Gut Microbiome Differences Across Mixed-Sex and Female-Only Social Rearing Regimes in Female Field Crickets Teleogryllus occipitalis (Orthoptera: Gryllidae)
Round 1
Reviewer 1 Report
Comments and Suggestions for AuthorsOverall, the study is highly relevant and meets the requirements of the Insects. The Authors obtained important data on the structural and functional organization of the gut microbiome of Teleogryllus occipitalis (Orthoptera: Gryllidae) crickets, taking into account the social environment.
Such studies are extremely important for the discussion of entomology and the ecological physiology of insects.
I have one important comment:
- I would recommend that the authors add an additional section in Section "2. Materials and Methods" about the method for removing host (Teleogryllus occipitalis) DNA data during bioinformatics analysis. What scripts were used for this? This information is important, as shotgun sequencing of the metagenome was used. This information will enable other researchers to obtain reproducible data in the future.
Author Response
Comments 1: Overall, the study is highly relevant and meets the requirements of the Insects. The Authors obtained important data on the structural and functional organization of the gut microbiome of Teleogryllus occipitalis (Orthoptera: Gryllidae) crickets, taking into account the social environment.
Such studies are extremely important for the discussion of entomology and the ecological physiology of insects.
Response 1: We thank the reviewer for the positive and supportive assessment of our work. We have carefully revised the manuscript in response to the comments provided, as detailed below.
Comments 2: I have one important comment:
I would recommend that the authors add an additional section in Section "2. Materials and Methods" about the method for removing host (Teleogryllus occipitalis) DNA data during bioinformatics analysis. What scripts were used for this? This information is important, as shotgun sequencing of the metagenome was used. This information will enable other researchers to obtain reproducible data in the future.
Response 2: Thank you for this important suggestion. We have added a new subsection, Section 2.3, “Removal of host-derived reads,” to clearly describe how host-derived reads were removed from the shotgun metagenomic data in the Materials and Methods section (lines 148, 170-185). To ensure reproducibility, we have also provided the complete workflow script, including database download and indexing steps and the exact command lines, in the Supplementary Materials as Script S1 (lines 499-500).
Reviewer 2 Report
Comments and Suggestions for AuthorsHirata et al. investigated the gut microbiome of female field crickets (Teleogryllus occipitalis) reared under mixed-sex and female-only social conditions using whole-genome shotgun metagenomics. The authors demonstrate that both the composition and predicted functional potential of the gut microbiome differ between social rearing regimes (mixed-sex versus female-only housing). Overall, the manuscript addresses an interesting and timely topic, and the experimental approach is generally sound. However, several issues need to be addressed before the manuscript can be considered suitable for publication, as detailed below.
Major concerns:
1. Throughout the manuscript, differences in the female gut microbiome are frequently attributed to the “presence or absence of males.” While this interpretation is intuitive, the experimental design does not allow the authors to disentangle which specific components of the social environment are responsible for the observed microbiome differences. In particular, potentially confounding factors—such as mating versus non-mating effects—cannot be separated under the current design.
2. The results of this study are largely descriptive, and the functional predictions of the gut microbiota are not supported by direct experimental validation. Therefore, it is recommended that additional experimental evidence be provided to substantiate these functional inferences. Alternatively, the limitations of the study should be more explicitly acknowledged and discussed in the Discussion section.
3. The number of biological replicates per group is limited to three, which may reduce statistical power and affect the robustness and reliability of the conclusions.
Minor comments:
Lines 89 and 99: T. occipitalis should be italicized. Please ensure that all taxonomic names are consistently italicized and properly formatted throughout the manuscript.
Line 101: Replace “LD = 16:8 h” with “L:D = 16:8 h.”
Figures: The panel label letters are too large and appear disproportionate to the images; reducing their size would improve visual balance and readability.
Figure 3: The numerical results of the diversity difference analyses among groups are not shown. It is recommended that these values be displayed directly in the figure for clarity.
