Diversity and saline-alkali resistance of Coleoptera endosymbiont bacteria in arid and semi-arid climate

ABSTRACT Soil salinization usually occurs in arid and semi-arid climate areas from 37 to 50 degrees north latitude and 73 to 123 degrees east longitude. These regions are inhabited by a large number of Coleopteran insects, which play an important role in the ecological cycle. However, little is known about the endosymbiotic microbial taxa and their biological characteristics in these insects. A study of endosymbiotic microorganisms of Coleoptera from Xinjiang, a typical arid and inland saline area, revealed that endosymbiont bacteria with salinity tolerance are common among the endosymbionts of Coleoptera. Functional prediction of the microbiota analysis indicated a higher abundance of inorganic ion transporters and metabolism in these endosymbiont strains. Screening was conducted on the tolerable 11% NaCl levels of Brevibacterium casei G20 (PRJNA754761), and differential metabolite and proteins were performed. The differential metabolites of the strain during the exponential and plateau phases were found to include benzene compounds, organic acids, and their derivatives. These results suggest that the endosymbiotic microorganisms of Coleoptera in this environment have adaptive evolution to extreme environments, and this group of microorganisms is also one of the important resources for mining saline and alkaline-tolerant chassis microorganisms and high-robustness enzymes. IMPORTANCE Coleoptera insects, as the first largest order of insect class, have the characteristics of a wide variety and wide distribution. The arid and semi-arid climate makes it more adaptable. By studying the endosymbiont bacteria of Coleoptera insects, we can systematically understand the adaptability of endosymbiont bacteria to host and special environment. Through the analysis of endosymbiont bacteria of Coleoptera insects in different saline-alkali areas in arid and semi-arid regions of Xinjiang, it was found that bacteria in different host samples were resistant to saline-alkali stress. These results suggest that bacteria and their hosts co-evolved in response to this climate. Therefore, this study is of great significance for understanding the endosymbiont bacteria of Coleoptera insects and obtaining extremophile resources (Saline-alkali-resistant chassis strains with modification potential for the production of bulk chemicals and highly robust industrial enzymes).

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Reviewer #1 (Comments for the Author): Comments and suggestions for the authors 1.You have written Coleoptera endophyte but endophyte take shelter in the plant tissue at least part of its life cycle.So, how can you tell the term 'endophyte' here?Have you found that those bacteria reside in the plant at least part of its life cycle?2. Have you performed any confirmative test which can assure that strain B. casei G20 is an endosymbiont of Coleoptera insects? 3. It will be better if you use only strain number instead of genus and species name in the methodology section.4. Manuscript is well written and in the present scenario identified strain is significant.Reviewer #2 (Comments for the Author): Thank you for the authors properly addressing all the comments from the reviewers.

Dear Editor and Reviewers,
We are very grateful for your careful review of the manuscript and for providing a summary and encouraging comments on the manuscript.According to the editor and reviewers' comments, we have made some changes marked in red in revised paper, which will not influence the content and framework of the paper.We appreciate for editors and reviewers hard work and hope that the corrections will meet with approval.
Once again, thank you very much for your comments and suggestions.
The reviewer comments are laid out below in italicized font, and specific concerns have been numbered.Our response is given in normal font and changes/additions to the manuscript are given in the blue text.

Reviewer #1 (Comments for the Author):
Comments and suggestions for the authors ① You have written Coleoptera endophyte but endophyte take shelter in the plant tissue at least part of its life cycle.So, how can you tell the term 'endophyte' here?Have you found that those bacteria reside in the plant at least part of its life cycle?
The author's response:Thank you for your meticulous scientific advice.Regarding the use of'endophyte'and'endophytic'， previously we wanted to use them to describe the strains we screened.There is relatively limited research on useful microorganisms in the order Coleoptera and detailed references are scarce.Therefore, we wanted to quote the professional terminology used in the study of plant endophytic bacteria.However, through your advice and some literature search, we found that the prefix'phy'indicates a connection with plants or something involving plants.This prefix usually comes from the Greek word root'phyto'meaning'plant'. Thus, our usage in this context is indeed inappropriate.Following your suggestion, we have replaced'endophyte'or'endophytic'with 'endosymbiont'throughout the entire text.
As for whether the screened strains colonize plants at some point in their life, we believe the following factors may affect their life stages.The research indicates that the Coleoptera insects often colonize plants during the larval stage.After a long period of coevolution with the plants and their endosymbionts, it has been found that the presence of plant DNA can also be detected in the intestines of the insects as they grow into their adult stage (Jacqueline et al., 2024).This may imply a certain relationship between the microorganisms isolated from Coleoptera and plants.
However, because our study focuses on exploring the coevolutionary relationship between hosts(Coleoptera)and endosymbionts in saline-alkali environments and some available resources, we did not extensively discuss the life habits of Coleoptera in its various growth stages.Of course, we are continuing to delve into the connection between insects and their microorganisms in the hope of discovering more intriguing scientific phenomena.

