The worldwide prevention of gestational diabetes mellitus (GDM) is a significant health challenge. Plant-based dietary patterns are a series dietary habits that emphasized foods derived from plant sources more and from animal foods less. Now, no consensus exists on the effects of plant-based dietary patterns on the incident of GDM.This study aimed to estimate the effects of plant-based dietary patterns on the risk of developing GDM.This systematic review was conducted following the checklist of PRISMA. Six electronic databases including PubMed, Embase, Web of Science, China National Knowledge Infrastructure, Wangfang, and Chinese Scientific Journals Database were searched from inception to November 20, 2022. A fixed or random effect model was used to synthesize results of included studies. Then, subgroup analysis, meta-regression and sensitivity analysis were performed to assure the reliability and stability of the results.I2 = 14.8%). The slightly stronger association between plant-based diets and the risk of developing GDM was found when healthy plant-based dietary pattern index was included in pooled estimate , compared with that unhealthy one was included .Ten studies including 32,006 participants were identified. The results of this study showed that the better adherence to the plant-based dietary patterns was related to the lower risk of developing GDM (RR = 0.88[0.81 to 0.96], The plant-based dietary patterns are associated with a lower risk of developing GDM. Furthermore, healthy plant-based dietary patterns are more recommended than unhealthy one. It is significant to help medical staff to guide pregnant women to choose reasonable diets. Gestational diabetes mellitus (GDM) refers to diabetes diagnosed during gestation that not detected glucose intolerance prior to pregnancy . GDM affDiet plays an important role during gestation . MaternaPlant-based dietary pattern is an umbrella term that refers to a habit that emphasized foods derived from plant sources more and from animal foods less, such as vegetarian diets ,21. Globhttps://www.crd.york.ac.uk/PROSPERO/) with registration No. CRD42022374542 and was conducted according to the checklist of PRISMA Reviewers' comments:Reviewer's Responses to Questions  Comments to the Author1. Is the manuscript technically sound, and do the data support the conclusions?  The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented. Reviewer #1: YesReviewer #2: YesReviewer #3: Partly**********   2. Has the statistical analysis been performed appropriately and rigorously?   Reviewer #1: YesReviewer #2: NoReviewer #3: Yes**********   3. Have the authors made all data underlying the findings in their manuscript fully available?PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.  The Reviewer #1: YesReviewer #2: YesReviewer #3: Yes**********   4. Is the manuscript presented in an intelligible fashion and written in standard English?  PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.Reviewer #1: YesReviewer #2: YesReviewer #3: Yes**********   5. Review Comments to the Author  Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. Reviewer #1: Zhu and colleagues conducted a meta-analysis exploring the evidence linking plant-based or vegetarian dietary patterns to the risk of developing gestational diabetes mellitus. Ten prospective cohort studies were found in multiple databases and selected for the analysis. After robust statistical analysis which included assessment of the dietary patterns (plant based indices verses adherence to predefined vegetarian/vegan diets) and adjustments for confounders such as BMI, the authors concluded that plant-based dietary patterns are associated with a lower risk for developing gestational diabetes. This association was stronger when "healthy plant-based indices" were used, and weaker after adjusting for BMI. The authors address the confounder of BMI and gestational weight gain, the latter of which was not included in the analysis or the selected studies. The authors also speculate the potential mechanisms of which plant-based diets decrease the risk for gestational diabetes; one being the impact of changing the gut microbiome with increased consumption of plant-based foods and the decrease for inflammation, which is associated with diabetes.Line 50-Led to should be "leads"Line 52: Macrosomia and large for gestational age seem redundant; perhaps pick one to mention.line 292: adversely might be changed to inverselyOverall, the authors did a thorough statistical analysis from various angles  to assess the strength of the studies included. The results and interpretation are nicely described with good discussion of confounding factors.Reviewer #2: In this paper, Yu Zhu and colleagues have done a systematic review and meta analysis on the effect of plant based dietary patterns on the risk of GDM. They have included 10 studies which included 32,006 participants. The results of the systematic meta analysis suggest that better adherence to plant based dietary patterns was related to lower risk of developing GDM. The authors have to be congratulated on a nice piece of work. This study has been done carefully. It has been registered at PROSPERO International Prospective Register of Systematic Reviews. Data extraction and assessment of risk of bias has been done carefully. The PRISMA flowchart of study selection presented clearly. Publication bias was tested by Egger's and Begg's test.Suggestions :It would have been nice if the composition of the plant based diets were examined in greater detail . Many plant based diets tend to be rich in carbohydrates which increase the risk of type 2 diabetes. In this context, if the authors could comment on why plant based diets are protective and how they differ from animal based diets, that would make the paper more interesting.In the ‘Discussion” on Page 22 and in the “Conclusion”, they do talk about this. But perhaps in the context of healthy carbohydrates and unhealthy carbohydrates, this section can be expanded a little bit more.Reviewer #3: I have several comments and suggestions regarding the methods section of the manuscript, specifically related to improving its clarity and comprehensibility. Please find below my detail comments:1. To enhance clarity, several points in the abstract require further clarification:a. Line 37-39: revisit the statement as the statement implies that both unhealthy and healthy plant-based diets are associated with a lower risk of GDM, with the healthy plant-based diet showing a slightly stronger association.b. Line 40-41: This statement should be included in the methodology section.2. Introduction:a. While the author highlights the benefits of a plant-based dietary pattern and its positive effects on maternal and infant outcomes, it would be valuable to consider mentioning the current prevalence or proportion of individuals adopting such a dietary pattern. Including this information would help support the rationale for conducting this study and shed light on the relevance and potential impact of investigating the association between plant-based diets and gestational diabetes mellitus.3. Materials and methods:a. Line 92: The statement "Vegetarian dietary patterns or vegan dietary patterns were also reckoned with" may not conform to typical scientific writing standards.b. Line 89-95: It would be beneficial to provide explicit definitions of both vegetarian and vegan dietary patterns to ensure clarity in understanding their distinctions. While both dietary patterns involve a focus on plant-based foods, vegetarian diets typically exclude meat, poultry, and seafood, while vegan diets abstain from all animal-derived products, including dairy, eggs, and honey. By clearly defining these terms, readers will have a better understanding of the specific dietary restrictions and considerations associated with each pattern, which is crucial for interpreting the study findings accurately.c. Line 109: Please verify the accuracy of the term 'VIP' as a database name. Please double-check its validity or provide additional clarification if it refers to a specific database or acronym.d. Please consider rechecking the PRISMA checklist to ensure its adherence to the guidelines. Upon review, it appears that certain items may not fully align with the recommended criteria. For example: Data items, list and define all outcomes for which data were sought. Specify whether all results that were compatible with each outcome domain in each study were sought e. Line 148-152: please provide additional elaboration on the details of the subgroup analysis and sensitivity analysis conducted in this study?4. Resultsa. Line 169-170: please provide explicit definitions for the pre-defined vegetarian diet and semi-vegetarian diet used in this study?b. Line 179: kindly review the text and add the appropriate units where necessary**********   6. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.If you choose “no”, your identity will remain anonymous but your review may still be made public.Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.  