the potential horizontal transfer of bacteria associated with the higher chlorophyll A concentration in white water sites.
Note.It is good example of horizontal transfer of bacterial taxa from aquatic compartments to fish gut.Similar observation was found by Parshukov et al. 2019.Probably this ms (Supporting Information File S4) will be useful: Variations of the intestinal gut microbiota of farmed rainbow trout, Oncorhynchus mykiss (Walbaum), depending on the infection status of the fish.2019.Journal of Applied Microbiology 127(2) DOI: 10.1111/jam.14302Lines 412 -414.However, as these taxa were not detected in the water microbiome sampled in each water type, it was not possible to link these discriminant features to a putative horizontal transfer from the water microbiome.Lines 544-546.Since the gut microbiota of fish is relatively isolated from the water, changes in physicochemical characteristics could have a limited influence on its communities.Lines 572-573.However, we cannot directly link these bacterial strains to the water bacterioplankton as we did not observe these in our water microbiome assessment.Lines 596-598.Overall, our results support a low but present effect of the environmental dissimilarity between black and white water environments on the taxonomic structure of the mid gut microbiota in M. festivus.
Note.Due to the author have not data associated with the bacterial community structure of water and fish gut estimated during several time points, low level of relationships between bacterial communities of water and fish gut is expected.Both type of bacterial communities (from water and fish gut) could be characterized by different time that is needed to change them.It could be expected that bacterial community in water is more sensitive and changed faster than the bacterial community in fish gut even if, as the authors pointed out, the gut bacterial community originated from the surrounding water.I understand that sample collection in nature is not the same and easy as in laboratory but the limited data from nature may led to erroneous conclusions.
Unfortunately, I did not find any speculations regarding to feeding features of M. festivus from black and white waters.The authors mentioned that this fish is detritivorous (line 157) and that host diet is very important factor (line 73) but there are no any analysis or speculations associated with potentially different microbiota of sediments itself in the bottom of white and black waters or differences in community of invertebrates inhabiting the bottom sediments of those places (both sediments and invertebrates may possess by specific microbiomes).That fact that the structure of community of invertebrates are different between black and white waters is partially supported by differences in the structure of fish parasites (as it was shown in the present ms) required invertebrates as the first intermediate hosts (for example, cestodes).Moreover, the authors may have such data because 18S rRNA analysis was conducted.It could be interesting to compare the gut content of fish from black and white water using 18 S data and, consequently, speculate about similarity of fish diets from studied different sites.
Probably these two papers about microbiota of fish parasites are also will be useful.

1)
The effect of diet on the structure of gut bacterial community of sympatric pair of whitefishes (Coregonus lavaretus): One story more.2019PeerJ 7( 9 Below you will find instructions from the Microbiology Spectrum editorial office and comments generated during the review.
ASM policy requires that data be available to the public upon online posting of the article, so please verify all links to sequence records, if present, and make sure that each number retrieves the full record of the data.If a new accession number is not linked or a link is broken, provide production staff with the correct URL for the record.If the accession numbers for new data are Lines 195-197.A total of 167 midgut samples were collected and processed.DNA was extracted from 20 mg of a midgut segment for each fish.We specifically selected the middle section of the intestine to be constant in the tissues analysed.
Comment 3: Please clarify the potential effect of content in midgut of fish on general bacterial structure of this gut segment.Did the authors estimate the midgut fullness?It is known that the diversity estimates in gut mucosa and gut content could be different.I guess that among 167 specimens there were fish with different levels of gut fullness.
Response: M. festivus is a herbivorous/detritivorous fish which mostly feed on periphyton a resource that is very abundant in Amazonian water in both wet and dry season.For this reason, most fish had their gut full.For this reason, we sliced the same midgut section in each fish including gut content.Including the gut content in the analysis was also favorable for detecting parasitic taxa and describing fish' diet.We did not consider gut fullness in the analysis and considered it a random factor affecting every site at the same rate.
