Altitude as a key environmental factor shaping microbial communities of tea green leafhoppers (Matsumurasca onukii)

ABSTRACT The tea green leafhopper, Matsumurasca onukii Matsuda, is the most destructive insect pest of tea plantations in East Asia. While several microbes in M. onukii have been characterized, the microbial community compositions in wild M. onukii populations and the environmental factors that shape them are mostly unknown. In this study, M. onukii populations were collected from major tea growing regions in China. Following high-throughput sequencing of 16S rRNA gene fragments for bacteria and the internal transcribed spacer region for fungi, association analyses were performed within the microbial communities associated with M. onukii and their environmental drivers. We found that the bacterial community structures differed in various regions, and the abundance of dominant bacteria such as Wolbachia, Pseudomonas, Acinetobacter, Pantoea, Enterobacter, and Methylobacterium varied widely. Moreover, wild populations of M. onukii can be infected with facultative symbionts from six genera (Wolbachia, Rickettsia, Asaia, Serratia, Arsenophonus, and Cardinium) with divergent relative abundances. Correlation analysis indicated that altitude was a key environmental factor that shaped bacterial communities of M. onukii. Furthermore, longitude, temperature, and rainfall are also significantly correlated with the bacterial communities. The fungal communities of M. onukii populations were dominated by Ascomycota and Basidiomycota, of which most genera are considered to be plant endophytes or plant pathogens, such as Cladosporium, Fusarium, Alternaria, and Gibberella. We demonstrated that M. onukii carry a complex and variable microbial community, which is influenced by altitude as well as climate-related factors. Our results provide novel insights into the bacteria and fungi of M. onukii. IMPORTANCE Host-associated microbial communities play an important role in the fitness of insect hosts. However, the factors shaping microbial communities in wild populations, including environmental factors and interactions among microbial species, remain largely unknown. The tea green leafhopper has a wide geographical distribution and is highly adaptable, providing a suitable model for studying the effect of ecological drivers on microbiomes. This is the first large-scale culture-independent study investigating the microbial communities of M. onukii sampled from different locations. Altitude as a key environmental factor may have shaped microbial communities of M. onukii by affecting the relative abundance of endosymbionts, especially Wolbachia. The results of this study, therefore, offer not only an in-depth view of the microbial diversity of this species but also an insight into the influence of environmental factors.

future.Zhang et al. analyzed the diversity of the microbiome of M. onukii in major tea producing regions in China, and analyzed the relationship between its diversity and environmental factors.This is of great significance for enriching the research on the microbiome and its functions of M. onukii.The following suggestions are provided for the authors in order to improve the manuscript: 1. L66-69: References required; 2. L70: The above statement cannot reach this conclusion.Please describe it more rigorously; 3. The purpose of the manuscript in the introduction is only to analyze the microbiome diversity of M. onukii and its altitude significance which are too descriptive, and it is recommended to further refine the purpose and significance of the study; 4. Figure S6 is meaningful information, and it is recommended to put it in the maintext; 5.There are many abbreviations in the ms that are not easy to understand and need further clarification; 6.The diversity of fungi in the study does not seem to vary significantly between different altitude, especially the core flora, so explanations need to be made during the discussion; 7.Although altitude is an important environmental factor that affects microbial diversity in the study, other factors also play a role, and explanations should also be made during the discussion to avoid misleading the reader.At the same time, appropriate additions and changes should be made in the abstract; 8. L391：Why only females?Because microbiome analysis did not distinguish between male and female; 9. L414-416: Why mix adults and nymphs?There are differences between them; 10.L417: Why are there only 9 samples?As there are 10 sampling points; 11. Figure legends are not detailed enough, additional information is needed.
Reviewer #2 (Comments for the Author): Insects frequently harbor a variety of symbiotic microbiota, and the interactions between them may have important effects on their evolution.However, the factors shaping microbial communities in wild populations remain largely unknown.Here, a comprehensive study is carried on altitude as a key environmental factor shaping microbial communities of tea green leafhopper.The experimental design is reasonable, and the findings are fascinating.More advanced metagenomes can be looked at for future work, and more novel data may be presented.