Reviewer #1: NoReviewer #2: NoReviewer #3: No**********https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email PLOS at figures@plos.org. Please note that Supporting Information files do not need this step.While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool,  25 Jul 2023Dear Editor Kent Lai and Reviewer #1, Reviewer #2, and Reviewer #3,Thank you so much for your work on our manuscript. Please accept our sincerely appreciation for your comments. On behalf of all authors in the article, I am sure that the scientific content of the article fully complies with all the journal requirements. Please read our answers about every question as following. We hope that our answers and corrections will be fine.For Editor's comments:Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.Our answer,Thank you so much for your work on our manuscript. Please accept our sincere appreciation. We had carefully revised related content in our manuscript. The response of each question had been carefully answered in the part of “For Reviewer’s comments” as following. Please see the new manuscript and Figures.1. Please ensure that your manuscript meets PLOS ONE’s style requirements, including those for file naming.Our answer,We had carefully checked our manuscript. And then, we corrected file naming of Figure so that controlling the its length within 15 words, and revised the footnote of Table 1 and cell lines of Table 2 for ensuring that our manuscript meets PLOS ONE’s style requirements. Please see the new manuscript and Figures.2. We note that the grant information you provided in the ‘Funding Information’ and ‘Financial Disclosure’ sections do not match. When you resubmit, please ensure that you provide the correct grant numbers for the awards you received for your study in the ‘Funding Information’ section.Our answer,We were very sorry that we did not do better in in providing match ‘Funding Information’ and ‘Financial Disclosure’. This research was funded by Joint Funds for the innovation of science and Technology, Fujian province (Grant number: 2020Y9133) and Fujian Maternity and Child Health Hospital (Grant number: YCXH 22-02). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. We were sure that we had provided the correct grant numbers of funding, and had clarified financial disclosure.http://journals.plos.org/plosone/s/submit-now. This information should be included in your cover letter; we will change the online submission form on your behalf.3. Thank you for stating the following in your Competing Interests section: “The authors declare that they have no competing interests.” Please complete your Competing Interests on the online submission form to state any Competing Interests. If you have no competing interests, please state “The authors have declared that no competing interests exist.”, as detailed online in our guide for authors at Our answer,We were sure that: “The authors declare that they have no competing interests”. 4. Please review your reference list to ensure that it is complete and correct. If you have cited papers that have been retracted, please include the rationale for doing so in the manuscript text, or remove these references and replace them with relevant current references. Any changes to the reference list should be mentioned in the rebuttal letter that accompanies your revised manuscript. If you need to cite a retracted article, indicate the article’s retracted status in the References list and also include a citation and full reference for the retraction notice.Our answer, We had carefully reviewed and checked our reference by searching in Web of Science, Pubmed and Wangfang database one by one. We are sure that all references are complete and correct. And we did not cite any papers that have been retracted. 5. Some of the figures were quite blurry. Please improve the resolution.Our correction,We were very sorry that we did not do better in processing resolution of Figures. We had improved the resolution of every Figure according to Figure format guidelines of PLOS ONE. Please see the new Figures.For reviewer #1’s comments:1. Line 50: Led to should be “leads”.2. Line 52: Macrosomia and large for gestational age seem redundant; perhaps pick one to mention.Our correction,Page 3 line 46 to 49: GDM affects about 5.8~12.9% of pregnancies globally depending on different diagnostic criteria and leads several adverse obstetrical outcomes including hypertensive disorders, cesarean section, neonatal hypoglycemia, and macrosomia [4-6].2. Zhu Y, Zhang C. Prevalence of Gestational Diabetes and Risk of Progression to Type 2 Diabetes: a Global Perspective. Curr Diab Rep. 2016;16(1):7.DOI: 10.1007/s11892-015-0699-x.3. Chiefari E, Arcidiacono B, Foti D, Brunetti A. Gestational diabetes mellitus: an updated overview. Journal of endocrinological investigation. 2017;40(9):899-909.DOI: 10.1007/s40618-016-0607-5.4. Ye W, Luo C, Huang J, Li C, Liu Z, Liu F. Gestational diabetes mellitus and adverse pregnancy outcomes: systematic review and meta-analysis. BMJ . 2022;377:e067946.DOI: 10.1136/bmj-2021-067946.5. Davenport MH, Ruchat SM, Poitras VJ, Jaramillo Garcia A, Gray CE, Barrowman N, et al. Prenatal exercise for the prevention of gestational diabetes mellitus and hypertensive disorders of pregnancy: a systematic review and meta-analysis. Br J Sports Med. 2018;52(21):1367-75.DOI: 10.1136/bjsports-2018-099355.6. Koivunen S, Torkki A, Bloigu A, Gissler M, Pouta A, Kajantie E, et al. Towards national comprehensive gestational diabetes screening - consequences for neonatal outcome and care. Acta Obstet Gynecol Scand. 2017;96(1):106-13.DOI: 10.1111/aogs.13030.3. Line 292: adversely might be changed to inversely.Our correction,Page 20 line 325 to 327: In this systematic review and meta-analysis, we included ten studies with moderate to high quality involving fourteen records and found that greater adherence to plant-based dietary patterns is inversely associated with the risk of developing GDM.For Reviewer #2’s comments:It would have been nice if the composition of the plant based diets were examined in greater detail . Many plant based diets tend to be rich in carbohydrates which increase the risk of type 2 diabetes. In this context, if the authors could comment on why plant based diets are protective and how they differ from animal based diets, that would make the paper more interesting. In the “Discussion” on Page 22 and in the “Conclusion”, they do talk about this. But perhaps in the context of healthy carbohydrates and unhealthy carbohydrates, this section can be expanded a little bit more.Our answer,Thank you so much for your work on our manuscript. According to your comments, we carefully reviewed the details of plant-based dietary patterns proposed by the included studies. Regretfully, the included studies did not analyze plant-based dietary patterns in view of the breakdown of carbohydrate, protein and fat. Thus, we may not be able to examine this facet in greater detail. However, in “Discussion” section, we discussed the carbohydrates quality, which demonstrated the necessity of distinguishing between healthy carbohydrates and unhealthy carbohydrates. Additionally, we highlighted the importance of the source of protein and fat  in plant-based dietary patterns. We hope that our discussion will be fine.Our correction,Page 22 line 380 to page 23 line 402: The stronger pooled RR was observed when we considered “healthful plant-based dietary indices”  instead of “unhealthy plant-based dietary indices”  in an included study[36]. The finding indicated an opinion that not all plant-based diets were beneficial, which has been emphasized in prior studies. Carbohydrates quality, a multidimensional index, usually be represented as some characteristics: glycaemic index, glycaemic load and dietary fiber content. It was considered as a crucial facet of plant-based diets[63]. For instance, plants with high glycaemic index, including potatoes, rice and refined bread, induced a sharp increase in glycaemia level that manifested the lower carbohydrates quality[64]. Plant-based diets dominated by such components were associated with higher risk of developing GDM, whereas plant-based diets with low glycaemic index were related to the lower prevalence of GDM[67]. Besides, Looman et al.[62] unexpectedly found that low-carbohydrate diets, characterized by high fat, high protein and low carbohydrates, were positive associated with the risk of developing GDM. Their further exploration revealed that women with higher fiber and fruits intake had a decreased risk of developing GDM, however, women with higher cereal intake had increased risk of developing GDM. It also underscored the importance of the carbohydrates source. Except for carbohydrates quality, the source of protein and fat in plant-based dietary patterns mattered, too. Pregestational low-carbohydrate diets with high protein and fat from animal foods were positively associated with the risk of developing GDM, nevertheless, low-carbohydrate diets with high protein and fat from vegetable foods were inversely related to the risk of developing GDM[70]. In detail, higher animal protein intake would increase the risk of developing GDM, whereas higher plant protein intake would do not[68]. In addition, higher fat intake, whether animal fat or plant fat, would increase the risk of developing GDM[69]. As such, plant-based dietary patterns with higher carbohydrates quality and plant protein consumption were more encouraged. Moreover, the concept of healthful plant-based diet was proposed by Satija et al.[33], who classified sugar-sweetened beverages, sweets and desserts, refined grains and juices as relatively unhealthy plant diets. These food groups were related to a higher risk of GDM[71], which may be interpreted by following potential mechanisms. Higher glycemic load from sugar-sweetened beverages and sweets and desserts may lead to increased weight gain through decreasing satiety or increased hunger signals, and disrupt metabolism via affecting neurochemical changes in the brain[72-74]. Milling and squeezing during the productive process of refined grains and juices reduced dietary fiber and bioactive phytochemicals, then potentially lowering dietary quality[75]. Significantly, it is necessary to distinguish whether the type of juice is homemade or commercial. The homemade juices might not be removed the pulp and edible skin, thus can provide some essential components for preventing GDM, such as fiber, vitamins, minerals, and phytochemicals[76]. Adversely, commercial juices provide only excessive sugars and energy that overload the glycometabolism. Overall, only healthy plant-based diets reduce the risk of GDM rather than unhealthy plant-based diets. We recommended that quality of foods should be emphasized when sticking to plant-based dietary patterns.33. Satija A, Bhupathiraju SN, Rimm EB, Spiegelman D, Chiuve SE, Borgi L, et al. Plant-Based Dietary Patterns and Incidence of Type 2 Diabetes in US Men and Women: Results from Three Prospective Cohort Studies. PLoS Med. 2016;13(6):e1002039.DOI: 10.1371/journal.pmed.1002039.36. Chen ZL, Qian F, Liu G, Li MY, Voortman T, Tobias DK, et al. Prepregnancy plant-based diets and the risk of gestational diabetes mellitus: a prospective cohort study of 14,926 women. Am J Clin Nutr. 2021;114(6):1997-2005.DOI: 10.1093/ajcn/nqab275.61. Satija A, Malik V, Rimm EB, Sacks F, Willett W, Hu FB. Changes in intake of plant-based diets and weight change: results from 3 prospective cohort studies. The American journal of clinical nutrition. 