Lines 458-462.For instance, we detected the presence of 135 nonhost-related ASVs from 53 different genera across our 167 Mesonauta festivus samples.In comparison, Minich et al. (2018) detected less than 50 Eukaryotic operational taxonomic units (OTUs) in their faeces and gut samples of aquaculture fish.
Comment 4: Sorry, I do not understand why the authors try to find relations between the level of parasite infestation in fish populations from nature and fish cultivated under aquaculture conditions?It is expected that these communities will be very different.
Response: Reviewer is right.The comparable studies are scarce and often use different primers or markers, target other organisms and different environments.We decided to remove this sentence.
Lines 487-501.Note.I very like that the authors discuss about the limitations of DNA-barcoding approach trying to explain observed results.For me, the most important limitation of this approach is impossibility to estimate the "size" of the factor "parasite infestation", I mean, such parameter as intensity level.Indeed, the lack of significant effect of parasite invasion in experiment where available information is categorized only as "yes" or "no" parasites in the fish gut is very vulnerable.
Comment 5: I guess that the most important factor is the present or absent the first intermediated host for given cestode species rather than the type of water.Perhaps, in black water there are no specific zooplanktonic crustations that could be used as the first intermediate hosts by cestodes.
Response: This is a very interesting comment.According to the study of Nakajiima et al. ( 2017), Rio Negro black water mean density and biomass of mesozooplankton including potential intermediate copepod hosts of cestode were higher compared to the Rio Solimoes white water, by 2.8 and 2.0 times, respectively.Therefore, such results would not support the hypothesis according to which the low prevalence of cestode intermediate copepod host would explain the lower cestode prevalence in fish.However, the taxonomic information provided in Nakajiima et al. ( 2017) is too limited to state whether water color has an impact on intermediated host for any given cestode species.
Lines 556-558.Similarly, we detected a higher abundance of Cyanobacteria in fish from white water, furthering the potential horizontal transfer of bacteria associated with the higher chlorophyll A concentration in white water sites.

Response:
We thank the reviewer for putting this publication to our attention.We added this sentence (Lines 564-566): "Dispersal of bacterioplankton taxa from water to fish gut were documented for discus fish (Sylvain et al. 2017), rainbow trout (Parshukov et al. 2019) and Atlantic Salmon (Lavoie et al. 2021).".
Lines 412 -414.However, as these taxa were not detected in the water microbiome sampled in each water type, it was not possible to link these discriminant features to a putative horizontal transfer from the water microbiome.Lines 544-546.Since the gut microbiota of fish is relatively isolated from the water, changes in physicochemical characteristics could have a limited influence on its communities.Lines 572-573.However, we cannot directly link these bacterial strains to the water bacterioplankton as we did not observe these in our water microbiome assessment.Lines 596-598.Overall, our results support a low but present effect of the environmental dissimilarity between black and white water environments on the taxonomic structure of the mid gut microbiota in M. festivus.
Comment 7: Due to the author have not data associated with the bacterial community structure of water and fish gut estimated during several time points, low level of relationships between bacterial communities of water and fish gut is expected.Both type of bacterial communities (from water and fish gut) could be characterized by different time that is needed to change them.It could be expected that bacterial community in water is more sensitive and changed faster than the bacterial community in fish gut even if, as the authors pointed out, the gut bacterial community originated from the surrounding water.I understand that sample collection in nature is not the same and easy as in laboratory but the limited data from nature may led to erroneous conclusions.