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Thank you for submitting your paper to Microbiology Spectrum.
Insects frequently harbor a variety of symbiotic microbiota, and the interactions between them may have important effects on their evolution.However, the factors shaping microbial communities in wild populations remain largely unknown.Here, a comprehensive study is carried on altitude as a key environmental factor shaping microbial communities of tea green leafhopper.The experimental design is reasonable, and the findings are fascinating.Before the paper is accepted, here are some minor comments: Many studies have shown that bacteria play an important role in symbiotic bacteria for stinging and sucking mouthparts.I saw that your Introduction also mainly discussed the influence of bacteria on some insects, but your experiment also designed the high-throughput sequencing of ITS.Can you explain why this experiment was designed?Line367-370: How to distinguish mountain tea plantaions and hilly tea plantations?What is the sampling point selection criteria?Line 391-392: What is the purpose of amplifying mitochondrial COI gene and 16sRNA gene ?
What is the niche of Matsumurasca onukii.

Results
Line168-169: The author emphasizes the ectosymbionts and endosymbionts.What is the reason for separating them for analysis.

References
In the references, there are some errors in abbreviations and ltalics of some journal names, such as 10, 11, and some Latin names are not in italics, such as 38, please correct them.

Dear Prof Chen and Reviewers
Thank you for your letter and for the reviewers' comments concerning our manuscript entitled "Altitude as a key environmental factor shaping microbial communities of tea green leafhoppers (Matsumurasca onukii) (Spectrum01009-23"). "Under Peer Review" on 2023-03-10 and "Manuscript in Revision" on 2023-06-06.Those comments are all valuable and very helpful for revising and improving our paper, as well as the important guiding significance to our researches.
We have studied comments carefully and have made correction which we hope meet with approval.The main corrections in the paper and the responds to the reviewer's comments are as flowing:

Q5. There are many abbreviations in the ms that are not easy to understand and need further clarification;
Responds: I am sincerely sorry for the writing mistakes that have caused you trouble.During the writing process, I inadvertently placed the "MATERIALS AND METHODS" section after the "INTRODUCTION", leading to some abbreviations being explained in the wrong section.However, according to the submission format for the manuscript, the "MATERIALS AND METHODS" section should be placed after the "CONCLUSION".As a result, some abbreviations in the "RESULTS" and "DISCUSSION" sections may not be easily understood.We have thoroughly reviewed and modified the abbreviations in this manuscript to address this issue."Altitude alone may not be the primary factor influencing fungal diversity in tea green leafhoppers.

Q11.
Other factors such as geographic location, host specificity, and ecological interactions could have a more substantial impact on shaping the fungal composition.These additional factors might outweigh the influence of altitude, resulting in a lack of significant differences observed in the fungal communities across different regions.

Q8. L391：Why only females? Because microbiome analysis did not distinguish between male and female;
Responds: The amplification of the COI fragment of the mtDNA and 16S rRNA from the female tea green leafhopper was conducted to confirm the identity of the insect samples used in this experiment as Matsumurasca onukii.This step helped ensure that the collected samples were indeed the target species.
Subsequently, for the microbiome analysis, a mixed sample comprising both nymphs and adults (including both males and females) of the tea green leafhopper was used.This approach allowed for a comprehensive assessment of the microbial community associated with the tea green leafhopper across different life stages and genders.