2019;110(3):574-82.DOI: 10.1093/ajcn/nqz049.62. Looman M, Schoenaker D, Soedamah-Muthu SS, Geelen A, Feskens EJM, Mishra GD. Pre-pregnancy dietary carbohydrate quantity and quality, and risk of developing gestational diabetes: the Australian Longitudinal Study on Women's Health. The British journal of nutrition. 2018;120(4):435-44.DOI: 10.1017/s0007114518001277.63. Filardi T, Panimolle F, Crescioli C, Lenzi A, Morano S. Gestational Diabetes Mellitus: The Impact of Carbohydrate Quality in Diet. Nutrients. 2019;11(7):1549.DOI: 10.3390/nu11071549.64. Brouns F, Bjorck I, Frayn KN, Gibbs AL, Lang V, Slama G, et al. Glycaemic index methodology. Nutrition research reviews. 2005;18(1):145-71.DOI: 10.1079/nrr2005100.65. Bao W, Tobias DK, Hu FB, Chavarro JE, Zhang C. Pre-pregnancy potato consumption and risk of gestational diabetes mellitus: Prospective cohort study. BMJ (Online). 2016;352:h6898.DOI: 10.1136/bmj.h6898.66. Guo F, Zhang Q, Jiang H, He Y, Li M, Ran JJ, et al. Dietary potato intake and risks of type 2 diabetes and gestational diabetes mellitus. Clin Nutr. 2021;40(6):3754-64.DOI: 10.1016/j.clnu.2021.04.039.67. Walsh JM, McGowan CA, Mahony R, Foley ME, McAuliffe FM. Low glycaemic index diet in pregnancy to prevent macrosomia (ROLO study): randomised control trial. BMJ . 2012;345:e5605.DOI: 10.1136/bmj.e5605.68. Yong HY, Mohd Shariff Z, Mohd Yusof BN, Rejali Z, Tee YYS, Bindels J, et al. Higher Animal Protein Intake During the Second Trimester of Pregnancy Is Associated With Risk of GDM. Frontiers in nutrition. 2021;8:718792.DOI: 10.3389/fnut.2021.718792.69. Feng Q, Yang M, Dong H, Sun H, Chen S, Chen C, et al. Dietary fat quantity and quality in early pregnancy and risk of gestational diabetes mellitus in Chinese women: a prospective cohort study. The British journal of nutrition. 2022:1-10.DOI: 10.1017/s0007114522002422.70. Bao W, Bowers K, Tobias DK, Olsen SF, Chavarro J, Vaag A, et al. Prepregnancy low-carbohydrate dietary pattern and risk of gestational diabetes mellitus: a prospective cohort study. The American journal of clinical nutrition. 2014;99(6):1378-84.DOI: 10.3945/ajcn.113.082966.71. Shin D, Lee KW, Song WO. Dietary Patterns during Pregnancy Are Associated with Risk of Gestational Diabetes Mellitus. Nutrients. 2015;7(11):9369-82.DOI: 10.3390/nu7115472.72. Chen L, Hu FB, Yeung E, Willett W, Zhang C. Prospective study of pre-gravid sugar-sweetened beverage consumption and the risk of gestational diabetes mellitus. Diabetes Care. 2009;32(12):2236-41.DOI: 10.2337/dc09-0866.73. Berthoud HR. The neurobiology of food intake in an obesogenic environment. The Proceedings of the Nutrition Society. 2012;71(4):478-87.DOI: 10.1017/s0029665112000602.74. Levine AS, Kotz CM, Gosnell BA. Sugars: hedonic aspects, neuroregulation, and energy balance. The American journal of clinical nutrition. 2003;78(4):834s-42s.DOI: 10.1093/ajcn/78.4.834S.75. Zhu Y, Olsen SF, Mendola P, Halldorsson TI, Yeung EH, Granström C, et al. Maternal dietary intakes of refined grains during pregnancy and growth through the first 7 y of life among children born to women with gestational diabetes. The American journal of clinical nutrition. 2017;106(1):96-104.DOI: 10.3945/ajcn.116.136291.76. Yong HY, Mohd Shariff Z, Mohd Yusof BN, Rejali Z, Tee YYS, Bindels J, et al. Beverage Intake and the Risk of Gestational Diabetes Mellitus: The SECOST. Nutrients. 2021;13(7):2208.DOI: 10.3390/nu13072208.For Reviewer #3’s comments:1. To enhance clarity, several points in the abstract require further clarification:a. Line 37-39: revisit the statement as the statement implies that both unhealthy and healthy plant-based diets are associated with a lower risk of GDM, with the healthy plant-based diet showing a slightly stronger association.Our answer,Thank you so much for your work on our manuscript. We were so sorry that we did not do better in clarifying this section. We had carefully revised related content in our manuscript. Please see the new Manuscript.Our correction,In the section of Abstract Page2 line 35 to 38: The slightly stronger association between plant-based diets and the risk of developing GDM was found when healthy plant-based dietary pattern index was included in pooled estimate , compared with that unhealthy one was included .b. Line 40-41: This statement should be included in the methodology section.Our correction,In the section of AbstractPage 2 line 31 to 32: Methods: This systematic review was conducted following the checklist of PRISMA. Six electronic databases including PubMed, Embase, Web of Science, China National Knowledge Infrastructure, Wangfang, and VIP database were searched from inception to November 20, 2022. A fixed or random effect model was used to synthesize results of included studies. Then, subgroup analysis, meta-regression and sensitivity analysis were performed to assure the reliability and stability of the results.2. Introduction:a. While the author highlights the benefits of a plant-based dietary pattern and its positive effects on maternal and infant outcomes, it would be valuable to consider mentioning the current prevalence or proportion of individuals adopting such a dietary pattern. Including this information would help support the rationale for conducting this study and shed light on the relevance and potential impact of investigating the association between plant-based diets and gestational diabetes mellitus.Our correction,In the section of Introduction:Page 4 line 70 to 76: Plant-based dietary pattern is an umbrella term that refers to a habit that emphasized foods derived from plant sources more and from animal foods less, such as vegetarian diets. Globally, the prevalence of adopting vegetarian diets varied according to different countries culture, whereas it was commonly less than 10%[22]. It indicated that current proportion of pregnant women who adhered to remains vegetarian diets may still relatively small. The Academy of Nutrition and Dietetics advocated that vegetarian diets for pregnant women could be nutritionally adequate in pregnancy and result in positive maternal and infant outcomes[23], and this viewpoint had got partial support from several previous systematic reviews[24-27]. Practically, women’s dietary patterns changed little from before to during pregnancy[28]. It is confirmed that plant-based dietary pattern is beneficial to decrease the risk of developing type 2 diabetes mellitus[29]. However, the conclusion that the effects of plant-based dietary patterns on developing GDM remains unclear. Therefore, in this systematic review and meta-analysis, we aimed to investigate the effects of plant-based diets on the risk of developing GDM.20. Gibbs J, Cappuccio FP. Plant-Based Dietary Patterns for Human and Planetary Health. Nutrients. 2022;14(8):1614.DOI: 10.3390/nu14081614.21. David LA, Maurice CF, Carmody RN, Gootenberg DB, Button JE, Wolfe BE, et al. Diet rapidly and reproducibly alters the human gut microbiome. Nature. 2014;505(7484):559-63.DOI: 10.1038/nature12820.22. Craig WJ, Mangels AR, Fresán U, Marsh K, Miles FL, Saunders AV, et al. The Safe and Effective Use of Plant-Based Diets with Guidelines for Health Professionals. Nutrients. 2021;13(11):4144.DOI: 10.3390/nu13114144.23. Craig WJ, Mangels AR. Position of the American Dietetic Association: vegetarian diets. Journal of the American Dietetic Association. 2009;109(7):1266-82.DOI: 10.1016/j.jada.2009.05.027.24. Raghavan R, Dreibelbis C, Kingshipp BL, Wong YP, Abrams B, Gernand AD, et al. Dietary patterns before and during pregnancy and birth outcomes: A systematic review. Am J Clin Nutr. 2019;109:729S-56S.DOI: 10.1093/ajcn/nqy353.25. Sebastiani G, Barbero AH, Borrás-Novel C, Casanova MA, Aldecoa-Bilbao V, Andreu-Fernández V, et al. The effects of vegetarian and vegan diet during pregnancy on the health of mothers and offspring. Nutrients. 2019;11(3):557.DOI: 10.3390/nu11030557.26. Tan C, Zhao Y, Wang S. Is a vegetarian diet safe to follow during pregnancy? A systematic review and meta-analysis of observational studies. Critical reviews in food science and nutrition. 2019;59(16):2586-96.DOI: 10.1080/10408398.2018.1461062.27. Schiattarella A, Lombardo M, Morlando M, Rizzo G. The Impact of a Plant-Based Diet on Gestational Diabetes: A Review. Antioxidants . 2021;10(4):557.DOI: 10.3390/antiox10040557.28. Crozier SR, Robinson SM, Godfrey KM, Cooper C, Inskip HM. Women's dietary patterns change little from before to during pregnancy. The Journal of nutrition. 2009;139(10):1956-63.DOI: 10.3945/jn.109.109579.29. Qian F, Liu G, Hu FB, Bhupathiraju SN, Sun Q. Association Between Plant-Based Dietary Patterns and Risk of Type 2 Diabetes: A Systematic Review and Meta-analysis. JAMA internal medicine. 