Response:
The reviewer raises another interesting point here.We modified the sentence accordingly (Lines 567-574): "However, it may be hazardous to directly link these bacterial strains to the water bacterioplankton in this study because we did not conduct a time series of sampling to account for a possible decoupling between their respective taxonomic dynamics in water and fish gut.".Unfortunately, I did not find any speculations regarding to feeding features of M. festivus from black and white waters.The authors mentioned that this fish is detritivorous (line 157) and that host diet is very important factor (line 73) but there are no any analysis or speculations associated with potentially different microbiota of sediments itself in the bottom of white and black waters or differences in community of invertebrates inhabiting the bottom sediments of those places (both sediments and invertebrates may possess by specific microbiomes).That fact that the structure of community of invertebrates are different between black and white waters is partially supported by differences in the structure of fish parasites (as it was shown in the present ms) required invertebrates as the first intermediate hosts (for example, cestodes).Moreover, the authors may have such data because 18S rRNA analysis was conducted.
Comparison of both results; Envfit : " chlorophyll a concentration in water: R 2 = 0.084, pvalue = 0.001; DOC: R 2 = 0.003, p-value = 0.76; Silicate: R 2 = 0.027, p-value = 0.11; Conductivity: R 2 = 0.027, p-value = 0.11; pH: R 2 = 0.024, p-value = 0.14).PERMANOVA : Results are very different in the PERMANOVA as they show a significant association between each environmental parameter and microbiome beta diversity.However, this is probably due to the very high number of comparisons performed by the PERMANOVA compared to the lower number of comparisons used in envfit which only considers environmental parameters and bray Curtis distances given to the function.Indeed, since this type of analysis cannot be easily plotted and shows all significant results, it is very difficult to highlight a pattern in the data.For these reasons, we think that the envfit analysis, while potentially less powerful in detecting significant interactions, leads to more interesting and interpretable results.
Comment 7: Line 399: Adonis is often found to be sensitive to the order in which the regression covariates are listed.Have the authors validated that reordering genotype and water type in their PERMANOVA regression model produces consistent results to those reported here?
Response: Yes, we previously did the PERMANOVA in both orders to consider this potential bias and, while results are not perfectly identical, they show the same patterns, as exemplified below with one of our main PERMANOVA ran at a low depth.
Comment 8: Line 567: Are the authors confident in their Cyanobacteria annotations?Could these annotations alternatively result from the presence of chloroplast DNA in the fish diet?
Response: Numerous plants were detected in the gut using 18S marker.However, given that divergence time between Cyanobacteria and the oldest vascular plant fossil (tracheophytes) is about 460 MYA (Kenrick and Crane, 1997), it is very unlikely that Cyanobacteria annotation was corrupted with chloroplast sequences, using E-values threshold as low as 10exp-30.Thank you for submitting your manuscript to Microbiology Spectrum.As you will see your paper is very close to acceptance.Please modify the manuscript along the lines I have recommended.As these revisions are quite minor, I expect that you should be able to turn in the revised paper in less than 30 days, if not sooner.If your manuscript was reviewed, you will find the reviewers' comments below.
I ask that you please revise the manuscript text to include your responses to Reviewer 2 at lines 390 and 399.I think these are acceptable responses, and as it is likely that peers will have similar questions, incorporating these responses into the text will improve the transparency and robustness of your arguments.
When submitting the revised version of your paper, please provide (1) point-by-point responses to the issues raised by the reviewers as file type "Response to Reviewers," not in your cover letter, and (2) a PDF file that indicates the changes from the original submission (by highlighting or underlining the changes) as file type "Marked Up Manuscript -For Review Only".Please use this link to submit your revised manuscript.Detailed instructions on submitting your revised paper are below.

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Editor, Microbiology Spectrum Reviewer comments: Reviewer #1 (Comments for the Author): Thank you for your complete replies to my comments.

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September 2, 2022 Mx.Nicolas Leroux Laval University Biology 1030 avenue de la Médecine Quebec, QC Canada Re: Spectrum02755-22 (Gut microbiota of an Amazonian fish in a heterogeneous riverscape: Integrating Genotype, Environment and Parasitic infections) Dear Mx.Nicolas Leroux: Thank you for submitting your manuscript to Microbiology Spectrum.It has been reviewed by two experts, and both viewed the manuscript positively and had some suggestions for revision.I have therefore decided to request 'Modifications', which means that if you choose to, you can submit a revised version of the text following the details outlined below, and that It is likely that I will send the revision out for a second round of review.