Author Response
Comments 1: Hirata et al. investigated the gut microbiome of female field crickets (Teleogryllus occipitalis) reared under mixed-sex and female-only social conditions using whole-genome shotgun metagenomics. The authors demonstrate that both the composition and predicted functional potential of the gut microbiome differ between social rearing regimes (mixed-sex versus female-only housing). Overall, the manuscript addresses an interesting and timely topic, and the experimental approach is generally sound. However, several issues need to be addressed before the manuscript can be considered suitable for publication, as detailed below.
Response 1: We thank the reviewer for the positive assessment of our work and for the constructive feedback. We have revised the manuscript accordingly and addressed each issue in detail. All revisions are highlighted in the resubmitted files, and our point-by-point responses are provided below.
Comments 2: Throughout the manuscript, differences in the female gut microbiome are frequently attributed to the “presence or absence of males.” While this interpretation is intuitive, the experimental design does not allow the authors to disentangle which specific components of the social environment are responsible for the observed microbiome differences. In particular, potentially confounding factors—such as mating versus non-mating effects—cannot be separated under the current design.
Response 2: We thank the reviewer for this important and constructive comment. We fully agree that our experimental design contrasts two group-rearing social regimes (mixed-sex vs. female-only) and that the mixed-sex regime can encompass multiple components of the social environment (e.g., mating and non-mating effects). Because individual mating events and specific male-associated cues were not monitored or independently manipulated, our data cannot disentangle mating effects from non-mating components of male exposure.
To address this concern, we revised the manuscript in three ways:
Terminology harmonization across the manuscript: We revised the wording throughout the manuscript to avoid attributing the observed microbiome differences specifically to “male presence/absence.” Instead, we consistently interpret the findings as differences associated with the social rearing regime (mixed-sex vs female-only). (lines 18-19, 22-29, 32-36, 59-62, 112, 126-127, 417-420, 447-448, 465)
Explicit clarification in Methods: We added a sentence in the Methods stating that　individual mating status was not monitored, and therefore, the mixed-sex regime should be interpreted as a composite social environment including exposure to males and the possibility of mating, rather than a controlled manipulation of mating status (lines 113-117).
Expanded limitations and future directions: We explicitly stated in the Discussion that, under the current design, we cannot separate mating effects from non-mating components of male exposure because mating status and male-derived cues were not independently controlled. We also added brief, concrete examples of future study designs that could help disentangle these components, such as controlled mating and/or barrier-cage experiments (lines 474-480).
We hope these revisions clarify the scope of inference supported by the current dataset while retaining the main result: under group-rearing conditions, the female gut microbiome differs consistently between mixed-sex and female-only social rearing regimes.
Comments 2: The results of this study are largely descriptive, and the functional predictions of the gut microbiota are not supported by direct experimental validation. Therefore, it is recommended that additional experimental evidence be provided to substantiate these functional inferences. Alternatively, the limitations of the study should be more explicitly acknowledged and discussed in the Discussion section.
Response 2: We thank the reviewer for this important comment. We agree that our functional interpretations are based on metagenomic gene-content annotations and therefore represent predicted functional potential rather than experimentally validated activity.
In response, we added explicit statements in the Discussion to clarify that our functional inferences are based solely on metagenomic gene-content annotations. Specifically, we inserted a cautionary note at the beginning of the Discussion and added a concluding limitations statement noting that putative functional shifts were inferred from gene-abundance profiles rather than direct measurements (e.g., gene expression or metabolites) (lines 409-411 and 480-484).
In addition, we revised the manuscript to temper functional language throughout. Specifically, we revised the text to consistently describe predicted functional differences as annotation-based variation in gene content rather than as confirmed functional activity across the manuscript (lines 24-26, 43-47, 75-77, 353, 356, 378, and 444).