2019;179(10):1335-44.DOI: 10.1001/jamainternmed.2019.2195.3. Materials and methods:a. Line 92: The statement “Vegetarian dietary patterns or vegan dietary patterns were also reckoned with” may not conform to typical scientific writing standards.Our answer,We were sorry about we used atypical terms. We reviewed the scientific writing terms and corrected “vegetarian dietary patterns” and “vegan dietary patterns” to “vegetarian diet” and “vegan diet” according to MeSH terms, respectively. Our correction,In the section of Materials and methods:Page 5 line 104 to 105: According to related studies that assessed dietary patterns, three methods mainly were used to define the plant-based dietary patterns: (a) prior designated diets that avoidance of animal-based foods, such as vegetarian diet and vegan diet; (b) a previous plant-based dietary indices scores; and (c) a posteriori factor analysis to extract dietary pattern.31. Storz MA. What makes a plant-based diet? a review of current concepts and proposal for a standardized plant-based dietary intervention checklist. Eur J Clin Nutr. 2022;76(6):789-800.DOI: 10.1038/s41430-021-01023-z.32. Gan ZH, Cheong HC, Tu YK, Kuo PH. Association between Plant-Based Dietary Patterns and Risk of Cardiovascular Disease: A Systematic Review and Meta-Analysis of Prospective Cohort Studies. Nutrients. 2021;13(11):3952.DOI: 10.3390/nu13113952.b. Line 89-95: It would be beneficial to provide explicit definitions of both vegetarian and vegan dietary patterns to ensure clarity in understanding their distinctions. While both dietary patterns involve a focus on plant-based foods, vegetarian diets typically exclude meat, poultry, and seafood, while vegan diets abstain from all animal-derived products, including dairy, eggs, and honey. By clearly defining these terms, readers will have a better understanding of the specific dietary restrictions and considerations associated with each pattern, which is crucial for interpreting the study findings accurately.Our answer, We had provided explicit definitions of both vegetarian and vegan diets. Moreover, to enhance clarity of article, we separated a single part to explain assessment of plant-based dietary patterns in our study.Our correction,In the section of Materials and methods,Page 4 line 90 to 92: The inclusion criteria were defined as following: (a) Population involving women with singleton pregnancy and without any acute or chronic diseases that impact dietary intake, such as cancer or kidney disease. (b) The exposure of interest was adherence to the plant-based dietary patterns, which generally were defined as diets that consuming higher consumption of planted-based foods and lower consumption or avoidance of animal-based foods. (c) The comparator depends on actual exposure. (d) The outcome of interest was the incident of GDM……Page 5 line 102 to page 6 line 130: Assessment of plant-based dietary patternsAccording to related studies that assessed dietary patterns, three methods mainly were used to define the plant-based dietary patterns: (a) prior designated diets that avoidance of animal-based foods, such as vegetarian diet and vegan diet; (b) a previous plant-based dietary indices scores; and (c) a posteriori factor analysis to extract dietary pattern.Especially, vegetarian diets typically included plant-based foods including grains, legumes, nuts, seeds vegetables and fruits, and excluded all kinds of animal-based foods such as meat, meat products, poultry, seafood, mollusks and crustaceans[25]. Vegetarian diets usually contain two main directions: (1) lacto-ovo-vegetarian diets, which excluded meat but included dairy products, eggs and honey; (2) vegan diets, which excluded meat, dairy products, eggs and honey[25]. In this study, non-vegetarian diets or omnivorous diets were considered as the comparators of vegetarian diets/vegan diets.When turns to previous plant-based dietary indices scores, Satija et al.[33] created a concept of plant-based dietary pattern indices to assess the adherence to plant-based dietary patterns including overall plant-based dietary indices (PDI), healthy plant-based dietary indices (hPDI), and unhealthy plant-based dietary indices (uPDI). Scoring was done according to quantile with the lowest food consumption receiving 1 point and quantile with highest food consumption receiving 5 points. For PDI, positive scores were assigned to plant food groups, and reverse scores were assigned to animal food groups. For hPDI, positive scores were assigned to healthy plant food groups , while reverse scores were assigned to animal food groups and unhealthy plant food groups. For uPDI, positive scores were assigned to unhealthy plant food groups , whereas reverse scores were assigned to animal food groups and healthy plant food groups. By above definition, the lowest PDI, hPDI and uPDI quantile, which indicated the poorest insistence on the plant-based dietary patterns, were used as a comparator of highest PDI, hPDI and uPDI quantile, respectively. Also, for the included studies that used factor analysis to extract plant-based dietary patterns, the lowest quantile was used as a comparator.25. Sebastiani G, Barbero AH, Borrás-Novel C, Casanova MA, Aldecoa-Bilbao V, Andreu-Fernández V, et al. The effects of vegetarian and vegan diet during pregnancy on the health of mothers and offspring. Nutrients. 2019;11(3):557.DOI: 10.3390/nu11030557.31. Storz MA. What makes a plant-based diet? a review of current concepts and proposal for a standardized plant-based dietary intervention checklist. Eur J Clin Nutr. 2022;76(6):789-800.DOI: 10.1038/s41430-021-01023-z.32. Gan ZH, Cheong HC, Tu YK, Kuo PH. Association between Plant-Based Dietary Patterns and Risk of Cardiovascular Disease: A Systematic Review and Meta-Analysis of Prospective Cohort Studies. Nutrients. 2021;13(11):3952.DOI: 10.3390/nu13113952.33. Satija A, Bhupathiraju SN, Rimm EB, Spiegelman D, Chiuve SE, Borgi L, et al. Plant-Based Dietary Patterns and Incidence of Type 2 Diabetes in US Men and Women: Results from Three Prospective Cohort Studies. PLoS Med. 2016;13(6):e1002039.DOI: 10.1371/journal.pmed.1002039.c. Line 109: Please verify the accuracy of the term ‘VIP’ as a database name. Please double-check its validity or provide additional clarification if it refers to a specific database or acronym.Our answer,‘VIP’ is the abbreviation of Chinese Scientific Journals Database. We have provided the complete description here for accuracy.Our correction,In the section of Abstract: Page 2 line 27 to 30: Methods: This systematic review was conducted following the checklist of PRISMA. Six electronic databases including PubMed, Embase, Web of Science, China National Knowledge Infrastructure, Wangfang, and Chinese Scientific Journals Database were searched from inception to November 20, 2022. A fixed or random effect model was used to synthesize results of included studies…….In the section of Materials and methods:Page 6 line 133 to 135: Six electronic databases including PubMed, Embase, Web of Science, China National Knowledge Infrastructure (CNKI), Wangfang, and Chinese Scientific Journals Database (VIP) were searched using a strategy of Mesh-term combined with text-word.d. Please consider rechecking the PRISMA checklist to ensure its adherence to the guidelines. Upon review, it appears that certain items may not fully align with the recommended criteria. For example: Data items, list and define all outcomes for which data were sought. Specify whether all results that were compatible with each outcome domain in each study were sought .Our correction,We carefully rechecked items of the PRISMA checklist and refilled it. Please see our new S3 Appendix. e. Line 148-152: please provide additional elaboration on the details of the subgroup analysis and sensitivity analysis conducted in this study?Our correction,Page8 ling 175 to 187: Subgroup analysis and meta-regression analysis were estimated for regulating effect sizes of the following possible variables: study country , method of dietary patterns assessment  and period of dietary patterns investigation (before pregnancy and during pregnancy). Sensitivity analysis was conducted by removing one study from overall analysis each time. Moreover, for studies that reported the results before and after BMI adjustment, we included those data to assess the pooled results changes before and after BMI adjustment. In the studies that defined adherence to plant-based dietary patterns using dietary overall or healthy/unhealthy plant-based dietary indices, the relationship for overall plant-based dietary indices was estimated in the pooled result evaluation, the relationship for healthful/unhealthy plant-based dietary indices were estimated in a sensitivity analysis. Besides, sensitivity analysis was conducted through excluding any single study.4. Resultsa. Line 169-170: please provide explicit definitions for the pre-defined vegetarian diet and semi-vegetarian diet used in this study?Our answer, In this study, we used the definitions for the pre-defined vegetarian diet and semi-vegetarian diet were defined by the authors of the included study. We had added the definitions in the Manuscript. Our correction,Page 9 line 203 to 207: Particularly, Yisahak et al.[35] reported two records that full vegetarian diet and semi-vegetarian diet from two investigations. They defined ‘full vegetarian diet’ as never ate meat, poultry and fish, or ate these foods less than once a month but had no restriction on fish; and ‘semi-vegetarian diet’ as ate meat, poultry and fish greater than once a month, but less than once a week.35. Yisahak SF, Hinkle SN, Mumford SL, Li MY, Andriessen VC, Grantz KL, et al. Vegetarian diets during pregnancy, and maternal and neonatal outcomes. International journal of epidemiology. 2021;50(1):165-78.DOI: 10.1093/ije/dyaa200.b. Line 179: kindly review the text and add the appropriate units where necessaryOur correction,Page 9 line 215 to page 10 line 217: Totally, 32,006 women were included, who were with mean pre-pregnancy/pregnancy BMI ranging from 20.4 to 25.8 kg/m2 and their age ranging from 26.7 to 32.3 years old.All the best.Thanks again.Yours sincerely,AuthorsAttachmentResponse to Reviewers .docxSubmitted filename: Click here for additional data file. 4 Sep 2023The effects of plant-based dietary patterns on the risk of developing gestational diabetes mellitus: A systematic review and meta-analysisPONE-D-23-15568R1Dear Dr. Jiang,We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.Within one week, you’ll receive an e-mail detailing the required amendments. 