You will see that each reviewer has suggested several places where you can clarify the methods or, if necessary, add additional analyses.I generally agree with their concerns.As you revise, I confirm to you that Spectrum's scope is that "rather than making subjective evaluations of potential impact, Microbiology Spectrum publishes research studies that are of high technical quality and are useful to the community."I will be supportive of revisions that meet this expectation, and encourage you to consider these criteria and explain them in your reviewer response during the revision.
When submitting the revised version of your paper, please provide (1) point-by-point responses to the issues raised by the reviewers as file type "Response to Reviewers," not in your cover letter, and (2) a PDF file that indicates the changes from the original submission (by highlighting or underlining the changes) as file type "Marked Up Manuscript -For Review Only".Please use this link to submit your revised manuscript -we strongly recommend that you submit your paper within the next 60 days or reach out to me.Detailed instructions on submitting your revised paper are below. https://spectrum.msubmit.net/cgibin/main.plex?el=A7QF1CBMh5A5FRxj3F2A9ftdjg991zN7RbO4ZYBFfBH0AZ Below you will find instructions from the Microbiology Spectrum editorial office and comments generated during the review.
ASM policy requires that data be available to the public upon online posting of the article, so please verify all links to sequence records, if present, and make sure that each number retrieves the full record of the data.If a new accession number is not linked or a link is broken, provide production staff with the correct URL for the record.If the accession numbers for new data are The authors present an interesting study of the gut microbiomes of M. festivus, an Amazonian wild fish, and the impact of genotype, water quality, and parasite incidence on the gut microbiome.The manuscript is well-written and then study and analyses are generally sound.That said, I do have several minor comments for the authors to consider: Comment 1: Line 98: Recommend replacing "horizontal transfer", which is a term that invoked genetic mechanisms, with "dispersal", which invokes the intended ecological mechanism.

Response:
The reviewer is right."horizontal transfer" was replaced with the terms "dispersal" or "recruitment".Lines 95-98: "Since the surrounding water may be a source of recruitment of environmental microbial strains to the fish gut microbiota (Sylvain et al. 2019, Lavoie et al. 2021), we posed the hypothesis that certain taxa of M. festivus' gut microbiome would be related to the water type (black or white).";Lines 543-544: "could be the result of dispersal from the environment to the microbiota of M. festivus." Comment 2: Line 145: I have trouble following the defined scenario here, because it is presented in the context of strong versus weak environmental effects.However, the expecations outlined as a result of these effects includes genetic components.It thus rather seems that the context of the spectrum being defined in this scenario is actually about the strength of the environmental effects relative to the strength of the genetic effects.If that's an accurate interpretation of this scenario, it would help readers to be explicit.If I'm mistaken, clarification in this passage would help.
Response 2: Reviewer is absolutely right.The paragraph was rewritten accordingly.See lines 141-148: "In any case, the interplay between genetic and environmental effect on gut microbiota taxonomic structure ranges between the following extreme scenario: strong environmental effects relative to weak genetic effects and vice versa.In the first case, fish should display similar microbial community shifts at similar environmental shifts (e.g.independent black water sites versus their respective connected white water sites).On the opposite, in the presence of a weak environmental effect and a strong genetic effect, fish from a same genetic population, but inhabiting contrasting environments, are expected to converge in terms of microbiota composition.".
Comment 3: Line 198: Does the DNeasy kit include mechanical disruption of the samples (e.g., bead beating)?If not, it would be worth articulating whether this could be a caveat impacting the results, as many taxa may be recalcitrant to other forms of cellular lysis.