Finally, we expanded the Future Directions to specify that integrating metatranscriptomics and metabolomics will be necessary to validate whether the observed gene-content differences are accompanied by corresponding changes in microbial transcription and metabolite outputs (lines 491-495).
Comments 3: The number of biological replicates per group is limited to three, which may reduce statistical power and affect the robustness and reliability of the conclusions.
Response 3: We thank the reviewer for raising this important concern. We agree that using only three biological replicates per group may limit statistical power and reduce the precision and generalizability of our estimates. In our study, each biological replicate corresponded to an independent pool of five individuals (15 individuals per group), and each pool was processed independently and treated as the experimental unit. Accordingly, we have revised the Discussion to explicitly acknowledge that the limited number of pooled biological replicates (n = 3 per group) reduces statistical power, limits the precision of effect-size estimates, and may affect the robustness and reliability of our conclusions (lines 484-486).
To avoid overinterpretation, we also revised the manuscript to temper causal language throughout (lines 288, 293, 297-298, 311-312, 382-383, 408, 487 and 489). Although replication is limited, multiple complementary analyses, including hierarchical clustering, Bray–Curtis-based ordination, and differential abundance analyses at both taxonomic and functional levels, showed consistent trends across the pooled replicates. Together, these analyses provide convergent support for the main patterns described in the manuscript.
Comments 4: Lines 89 and 99: T. occipitalis should be italicized. Please ensure that all taxonomic names are consistently italicized and properly formatted throughout the manuscript.
Response 4: We thank the reviewer for this comment. We have italicized T. occipitalis (lines 92 and 105) and checked the manuscript to ensure consistent italicization and formatting of all taxonomic names throughout (lines 543-659).
Comments 5: Line 101: Replace “LD = 16:8 h” with “L:D = 16:8 h.”
Response 5: We thank the reviewer for this helpful correction. We replaced “LD = 16:8 h” with “L:D = 16:8 h” at line 107 in the revised manuscript.
Comments 6: Figures: The panel label letters are too large and appear disproportionate to the images; reducing their size would improve visual balance and readability.
Response 6: We thank the reviewer for this helpful suggestion. We reduced the size of the panel label letters in all figures to improve visual balance and readability in the revised manuscript.
Comments 7: Figure 3: The numerical results of the diversity difference analyses among groups are not shown. It is recommended that these values be displayed directly in the figure for clarity.
Response 7: Thank you for this helpful suggestion. We revised Figure 3B to display the numerical results of the between-group comparisons by annotating the P-values for each alpha-diversity index (Chao1, Shannon, Simpson, and Pielou) directly on the figure.
Reviewer 3 Report
Comments and Suggestions for AuthorsThis manuscript utilized metagenomic sequencing technology to identify the gut microbiota of the field cricket Teleogryllus occipitalis, and compared the gut microbiome of females in mixed-sex rearing with those of females in female-only rearing. The results found that the females raised with males harbored different gut microbiota and microbial genes from females reared without males. The finding revealed that the social rearing conditions, including the presence or absence of males, are associated with the gut microbiota of female crickets.
The manuscript has a novel conception, clear language expression, correct methods and definite result analysis. But I have a question, why didn't the author use a sample of male-only rearing?
Other minor mistakes need to be corrected, such as,
Line 15, “The cricket is one of the useful hemimetabolous model” is not correct, should be “incomplete metamorphosis or paurometamorphosis”.
Line 99, “T. occipitalis” should be italic.
Line 234-235, this sentence should be put in the according method part.
Line 263, from the Figure 1, I think “…in at least one sample are shown” is not right, it seems “…are shown in all samples”.
Line 268-270, The description of Figure 2 is too simplistic, making it difficult for readers to understand why the male samples did not gather together but were separated.
Comments on the Quality of English LanguageMy native language is not English, so I can't offer good suggestions on English language expression.