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Nosema is primarily known to infect insects of economic importance stimulating high research interest, while other hosts remain understudied. Nosema granulosis is one of the formally described Nosema species infecting amphipod crustaceans, being known to infect only two host species. Our first aim was to characterize Nosema spp. infections in different amphipod species from various European localities using the small subunit ribosomal DNA (SSU) marker. Second, we aimed to assess the phylogenetic diversity, host specificity and to explore the evolutionary history that may explain the diversity of gammarid-infecting Nosema lineages by performing a phylogenetic reconstruction based on RNA polymerase II subunit B1 (RPB1) gene sequences. For the host species Gammarus balcanicus, we also analyzed whether parasites were in excess in females to test for sex ratio distortion in relation with Nosema infection. We identified Nosema spp. in 316 individuals from nine amphipod species being widespread in Europe. The RPB1-based phylogenetic reconstruction using newly reported sequences and available data from other invertebrates identified 39 haplogroups being associated with amphipods. These haplogroups clustered into five clades (A-E) that did not form a single amphipod-infecting monophyletic group. Closely related sister clades C and D correspond to Nosema granulosis. Clades A, B and E might represent unknown Nosema species infecting amphipods. Host specificity seemed to be variable with some clades being restricted to single hosts, and some that could be found in several host species. We show that Nosema parasite richness in gammarid hosts is much higher than expected, illustrating the advantage of the use of RPB1 marker over SSU. Finally, we found no hint of sex ratio distortion in Nosema clade A infecting G. balcanicus. This study shows that Nosema spp. are abundant, widespread and diverse in European gammarids. Thus, Nosema is as diverse in aquatic as in terrestrial hosts.The microsporidian genus  Nosema are important pathogens as they infect insects of economic importance such as pollinators or domestic silk moths. However, they have been overlooked in other groups of invertebrates such as crustaceans, where only few Nosema spp. were detected, including Nosema granulosis in two gammarid amphipod hosts. Here, using molecular tools, we detected infections by Nosema spp. in nine freshwater amphipod species from various European localities. These infections are common, widespread, and the phylogenetic analysis revealed that some Nosema lineages are specific to certain host species, while the others are not restricted to single hosts. We also confirms that there are several Nosema lineages infecting gammarid crustaceans, not only N. granulosis, that can be new candidate species of Nosema. We conclude that Nosema is as diverse in aquatic as in terrestrial hosts.The intracellular microsporidian parasites of the genus  Nosema (Nosematida) holds in a seminal place. The first formally described Microsporidium was Nosema bombycis [Nosema spp. or closely-related species have been described infecting insects [Nosema that are horizontally transmitted (HT) [Since the mid-19th century, Microsporidia, a large group of obligate intracellular eukaryotic microparasites related to fungi, has aroused interest in both basic and applied studies . In thisbombycis . Since t insects . All are insects ,7. Thereted (HT) , while oNosema spp. are known to infect freshwater decapod or amphipod crustaceans based on morphological and molecular data [Dictyocoela spp. and Cucumispora spp. [summarised in Nosema species, N. granulosis, which has been reported from only two amphipod species. This parasite, unlike Nosema of insects, has not been recognized as a pathogen so far, while in other crustaceans the impact on host fitness is unknown. Nosema granulosis was first described in the amphipod Gammarus duebeni [G. duebeni females transmit N. granulosis directly to their offspring through infected eggs [N. granulosis can live and be transmitted [G. duebeni and Gammarus roeselii [N. granulosis in G. roeselii (presumably resulting from failed feminisation) [G. roeselii [N. granulosis strains are predicted to have higher growth rate and may help the invasive host G. roeselii in colonizing new territories [et al. [N. granulosis strain of G. roeselii is associated with the only one host mitochondrial genotype that invaded Western Europe after the last glaciation [Dictyocoela microsporidians vary in their feminizing ability [N. granulosis infecting G. duebeni and G. roeselii varies between parasite strains and/or host species. The recent detection of Nosema spp. in populations of different amphipod species with highly variable prevalence fuels this hypothesis [N. granulosis diversity in amphipods. Studying microsporidian infections in amphipods is facilitated by the fact that some hosts are subjected to intensive phylogenetic and biogeographical research. For example, a study on New Zealand amphipods suggested that amphipod-Dictyocoela associations could have preceded the split of Pangea some 180 million years ago (MYA) and that these parasites co-differentiated with their hosts [Gammarus balcanicus and G. roeselii [Dictyocoela [Nosema infections remain too scarce in European gammarids to allow a general analysis of their diversification. Gammarus balcanicus is a potential host of particular interest, having begun its diversification in the early Miocene (c.a. 17 MYA) in the central Balkans, and for which cryptic lineages are locally endemic due to paleo-habitat fragmentation and a complex phylogeographic history [Gammarus pulex [Gammarus fossarum [Gammarus itself has been estimated at 65 MYA [Nosema diversity to a wider set of European gammarid species and to decipher the age of infection in this group of hosts.Few lar data –18. Many duebeni . Infecteted eggs –21. Vertnsmitted . Under tnsmitted ,23–26. Aroeselii ,21,23,27isation) ,23,28,29ritories . Support [et al.  found thaciation . Howeverpothesis ,34, but ir hosts . Similarroeselii ,36–38 altyocoela ,39,40. Hus pulex –43 and Gfossarum  show higt 65 MYA . All thie.g. genera) [Dictyocoela duebenum / muelleri [Nosema genus. On the one hand, it enabled formal redefinition of the two genera Nosema and Vairimorpha within the family Nosematidae [N. granulosis in G. roeselii based on SSU showed a low level of genetic variation despite a pan-European sampling [N. granulosis and the evolution of Nosema-amphipod associations. The genomes of the few economically relevant insect-infecting Nosema spp. have been sequenced [N. granulosis [Besides the aforementioned advantages, the study of the phylogenetic diversity of microsporidian parasites faces some problems with the resolution of available genetic markers. The small subunit ribosomal DNA marker (SSU) is routinely used to PCR-screen hosts for microsporidian infections. SSU sequencing data proved to be very useful for phylogenetic reconstructions at higher taxonomic levels and for distinguishing major microsporidian clades ( genera) , and som genera) ,46. Howeematidae . On the sampling . Therefoequenced –49, as wanulosis . Based oanulosis ,51–53. Ranulosis , displayNosema lineages infecting amphipod crustaceans, focusing mainly on Gammarus species, by using both SSU and RPB1 markers. Specifically, we first aimed at elucidating the extent of molecular variation associated with Nosema infections in amphipods . Second, we wanted to infer host specificity and explore evolutionary history scenarios that may explain the diversity of gammarid-infecting Nosema. Finally, as new divergent lineages of Nosema were detected over the entire distribution area of a new host, Gammarus balcanicus, the possible existence of sex ratio distortion associated with vertical transmission was tested, using the crude proxy of an infection bias in females . Such aNosema-infected amphipod individuals gathered from three sources. The first source was data from the literature for which both SSU and RPB1 sequences were available. The second source was data for which SSU information is available from the literature or GenBank and for which RPB1 information was tentatively gained from the original DNA samples as part of this study. The third source was de novo sequencing data of parasite SSU and RPB1 from individuals detected as infected by Nosema by SSU PCR screenings , G. pulex and G. fossarum from Germany [G. duebeni from Ireland [G. roeselii from France [Dikerogammarus villosus [G. roeselii [G. balcanicus [G. duebeni , G. balcanicus (site SK in Slovakia), and G. pulex . In addition, host individuals from two large-scale surveys we conducted on G. fossarum and G. balcanicus were included , the sex of individuals was noted during dissection  were selected for RPB1 amplification . As all published primers tested [N. granulosis sequences  and 5’-TCT ACT CTC TTM CCC ATA AG-3’ , generating a 520 bp-long amplicon. The F4 primers were 5’-GAA AGA CAC ATG CAG RAT G-3’  and 5’-TTC CWG ACA TGA TYT CTC C-3’ , generating a 640 bp-long amplicon.All s tested ,52,62,63EF119339 , and G. 01000442 ), and twDQ996231 ) and NosDQ996232 ). VariabDQ996232 , targeti2, 0.5 units of 5 PRIME HotMaster Taq DNA polymerase , 0.2 μM dNTPs , 0.2 μM of forward and reverse primers each  and 2 ng DNA template. Amplification conditions were as follows: an initial denaturing phase at 94°C for 2 min, 35 cycles at 94°C for 20s, annealing temperature was 50°C for each fragment for 20 s and extension was at 65°C for 30 s, with a final extension step at 65°C for 5 min. PCR products were purified and sequenced directly with the BigDye technology  using the forward PCR primers. The chromatograms showed no ambiguity in sequences and all the detected double peaks were of