② Have you performed any confirmative test which can assure that strain B. casei G20 is an endosymbiont of Coleoptera insects?
The author's response:Thank you for your constructive feedback.Based on this scientific evidence, we conducted a microbial diversity analysis on the insect bodies.
When we collected samples, these insects were in their natural living state.During transportation, storage, and experimental operation processes, we minimized human experimental errors to the greatest extent possible.Subsequently, we conducted microbial diversity testing on the insect bodies, and the diversity data results of each sample are consistent with the screening results, mutually verifying each other.According to the research method of gut content analysis of a phloem-feeding insect (Cooper et al., 2016),we conducted surface cleaning of the insect bodies by soaking them in a 75% ethanol solution for 30 seconds.After removing the insect bodies, they were soaked in a 1% sodium hypochlorite solution for 5 minutes and rinsed 10 times with sterile water.To assess the effectiveness of surface disinfection, the sterile water from the final rinse was spread on LB solid medium and incubated at 37°C for two days.If no colonies appeared on the plate, it indicated thorough sterilization of the insect body surfaces.
During the screening of strains, we dissected the insect bodies using the most rigorous operational methods to ensure the scientific rigor of the screening process and to avoid any potential errors caused by the external environment.The insects we studied were all in the adult stage.We believe that studying insects in their adult stage can better illustrate the evolutionary relationship between internal microorganisms and insect bodies, thus addressing scientific questions.
Therefore, we can only confirm that the strains originate from inside the insects, as we did not track the life history of the insects, and we did not conduct in-depth research on specific tissue locations.

③ It will be better if you use only strain number instead of genus and species name in the methodology section.
The author's response:We sincerely thank the reviewer for careful reading.As suggested by the reviewer, we have corrected the 'Brevibacterium casei G20 'into 'Brevibacterium linens YS ' .(Line195-196,Line210-21and Line226) ④ Manuscript is well written and in the present scenario identified strain is significant.The author's response:Thank you for the encouragement from the reviewers.We take all the feedback seriously and look forward to making new discoveries in our ongoing research.

Reviewer #2 (Comments for the Author):
① Thank you for the authors properly addressing all the comments from the reviewers.
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Sincerely, Jing Han Editor Microbiology Spectrum
Reviewer #2 (Comments for the Author): I recently had the opportunity to review your abstract titled "Diversity and Saline-alkali Resistance of Coleoptera Endosymbiont Bacteria in Arid andSemi-arid Climate" and I must commend you on the intriguing insights you have provided regarding the endosymbiotic microorganisms of Coleoptera in arid and saline environments.Your study sheds light on an underexplored area of research and offers valuable implications for understanding microbial adaptation to extreme conditions.While I found your findings to be highly compelling, I believe that there is significant potential for enhancing the impact of your study through further refinement of the data analysis section.Specifically, I would like to suggest some areas for major improvement: While you have presented results regarding the abundance and functional predictions of endosymbiont bacteria in Coleoptera, a more comprehensive statistical analysis would greatly strengthen the robustness of your findings.Incorporating appropriate statistical tests to assess the significance of observed differences and correlations would provide greater confidence in the reliability of your conclusions.The differential metabolite and protein analysis of Brevibacterium casei G20 during different growth phases offer valuable insights into its adaptive responses to saline environments.However, to fully leverage the potential of multi-omics data, I recommend integrating these findings with other omics datasets (e.g., transcriptomics, metagenomics) to gain a more holistic understanding of microbial adaptation mechanisms.While you have highlighted the presence of inorganic ion transporters and metabolism-related pathways in the endosymbiont strains, further elaboration on the functional significance of these findings would enhance the interpretability of your results.Providing insights into how these metabolic pathways contribute to salinity tolerance and microbial adaptation would enrich the discussion and broaden the impact of your study.Including a discussion of methodological considerations, such as sample processing techniques, quality control measures, and potential limitations of the analytical methods employed, would provide important context for interpreting the results and help readers assess the robustness of the findings.Incorporating these suggestions into the data analysis section of your study has the potential to significantly enhance its scientific rigor and impact.I am confident that with these improvements, your study will make an even greater contribution to the field of microbial ecology and adaptation to extreme environments.