Q9. L414-416: Why mix adults and nymphs? There are differences between them;
Responds: This is an important issue to consider.The decision to mix nymphs and adults together in our study was based on the following reasons: 1) Neither nymphs nor adults alone can fully represent the population of leafhoppers.By mixing both nymphs and adults together, we aimed to obtain a more comprehensive representation of the leafhopper population.Additionally, processing all samples in the same manner ensures consistency and comparability of the data.
2) The tea green leafhopper undergoes incomplete metamorphosis, and the nymphs and adults share similar morphology and feeding patterns.As a result, we anticipated that there would be minimal differences in the microbiome between the two groups.We also investigated the bacterial component of the microbiome carried by tea green leafhoppers at different developmental stages, and the corresponding results are presented in Table 1, Figure 1 and Figure   CS and FR sampling points: These two points were in close proximity, and based on preliminary analysis, there were no significant differences observed in the bacterial composition between them.Therefore, to ensure representative sampling without redundancy, a random selection was made from these points.
CQ and BN sampling points: Similarly, these two points showed similarities in bacterial composition, and no significant differences were identified during preliminary analysis.Thus, a random sampling approach was employed to capture representative samples without duplication.Publication Fees: We have partnered with Copyright Clearance Center to collect author charges.You will soon receive a message from no-reply@copyright.com with further instructions.For questions related to paying charges through RightsLink, please contact Copyright Clearance Center by email at ASM_Support@copyright.com or toll free at +1.877.622.5543.Hours of operation: 24 hours per day, 7 days per week.Copyright Clearance Center makes every attempt to respond to all emails within 24 hours.For a complete list of Publication Fees, including supplemental material costs, please visit our website.
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Thank you for submitting your paper to Spectrum. Sincerely,

Wenli Chen
Editor, Microbiology Spectrum Journals Department American Society for Microbiology 1752 N St., NW Washington, DC 20036 E-mail: spectrum@asmusa.org you for your reminding.This example is a detailed description of the literature cited in the previous sentence (i.e., "For example, microbiome infection frequencies determine the geographic distribution of the chestnut weevil, Curculio sikkimensis").Q2.L70: The above statement cannot reach this conclusion.Please describe it more rigorously; Responds: We have deleted this sentence.At the same time, the paragraph has been slightly modified.Please check in on Line 71-73 Q3.The purpose of the manuscript in the introduction is only to analyze the microbiome diversity of M. onukii and its altitude significance which are too descriptive, and it is recommended to further refine the purpose and significance of the study; Responds: Thank you very much for your suggestion, we modify it in the last paragraph of the Introduction part.Please check it on Line 115-120 "Not only that, but the factors influencing microbial communities in wild populations, including environmental factors and interactions among microbial species, are still poorly understood.Given the tea green leafhopper's broad geographical distribution and high adaptability, it serves as a suitable model for studing the impact of ecological drivers on microbiomes."Results Q4. Figure S6 is meaningful information, and it is recommended to put it in the maintext; Responds: Thank you for your reminding.We have put Figure S6 in the maintext of the manuscript.Please check it on Line 173.
legends are not detailed enough, additional information is needed.Responds: Thank you very much for your suggestion, we have carefully reviewed and rewritten Figure legends.Please check it on Line 681-734 Discussion Q6.The diversity of fungi in the study does not seem to vary significantly between different altitude, especially the core flora, so explanations need to be made during the discussion; Responds: We have revised it.Please check it on Line Line 363-365.

Q7.
Although altitude is an important environmental factor that affects microbial diversity in the study, other factors also play a role, and explanations should also be made during the discussion to avoid misleading the reader.At the same time, appropriate additions and changes should be made in the abstract; Responds: We have revised it.Please check it on Line 336-342.

Figure 3
Figure 3 Composition of gut bacteria in the tea green leafhopper at different developmental staes at six bacterial levels: phyla, class, order, family, genus and species

2 Table 1
Analysis of alpha diversity of gut bacteria in tea green leafhoppers at different developmental stages The diversity index values were compared with Kruskal-Wallis test results after false discovery (FDR) correction; P > 0.05 represent no significantly difference Figure 1 Non-metric Multidimensional Scaling (NMDS) analysis based on Weighted_Unifrac and

Index Low-instar (L) High-instar (H) Adults (A) P value
Unweighted_Unifrac distance (D) calculation the Beta diversity of the gut bacterial community Significant differences were detected by Adonis test; P > 0.05 represent no significantly difference

key environmental factor shaping microbial communities of tea green leafhoppers (Matsumurasca onukii))
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