high quality. Nevertheless, seven PCR products were bi-directionally sequenced to test for uncertain nucleotide position associated with double peaks in chromatograms, and 17 products  in our analysis: Nosema cheracis, infecting the Australian crayfish species Cherax destructor introduced in Europe and Nosema austropotamobii, infecting Austropotamobius pallipes, a western European freshwater crayfish. Ordospora colligata served as the outgroup [For phylogenetic analyses, sequences of ision of , for whioutgroup . All detPhylogenetic reconstructions were performed separately for SSU and RPB1 using Bayesian inference implemented in MrBayes  integratNosema clade, we applied two methods: Assemble Species by Automatic Partitioning (ASAP) [https://bioinfo.mnhn.fr/abi/public/asap/ accessed on June 2, 2023) based on the distance matrix generated through IQ-TREE analysis [To explore the number of molecular operational taxonomic units (MOTUs) that may represent potential species within the g (ASAP)  and the g (ASAP) . ASAP waanalysis . ASAP dianalysis .https://species.h-its.org/; accessed on June 1, 2023) with the Bayesian implementation (bPTP), which adds Bayesian support (pp) values for putative species to branches in the input tree. The PTP method infers speciation events based on a shift in the number of substitutions between internal nodes [PTP modelling was performed using the PTP web server    Tables.Nosema spp. found in amphipods also included a crayfish-infecting species (The phylogenetic reconstruction inferred from the SSU marker was to a large extent in line with previously published phylogenetic reconstructions (e.g. ), except species . Most haG. roeselii, 45 from G. balcanicus, 38 from G. fossarum, 15 from G. duebeni and 4 from G. pulex. To this set, two RPB1 sequences of VT, feminizing, N. granulosis infecting G. duebeni from Scotland and Whales [Amplification success of the F2 and F4 fragments of RPB1 gene was variable, including no success , single fragment amplified  or both fragments amplified . Therefore, we newly obtained RPB1 sequence information for 163 individuals (depending on DNA availability and sequencing success), including 60 from d Whales  were addNosema infecting amphipods revealed 39 haplogroups . Based od hosts . All butG. balcanicus  of the whole model was quite high , so the interaction was removed from the model. The non-significance of this interaction means that globally there was no difference in the infection rate between sexes among the different populations. The model including sex and population was globally significant . The infection rate significantly differed among populations, ranging from 4.2% (in populations AL57 and AL69) to 64.6% (in population UA03)  . In totaG. pulex and G. roeselii hosts. Gammarus roeselii parasites were restricted to south-eastern Europe (Greece and Albania), while the single haplogroup from G. pulex was recorded in France  sampled at French and Swiss sites , mostly overlapping with the aforementioned clades, but C and D grouped as one species, here interpreted as Nosema granulosis sensu stricto , or a freshwater origin of this Nosema clade, with horizontal transfers between host species. The divergence among crustacean Nosema strains reported here could appear low compared to host divergence  seemed to have initiated their diversification simultaneously several MYA, but their genetic divergence appears to have reached different levels [N. empoascae within the Nosema lineage infecting crustaceans is more puzzling and may reflect horizontal transfer between crustaceans and insects. Consistently, horizontal transfer of Wolbachia intracellular microorganisms between insects and crustaceans has previously been suggested [N. empoascae . Ind. IndNosea (sensu ). Howeve 300 MYA ). Crusta 300 MYA . If so, t levels . The preuggested . Howevere family ) a recene family , which mcheracis , even ifNosema clades infecting amphipods [G. balcanicus, whose biogeographical history is well documented for both host and other microsporidian parasites [Gammarus spp., conversely to most other Nosema parasites revealed in this study. An infection by Nosema clade A therefore possibly occurred anciently in G. balcanicus and subsequently spread further during host radiation. However, unlike other microsporidian parasites [Nosema clade A and G. balcanicus . As noted in [N. granulosis infection might have facilitated the recent spread of the host species, G. roeselii, in western Europe [Crangonyx pseudogracilis and its VT-parasite, Fibrillanosema crangonycis [Nosema belonging to clade C are genetically very similar to the originally described N. granulosis , we propose that all parasites clustering into clade C should be referred to as N. granulosis. Furthermore, because i) clades C and D are in a close sister group relationship, ii) clade C contains sequences linked to type material used for N. granulosis description [Nosema granulosis sensu stricto. By contrast, we suggest that parasites grouped into clades A, B and E, and lineage F may be candidates to the status of new Nosema species, based on their genetic divergence supported by the species delimitation analysis. Remarkably, this divergence is larger than the range of divergence observed for Nosema taxa from insects. It may be explained by the differences in mutation rate and evolutionary dynamics between Nosema spp. infecting gammarids and insects. In our interpretation of the number of putative Nosema species infecting gammarids, we followed a more conservative approach: we based our outcomes on the ASAP results assuming fewer putative species than the bPTP analysis. There are many studies demonstrating that ASAP is often in congruence with morphological/biological species delimitation while bPTP method tends to overestimate the species number [e.g. Nosema spp. already described from different amphipods, including G. pulex, based solely on morphology (light microscopy for older observations) [Nosema can be misleading [Prior to this study, microsporidians from gammarids showing high score values by BlastNN with anulosis ,34, to Nrpha sp. . Here we duebeni ,19–21 coparasite ,24. Thesnoted in , all thenoted in . Theoretnoted in . Thus, tn Europe ,37, by dngonycis ,84. Becacription , iii) thber e.g. ,85,86. Hvations) –17,87. Hsleading .Nosema from clades B, D and E are present in a single or few individuals within the same population. Because of the sharp contrast of this infection pattern compared to clade C, it does not seem reasonable to us to propose that parasites of clades B, D, and E are feminizing and VT microsporidia. Contrastingly, infections in G. balcanicus hosts belonging to clade A show an infection pattern similar to those of clade C. For example, sequences in populations from Slovakia, Hungary and Romania show closely-related or similar RPB1 haplogroups infecting numerous individuals (> 70) in a dozen of populations all over the host geographic range. This pattern therefore resembles the VT-feminizing N. granulosis infection patterns in G. roeselii  [Nosema haplogroups across the geographic range of G. balcanicus is therefore puzzling. Based on this survey and previous evidence, VT and feminization in Nosema infecting amphipods therefore currently appear to be limited to N. granulosis belonging to clade C. It is noteworthy that clade C also contains parasites infecting G. fossarum individuals, which opens the possibility that VT-feminization might be found in a larger spectrum of amphipod species than previously thought.Interestingly, most haplogroups involving lii Figs  and 3. Hfemales) ,23,89. TNosema parasites, allowing elucidating the parasitic richness in their gammarid hosts. We found that Nosema diversity is much higher in amphipods than previously thought, and that the association appears to be ancient in this host group. Furthermore, the new host species found to be infected by N. granulosis sensu stricto opens the possibility that VT-feminization may be present in a larger spectrum of amphipod species. Finally, given that gammarids are keystone species in the functioning of freshwater environments, with their functions being affected by microsporidian infections [In conclusion, our study illustrates the interest of using the RPB1 marker for the study of fections –93, it iS1 FigA. Overview of the amino-acid alignment for the RNA polymerase II largest subunit (RPB1) gene haplogroups identified in amphipods. Grey colour stands for absence of amino acids. B. Overview of the nucleotide alignment for the RPB1 gene haplogroups, for two fragments (F2 and F4). Grey colour stands for either absence of PCR product or shorter sequences for a given fragment.(PDF)Click here for additional data file.S2 FigA. Details of Clade A of Nosema phylogenetic reconstruction. Names in black are Nosema haplogroups, names in red or blue are names of host clades to which they are associated. Legends are similar to Gammarus balcanicus phylogenetic tree simplified from [B). N and S represent the two major G. balcanicus groups that differentiated around 18 MYA. C. Map showing sites where host and parasites are coming from, where the limit between N and S host groups were redrawn after [Nosema haplogroups on host phylogenetic tree. The sites were plotted on a map from Natural Earth resources in QGIS 3.32.0-Lima [ied from  presentewn after . Lines b2.0-Lima .(PDF)Click here for additional data file.S1 TableThe names of the SSU haplogroups are as in (XLSX)Click here for additional data file.S2 TableNosema infections outside the amphipod hosts, used for the phylogenetic reconstructions. **sequences too short to be deposited in GenBank (see Sequences in blue are samples of Bank see . Referen(XLSX)Click here for additional data file.S3 TableThe number of base substitutions per site from averaging over all sequence pairs within each group are shown. Analyses were conducted using the Kimura 2-parameter model. This analysis involved 36 nucleotide sequences. Codon positions included were 1st+2nd+3rd+noncoding. All ambiguous positions were removed for each sequence pair (pairwise deletion option). There were 1025 positions in the final dataset. Evolutionary analyses were conducted in MEGA11. The presence of n/c in the results denotes cases in which it was not possible to estimate evolutionary distances. SE—standard error. References: Refence list is provided in the spread-sheet REFERENCES.(XLSX)Click here for additional data file.S1 Appendix(TXT)Click here for additional data file.S2 Appendix(TXT)Click here for additional data file.S3 Appendix(TXT)Click here for additional data file.