Response:
We did not use mechanical disruption of the samples by bead beating.Instead, samples were cut into small pieces and digested with lysis buffer.We agree that this may have limited the completeness of detection of microbial and parasitic taxonomic diversity.However, this bias remains the same for each sample, which should not affect comparisons between groups.We have included a sentence in the methods to emphasize this point.See lines 192 " The middle section of the intestine was specifically selected and samples were cut into small pieces using sterile blades and digested with lysis buffer overnight.Although an additional step of mechanical disruption would have improved the detection of both microbial and parasitic taxonomic diversity, this potential bias should not affect comparisons between experimental groups." Comment 3: Line 222: Please specify the read length and whether the data produced by the MiSeq run was paired end.
Response: This information was provided.See lines 231-236: "The demultiplexed fastq sequence files were processed through DADA2 (Callahan et al. 2016)  This is a pair end sequencing for both 16S and 18S.This is now clarified line 221: "Multiplex paired-end sequencing was performed using the MiSeq platform".We omitted the function "mergePairs" for read merging.This is now corrected lines 236-240: "The filtered reads were then fed to the error rate learning, dereplication, merging and Amplicon Sequence Variant (ASV) inference steps using the functions "learnErrors", "derepFastq" mergePairs and "dada".Chimeric sequences were removed using the "removeBimeraDenovo" function with the "pseudo" method parameter.".
Comment 4: Line 281: Phylogenetic diversity metrics are used, but it is not clear how the phylogeny was assembled.Please provide these details in the Methods.
Response: Phylogenetic diversity was computed with Phyloseq using estimate_pd(phylo) function.The tree was computed with the simple agglomerative (bottom-up) hierarchical clustering method (UPGMA).Chao1 and Shannon were computed using estimate_richness function.Lines 282-286: "Using the 16S rRNA Phyloseq object previously produced, we estimated alpha diversity indexes such as the observed number of ASVs (Chao1), and Shannon entropy (Shannon 1949) using estimate_richness function.Then, phylogenetic diversity (Faith 1992) was computed with using estimate_pd(phylo) function.The tree was computed with the simple agglomerative (bottom-up) hierarchical clustering method (UPGMA).".
Comment 5: Line 323: It isn't clear what is meant by "pooling does not influence the analysis of the results in the discussion".

Response:
We clarified the sentence line 328-329 as: "We previously analysed independently the impact of each type of helminth detected and their pooling did not have any impact on the general significance of the results.".
Comment 6: Line 390: The methods seem to indicate that PERMANOVA was used to evaluate the association between sample covariates and beta-diversity, yet here it seems that an ordination based correlation analysis was used.Can the authors clarify why this approach was used to address this analysis and not PERMANOVA, which tends to be more robust for these types of investigations since it is not dependent upon the ordination?
Response: PERMANOVA was used to evaluate associations between covariate genotype and water types and the gut microbiome.However, we opted for an ordination-based method for the comparison of the 5 environmental parameters.We have now separated the two paragraphs in the methods to better separate both analyses.
We selected an ordination-based method as it allowed us to share the environmental vectors and the data together as a graphical output highlighting the relative influence of each parameter on the gut microbiome structure.Considering your comment, we decided to run the proposed analysis on a PERMANOVA to make sure we did not miss any important results.Here are the results: Comparison of both results; Envfit : " chlorophyll a concentration in water: R 2 = 0.084, pvalue = 0.001; DOC: R 2 = 0.003, p-value = 0.76; Silicate: R 2 = 0.027, p-value = 0.11; Conductivity: R 2 = 0.027, p-value = 0.11; pH: R 2 = 0.024, p-value = 0.14).PERMANOVA : Results are very different in the PERMANOVA as they show a significant association between each environmental parameter and microbiome beta diversity.However, this is probably due to the very high number of comparisons performed by the PERMANOVA compared to the lower number of comparisons used in envfit which only considers environmental parameters and bray Curtis distances given to the function.Indeed, since this type of analysis cannot be easily plotted and shows all significant results, it is very difficult to highlight a pattern in the data.For these reasons, we think that the envfit analysis, while potentially less powerful in detecting significant interactions, leads to more interesting and interpretable results.