Author Response
Comments 1: This manuscript utilized metagenomic sequencing technology to identify the gut microbiota of the field cricket Teleogryllus occipitalis, and compared the gut microbiome of females in mixed-sex rearing with those of females in female-only rearing. The results found that the females raised with males harbored different gut microbiota and microbial genes from females reared without males. The finding revealed that the social rearing conditions, including the presence or absence of males, are associated with the gut microbiota of female crickets.
The manuscript has a novel conception, clear language expression, correct methods and definite result analysis.
Response 1: We are grateful to the Reviewer for the favorable assessment of our manuscript. In response to the Reviewer’s comments, we have carefully revised the manuscript where appropriate. Detailed responses and the corresponding revisions are provided below.
Comments 2: But I have a question, why didn't the author use a sample of male-only rearing?
Response 2: We thank the reviewer for this insightful suggestion. The primary objective of this study was to test whether the presence of males is associated with variation in the gut microbiome of female Teleogryllus occipitalis. Therefore, our key comparison focused on females reared under two social conditions: mixed-sex rearing vs. female-only rearing.
We agree that a male-only rearing treatment would be informative and would help broaden the interpretation of sex-specific social effects. However, such a treatment would address a different question, specifically how the absence or presence of females affects the gut microbiome of males, and it would require an additional experimental group and study design beyond the scope of the present work.
In the current dataset, we included males sampled from the mixed-sex cages only as a reference for sex-related variation under the same rearing environment as mixed-sex females, not as a treatment to evaluate rearing-regime effects in males. To avoid any confusion, we have clarified this rationale in the Introduction/Methods (lines 96-100 and 128-130).
Comments 3: Other minor mistakes need to be corrected, such as, Line 15, “The cricket is one of the useful hemimetabolous model” is not correct, should be “incomplete metamorphosis or paurometamorphosis”.
Response 3: We thank the reviewer for this important correction. We revised the sentence at line 15-16 to use the appropriate terminology and now state: “Crickets are useful model organisms with incomplete metamorphosis for understanding insect physiology and behavior.”
Comments 4: Line 99, “T. occipitalis” should be italic.
Response 4: We thank the reviewer for this correction. We italicized T. occipitalis at lines 92 and 105 in the revised manuscript and checked the manuscript to ensure consistent italicization and formatting of all taxonomic names throughout (lines 543-659).
Comments 5: Line 234-235, this sentence should be put in the according method part.
Response 5: We thank the reviewer for this helpful suggestion. Because the same information was already described in the Materials and Methods section, we deleted this sentence from the Results to avoid redundancy.
Comments 6: Line 263, from the Figure 1, I think “…in at least one sample are shown” is not right, it seems “…are shown in all samples”.
Response 6: Thank you for this helpful comment. We apologize for the confusion. In Figure 1, taxa were selected using the criterion that they were exceeded the abundance threshold in at least one sample across the dataset, in order to keep the figure readable. After selecting this set of taxa, we plotted the same taxa set for all samples to facilitate direct comparison among groups (taxa not meeting the criterion were not displayed). To avoid misunderstanding, we have revised the legend to clarify that “in at least one sample” refers to the selection criterion. The changes appear on lines 281-282, 324-325 and 506-507 in the revised manuscript.
Comments 7: Line 268-270, The description of Figure 2 is too simplistic, making it difficult for readers to understand why the male samples did not gather together but were separated.
Response 7: Thank you for this helpful comment. We agree that our original description of Figure 2 was too brief and did not adequately describe the male clustering pattern. We have revised the Results section (lines 287-292) to explicitly state that females from mixed-sex and female-only rearing formed distinct clusters across taxonomic levels, whereas the male replicates did not form a single cohesive cluster. We also added a sentence indicating that this pattern suggests overlap of male gut community profiles with both female groups and higher among-replicate variability.
Round 2
Reviewer 2 Report
Comments and Suggestions for AuthorsI would like to acknowledge the author's diligent work in revising the manuscript. I have no additional feedback at this stage.