The biological control of aphids by aphid-eating lady beetles (coccinellids) is often hampered by aphid-tending ants or aggressive, invasive ants foraging for food; aggressive species, such as the imported fire ant, may attack and kill lady beetle larvae. This study tested the hypothesis that wax-secreting coccinellid larvae are less susceptible than non-wax-secreting coccinellid larvae to fire ant aggression. Laboratory experiments were set up using bird cherry-oat aphid nymphs and adults (as prey for coccinellids) on barley leaves with either coccinellid species with or without fire ant workers. Results revealed that the presence of fire ants reduced aphid predation by non-wax-secreting but not wax-secreting larvae. The frequency of fire ant attacks was greater on non-wax-secreting larvae; the mortality was significantly greater for non-wax-secreting larvae. The wax covering over coccinellid larvae reduced fire ant aggression. Unexpectedly, coccinellid larvae with the wax cover removed did not suffer greater fire ant attacks or increased mortality. In conclusion, the wax cover and, perhaps, the volatile or non-volatile compounds in the wax and on the integument of wax-secreting coccinellid larvae reduce fire ant aggression. Further research could identify the wax compounds and determine their role as potential repellents or irritants against fire ants.Solenopsis invicta Buren may attack and kill coccinellid larvae. This study tested the hypothesis that wax-secreting Scymnus creperus Mulsant larvae are less susceptible than non-wax-secreting Coleomegilla maculata (DeGeer) larvae to S. invicta aggression. Laboratory experiments were set up using bird cherry-oat aphid Rhopalosiphum padi (L.) nymphs and adults (as prey for coccinellids) on barley leaves in arenas with either coccinellid species and with or without S. invicta workers. The presence of S. invicta reduced aphid predation by C. maculata but not Sc. creperus. The frequency of S. invicta attack was greater for C. maculata than Sc. creperus; mortality was significantly greater for C. maculata than Sc. creperus. The wax covering on Sc. creperus reduced S. invicta aggression. Unexpectedly, Sc. creperus larvae with the wax cover removed did not suffer more S. invicta attacks or increased mortality. In conclusion, the wax cover and, perhaps, the volatile or non-volatile compounds in the wax and on the integument of Sc. creperus larvae reduce S. invicta aggression. Further research could identify the wax compounds and determine their functionality as semiochemicals against S. invicta.The biological control of aphids by aphidophagous coccinellids is often hampered by aphid-tending ants or aggressive, invasive ants foraging for food. Aggressive species such as the imported fire ant  Solenopsis invicta Buren (Hymenoptera: Formicidae), attack and kill coccinellid larvae that attempt to prey on tended aphids or mealybugs [Solenopsis invicta is known to tend to the mealybug Phenacoccus solenopsis Tinsley (Hemiptera: Pseudococcidae) and defend them from predation by the coccinellid Menochilus sexmaculata F. [S. invicta workers attack pests and beneficial insects, including predators, e.g., coccinellids [S. invicta workers attacked and killed 3rd instars and adults of the coccinellid Hippodamia convergens Guerin-Meneville on cotton plants infested with cotton aphid Aphis gossypii Glover; 3rd instar larvae of Scymnus louisianae Chapin were attacked but occasionally escaped predation [The biological control of hemipterans, such as aphids, mealybugs, and scale insects, by coccinellids has been hampered by tending ants or aggressive, invasive ants foraging for food ,2. Ants ealybugs . Solenopulata F. . In cottinellids . In laboredation .Scymnus nubilus Mulsant larvae serves to reduce intraguild predation [Scymnus interruptus (Goeze) and Scymnus nigrinus Kugelann larvae attenuated aggression by the ants Lasius niger L. and Formica polyctena Forster, respectively [Sc. louisianae reduced aggression by the ant Lasius neoniger Emery [Coccinella magnifica Redtenbacher, which co-exist with Formica spp., in their nests [Coccinellids may or may not possess effective behavioral, morphological, or chemical defense mechanisms to thwart attacks from aggressive ants . Speciesredation . The waxectively . Wax strer Emery  and chemer Emery ,16. Specer Emery . Note thir nests ,18.Scymnus creperus Mulsant (Coleoptera: Coccinellidae: Scymnini) is native to North America and distributed primarily in the southern regions of the USA [Coleomegilla maculata (DeGeer) (Coleoptera: Coccinellidae: Coccinellini) is native to North, Central, and South America [S. invicta is native to South America but is well established in the southern USA and northeastern Mexico and parts of the western USA [ the USA . It is a the USA ,21. The  America ,22. It i America ,24,25. Ttern USA ,27. C. maculata larvae has been challenged by the occupation of potted strawberry plants by ants, including S. invicta. In contrast, the larvae of an unidentified Scymnus species attacked and consumed sugarcane aphid Melanaphis sacchari (Zehntner) on potted sorghum plants in the presence of tending S. invicta in Stoneville, Mississippi . This study tested the hypothesis that Sc. creperus larvae are less susceptible than C. maculata larvae to S. invicta aggression. The objectives of this study were to determine the predation potential of C. maculata and Sc. creperus larvae in the presence or absence of S. invicta workers and estimate the frequency of S. invicta attacks and incidence of mortality to C. maculata, Sc. creperus, or Sc. creperus with the wax covering removed. This research will help select the most effective coccinellid species for aphid biological control on crop plants in areas where S. invicta and other aggressive ants are problematic.Our preliminary research on aphid control by mass-reared Rhopalosiphum padi (L.) nymphs and adults (in banker plant system) were purchased from IPM Laboratories Inc., Locke, NY, USA (https://www.ipmlabs.com). Barley seeds were also purchased from IPM Laboratories to establish barley host plants to maintain a continuous culture of R. padi at the National Biological Control Laboratory (NBCL), ARS, USDA in Stoneville, MS, USA.Barley seedlings infested with bird cherry-oat aphid Coleomegilla maculata 3rd instars used in this study were reared from eggs collected randomly from a stock culture maintained in an environmental rearing room  in the NBCL. This culture has been maintained for over 30 consecutive generations without any introduction of “feral” individuals. Larvae and adults were reared on factitious food based on a proprietary mixture of brine shrimp (Artemia franciscana Kellogg) eggs, microalgae (Chlorella vulgaris Beijerink), and fatty acids [The ty acids ,29.Scymnus creperus adults were purchased from IPM Laboratories Inc. Additional adults were obtained from an established laboratory colony at the University of Florida, in Gainesville, FL, USA. The adults and larvae were fed R. padi nymphs and adults, which had been reared on barley seedlings in the laboratory, for one generation before experimentation. Just before the experiments, the R. padi nymphs and adults, 3rd instar C. maculata, and 3rd instar Sc. creperus were maintained in a plant growth chamber .S. invicta were collected near roadsides and agricultural fields in Washington Co., Stoneville, MS, USA, in the spring and summer of 2018. Fire ants are mound-building ant species. Ants with mound soil were collected by shoveling mound soil into 5-gallon buckets. The water-drip method was then used to separate the ants from the soil [Acheta domesticus (L.) were used as a food source. Before experimentation, rearing trays were held on the laboratory benchtop and S. invicta workers were gently removed and placed in smaller containers coated with fluon. Workers actively moving around in the containers were selected at random for experiments.Imported fire ants the soil . Queen-rC. maculata or Sc. creperus on R. padi nymphs and adults on barley leaves in arenas with or without S. invicta workers. Rhopalosiphum padi mid-to-late instar nymphs and adults were randomly selected from colony cages and placed on a barley leaf in the center of 10 arenas . Because 3rd instar C. maculata are larger than 3rd instar Sc. creperus (and can purportedly consume more prey), 60 and 50 R. padi were randomly placed in arenas in the C. maculata and Sc. creperus trials, respectively. In preliminary observations, these prey densities exceeded the 7-h killing rate of the coccinellids. Next, five S. invicta workers, presumably of approximately the same age but variable in size, were randomly selected from an in-house colony  and carefully added into 5 of the 10 arenas in the C. maculata tests. Note that 10 S. invicta workers were used in the Sc. creperus tests because preliminary observations indicated that S. invicta were typically not aggressive to Sc. creperus larvae. In addition, S. invicta workers had no prior exposure to C. maculata or Sc. creperus larvae at any time in the laboratory. Within 1 h, a single 3rd instar C. maculata or 3rd instar Sc. creperus was gently added onto the barley leaf in each arena. At approximately 30-min intervals, within a 7-h time frame, the number of R. padi killed  by