Comment 7: Line 399: Adonis is often found to be sensitive to the order in which the regression covariates are listed.Have the authors validated that reordering genotype and water type in their PERMANOVA regression model produces consistent results to those reported here?
Response: Yes, we previously did the PERMANOVA in both orders to consider this potential bias and, while results are not perfectly identical, they show the same patterns, as exemplified below with one of our main PERMANOVA ran at a low depth.
Comment 8: Line 567: Are the authors confident in their Cyanobacteria annotations?Could these annotations alternatively result from the presence of chloroplast DNA in the fish diet?
Response: Numerous plants were detected in the gut using 18S marker.However, given that divergence time between Cyanobacteria and the oldest vascular plant fossil (tracheophytes) is about 460 MYA (Kenrick and Crane, 1997), it is very unlikely that Cyanobacteria annotation was corrupted with chloroplast sequences, using E-values threshold as low as 10exp-30.Your manuscript has been accepted, and I am forwarding it to the ASM Journals Department for publication.You will be notified when your proofs are ready to be viewed.
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Thank you for submitting your paper to Spectrum.

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Editor, Microbiology Spectrum Journals Department American Society for Microbiology 1752 N St., NW Washington, DC 20036 E-mail: spectrum@asmusa.org • Manuscript: A .DOC version of the revised manuscript • Figures: Editable, high-resolution, individual figure files are required at revision, TIFF or EPS files are preferred

Comment 6 :
It is good example of horizontal transfer of bacterial taxa from aquatic compartments to fish gut.Similar observation was found by Parshukov et al. intestinal gut microbiota of farmed rainbow trout, Oncorhynchus mykiss (Walbaum), depending on the infection status of the fish.2019.Journal of Applied Microbiology 127(2) DOI: 10.1111/jam.14302.
-22R1 (Gut microbiota of an Amazonian fish in a heterogeneous riverscape: Integrating Genotype, Environment and Parasitic infections) Dear Mx.Nicolas Leroux: -22R2 (Gut microbiota of an Amazonian fish in a heterogeneous riverscape: Integrating Genotype, Environment and Parasitic infections) Dear Mx.Nicolas Leroux: Thank you for submitting your manuscript to Microbiology Spectrum.It has been reviewed by two experts, and both viewed the manuscript positively and had some suggestions for revision.I have therefore decided to request 'Modifications', which means that if you choose to, you can submit a revised version of the text following the details outlined below, and that It is likely that I will send the revision out for a second round of review.
You will see that each reviewer has suggested several places where you can clarify the methods or, if necessary, add additional analyses.I generally agree with their concerns.As you revise, I confirm to you that Spectrum's scope is that "rather than making subjective evaluations of potential impact, Microbiology Spectrum publishes research studies that are of high technical quality and are useful to the community."Iwill be supportive of revisions that meet this expectation, and encourage you to consider these criteria and explain them in your reviewer response during the revision.When submitting the revised version of your paper, please provide (1) point-by-point responses to the issues raised by the reviewers as file type "Response to Reviewers," not in your cover letter, and (2) a PDF file that indicates the changes from the original submission (by highlighting or underlining the changes) as file type "Marked Up Manuscript -For Review Only".Please use this link to submit your revised manuscript -we strongly recommend that you submit your paper within the next 60 days or reach out to me.Detailed instructions on submitting your using the function "ytfilterAndTrim" with the following parameters: two as the phred score threshold for total read removal, a maximum expected error of two for forward reads and four for reverse reads, a truncation length of 280 base pairs for forward reads and 200 base pairs for reverse reads for 16S rRNA library, and a length of 275 base pairs for forward reads and 250 base pairs for reverse reads for 18S rRNA library.".