C. maculata or Sc. creperus was determined from each arena. This experiment was replicated four times, i.e., four trials, and the total sample size was 40 observations (or arenas) for C. maculata and Sc. creperus. Trials with C. maculata were conducted on 7, 11, 25 June, and 6 July 2018; those with Sc. creperus were on 16, 27, 31 July, and 9 August 2018. All arenas were maintained in a plant growth chamber , but removed on the day of experimentation. Experiments were performed on a laboratory bench at room temperature .Using a completely randomized design, this laboratory experiment was set up to estimate predation, i.e., the killing capacity, of 3rd instar S. invicta attack rate and mortality of 3rd instar C. maculata versus 3rd instar Sc. creperus. Approximately 20–30 R. padi nymphs and adults (randomly selected from the colony) were placed on a barley leaf, positioned inside a medium-sized Petri dish arena , and replicated 10 times; five arenas had one C. maculata and the other five arenas had one Sc. creperus. Next, 10 S. invicta workers  were gently added to each arena. Note that the S. invicta workers had no prior exposure to C. maculata or Sc. creperus larvae at any time in the laboratory. Within 1 h, a single 3rd instar C. maculata or 3rd instar Sc. creperus was gently added onto the barley leaf in each arena. At approximately 30-min intervals, the number (and proportion) of C. maculata versus Sc. creperus attacked and subsequently killed by S. invicta was recorded over a 7-h time frame. This experiment was replicated three times, i.e., three trials, and the total sample size was 30 observations (or arenas). Trial dates were on 1, 2, and 3 August 2018. All arenas were maintained in a plant growth chamber . On the day of experimentation, the arenas were removed and the experiments were performed on a laboratory bench at room temperature .Using a completely randomized design, this laboratory experiment was carried out to estimate the Sc. creperus affected the S. invicta attack rate and mortality of Sc. creperus. The wax filaments on the dorsal and lateral integument of sample 3rd instar Sc. creperus were carefully removed with a fine camel’s hair paintbrush. The three coccinellid treatments in this experiment were 3rd instar C. maculata, 3rd instar Sc. creperus, or 3rd instar Sc. creperus without wax. The prey density (20–30 R. padi) and arenas (medium-sized Petri dishes) were the same as before. The S. invicta density was 10 workers per arena for all three treatments. The procedures of adding R. padi onto a barley leaf, then S. invicta into each arena were the same. At approximately 30-min intervals, the numbers (and proportion) of C. maculata versus Sc. creperus versus Sc. creperus without wax attacked and subsequently killed by S. invicta were recorded over a 7-h time frame. A total of 5 replicate arenas were used for each treatment, for 15 arenas per trial. Trial dates were 16, 17, and 21 August 2018. The three trials resulted in a sample size of 45 observations  for the entire experiment. All arenas were maintained in a plant growth chamber . On the day of experimentation, the arenas were removed, and the experiments were conducted on a laboratory bench at room temperature .This experiment was identical to Expt. 2, except an additional treatment was added to determine if removing the waxy material from the integument of t-test was used to test the significance of S. invicta presence in arenas on predation of R. padi by C. maculata or Sc. creperus. A Pearson Product Moment Correlation Analysis was used to detect any correlation between the approximate time (within a 7-h time frame) that S. invicta killed C. maculata and the number of R. padi killed by C. maculata. A Student’s t-test was used to test the significance of S. invicta attack frequency and subsequent mortality of C. maculata and Sc. creperus in the same experiment. A one-way analysis of variance (one-way ANOVA) was used to test the significance of the S. invicta attack frequency and subsequent mortality of C. maculata, Sc. creperus, or Sc. creperus without wax covering in the same experiment. The Holm–Sidak Method was used after the one-way ANOVA to separate the mean values. Means were considered significantly different at p < 0.05. Computer software programs SigmaPlot 12.0 interfaced with SigmaStat  and JMP® 14.2.0  were used for data analysis.Before analysis, all data were checked for normality (using the Shapiro–Wilk test) and equal variances. For almost all datasets, the assumptions of normality and equal variances were met. Therefore, the square root transformation of absolute data and arcsine transformation of proportional data were not performed. In separate experiments, a Student’s R. padi nymphs and adults on a barley leaf. Companion images illustrate R. padi being consumed by 3rd instar C. maculata, S. invicta stinging and biting 3rd instar C. maculata, and S. invicta not attacking 3rd instar Sc. creperus.R. padi killed by 3rd instar C. maculata was significantly lower in the presence of S. invicta  proportion of R. padi killed during the 7-h test period was 0.46 (± 0.05) and 0.66 (± 0.02) in the C. maculata and S. invicta treatment versus C. maculata alone treatment, respectively. There were 60 R. padi nymphs and adults in each arena at the onset of the experiment with C. maculata. Solenopsis invicta worker aggression reduced the predation capacity of C. maculata. Moreover, the number of R. padi killed by C. maculata decreased as the time required for S. invicta to detect and kill C. maculata decreased  and 0.53 (±0.01) in the Sc. creperus and S. invicta treatment or Sc. creperus alone, respectively. There were 50 R. padi nymphs and adults in each arena at the onset of the experiment with Sc. creperus. Generally, S. invicta workers were not aggressive to Sc. creperus; no Sc. creperus were killed in any of the arenas.The mean ± SE number of S. invicta attacked and killed 3rd instar C. maculata rather than Sc. creperus in experimental arenas. The attack frequency was significantly greater on C. maculata than Sc. creperus . Mortality resulting from S. invicta attacks was significantly different between C. maculata and Sc. creperus . The attack frequency and mortality of C. maculata were approximately the same, indicating that S. invicta killed approximately all the C. maculata they attacked. The mortality of 3rd instar C. maculata was from the biting and stinging behavior of S. invicta. Moreover, in any of the three trials, S. invicta never consumed C. maculata during the 7-h test period. Although S. invicta attacked a small proportion of Sc. creperus during the 7-h test period  tended to the soybean aphid Aphis glycines Matsumura in the laboratory and consequently hindered predation by the coccinellid Harmonia axyridis  [Ha. axyridis are devoid of a wax covering and are members of the tribe Coccinellini. In another study, S. invicta reduced the predation efficiency of H. convergens (tribe Coccinellini) 3rd instars more than Sc. louisianae (tribe Scymnini) 3rd instars in laboratory experiments with the cotton aphid A. gossypii [This laboratory study clearly observed the differential susceptibility of for food ,17. Cocc . Larvae gossypii . The susC. maculata larvae possess morphological defenses  on the dorsal surface of their integument and chemical defenses  in their hemolymph, these defenses provide little protection from S. invicta aggression. Alkaloids discovered in C. maculata adults might function as semiochemicals [C. maculata larvae has not been determined.Although hemicals ,32. Prechemicals , whereasSc. louisianae larvae provided a morphological and perhaps chemical defense against attacks from the ant L. neoniger; removal of wax from the integument of Sc. louisianae larvae did not attenuate L. neoniger aggression [Sc. louisianae larvae reduced ant aggression, suggesting that chemical cues in the wax were partly responsible for lowering L. neoniger aggression [S. invicta attacks or cause mortality of Sc. creperus, suggesting that chemical compounds on the surface of their integument, in addition to the wax, might play a defensive role, perhaps as a repellent or an irritant. Moreover, S. invicta workers rubbed their mandibles against the substrate immediately after biting, then released wax-covered Sc. creperus larvae, apparently cleaning their mandibles. This S. invicta cleaning behavior further suggests a repellent or irritant functionality of the wax. Future research could identify the chemical compounds in the wax covering and on the surface of the integument of Sc. creperus larvae. In addition, determining the function of the isolated compounds in bioassays against S. invicta workers would be a worthwhile investigation potentially leading to the discovery of novel repellents.Wax structures on the integument of gression . Dissolvgression . In thisS. invicta is needed. Perhaps, combining tactics to control aphids by releasing the most effective coccinellids and using baits and repellents to keep S. invicta workers away from crop plants in cultured systems, e.g., greenhouses, would be the best approach.Complimentary research to develop effective baits and bio-based insecticides against C. maculata and Sc. creperus larvae to aggressive behavior from S. invicta workers in laboratory arenas. Wax covering the dorsal and lateral surfaces of the Sc. creperus integument provides protection and prevents mortality from S. invicta attacks. The selection of the most effective aphidophagous coccinellids would ensure the success of the biological control of aphids on crop plants. The selection of wax-covered larvae (Scymnini) rather than non-wax-covered larvae (Coccinellini) would be the best choice in areas, such as greenhouses or glasshouses, where aphid-tending ants or aggressive ants foraging for food on or near crop plants are problematic [In conclusion, this study provides evidence for the differential susceptibility of blematic .