Agri-environment scheme nectar chemistry can suppress the social epidemiology of parasites in an important pollinator

Emergent infectious diseases are one of the main drivers of species loss. Emergent infection with the microsporidian Nosema bombi has been implicated in the population and range declines of a suite of North American bumblebees, a group of important pollinators. Previous work has shown that phytochemicals found in pollen and nectar can negatively impact parasites in individuals, but how this relates to social epidemiology and by extension whether plants can be effectively used as pollinator disease management strategies remains unexplored. Here, we undertook a comprehensive screen of UK agri-environment scheme (AES) plants, a programme designed to benefit pollinators and wider biodiversity in agricultural settings, for phytochemicals in pollen and nectar using liquid chromatography and mass spectrometry. Caffeine, which occurs across a range of plant families, was identified in the nectar of sainfoin (Onobrychis viciifolia), a component of UK AES and a major global crop. We showed that caffeine significantly reduces N. bombi infection intensity, both prophylactically and therapeutically, in individual bumblebees (Bombus terrestris), and, for the first time, that such effects impact social epidemiology, with colonies reared from wild-caught queens having both lower prevalence and intensity of infection. Furthermore, infection prevalence was lower in foraging bumblebees from caffeine-treated colonies, suggesting a likely reduction in population-level transmission. Combined, these results show that N. bombi is less likely to be transmitted intracolonially when bumblebees consume naturally available caffeine, and that this may in turn reduce environmental prevalence. Consequently, our results demonstrate that floral phytochemicals at ecologically relevant concentrations can impact pollinator disease epidemiology and that planting strategies that increase floral abundance to support biodiversity could be co-opted as disease management tools.


General interest: Is the paper of sufficient general interest? Excellent
Quality of the paper: Is the overall quality of the paper suitable? Good Is the length of the paper justified? Yes Should the paper be seen by a specialist statistical reviewer? No Do you have any concerns about statistical analyses in this paper? If so, please specify them explicitly in your report. No It is a condition of publication that authors make their supporting data, code and materials available -either as supplementary material or hosted in an external repository. Please rate, if applicable, the supporting data on the following criteria.

Do you have any ethical concerns with this paper? No
Comments to the Author The authors Folly et al, explore the phytochemicals present in the nectar and pollen of plants planted as part of Agri-environmental schemes (AES) and determine if caffeine, a chemical found in one of the screened plants (Sainfoin), can have a beneficial effect on bumblebees exposed to the parasite Nosema bombi. Manipulative experiments examined the prophylactic and therapeutic effects of caffeine on queen less microcolonies established from commercially sourced Bombus terrestris colonies and exposed to Nosema bombi. In addition, whole colony effects were explored using wild caught queen B.terrestris queens. The results show that caffeine can have both a prophylactic and therapeutic effect on N. bombi infection and in whole colony caffeine can reduce infection intensity particularly in younger bumblebees.
Overall, this was an enjoyable paper to read with well laid out justification for the experimental work undertaken with clearly outlined methods and analysis employed and clearly presented results.

Introduction
The introduction is succinct and informative, however, the relevance of N. bombi to bumblebee populations, which is not mentioned until its brief appearance in the discussion could be expanded upon to help provide better context for the importance of the study.

Methods
The methods should better integrate the chemistry work. These are currently as supplementary methods but there is no clear link to them in the main manuscript. The manuscript would benefit from at least an overview of what has been done included in the methods and reference to the supplementary methods.

Results
The 'Phytochemical identification' column, which is differently named in supplementary table 3, despite referring to the same concept, should be renamed to 'Phytochemical identified' for clarity. Additionally, abbreviations are used in both supplementary table 2 and supplementary table 3 without being included in the table legends. In Figure 1, poor resolution and small size make reading the y-label difficult. Additionally, it is not clear why in line 135 there is reference to Figure 3. For Figure 3, the in-figure legend is unnecessary. In line 417, the word 'number' is misspelled.

Discussion
Throughout the report it does not become clear what the relevance of CRP is to the manuscript, as to my understanding, only plants involved in the AES scheme are included. Is it only included because of its similar approach to AES? Clarification needed. While the discussion briefly mentions field applicability, a broader exploration of this could be beneficial. This could involve a brief mention of potential interactions with other phytochemicals, exposure to different doses of caffeine or a further exploration of what is meant by increased potential for nutritional deficiencies. While it is clear that in the experiment food is freely provided, which would not be the case for field conditions, it is not clear why the presence of flowers which have the alkaloid would be more likely to lead to nutritional deficiencies. Perhaps, if such flowers are less nutritious themselves or through influencing foraging choices? In any clarity on this could improve the overall understanding of the topic for the reader.
Line 52: font size changes Line 94: may benefit from some system specific references 217-232: what about possible negative effects of phytochems? 254: clarify its not species specific within Bombus. 271-272: are the planting schemes in the US? 307: what was the concentration of the inoculant? How was the inoculant also deemed clear of other parasites? 308-309: mention the use of UV irradiation to remove microbes, a necessary step for the experiment to ensure that the bees are only inoculated with the pathogen being assessed in the study. However, the sterilisation of different bee pathogens require different doses of irradiationis it possible to include details on this or the supplier of the pollen. 309: was the pollen of a known flower? Was it tested for phytochemicals? 315: nice to see you checked repeatedly for adult parasite presence 329: to clarify, was caffeine placed on pollen at all? 330: though you describe the volume of MeOH added to the sucrose, you haven't noted the volume of sugar water to interpret what the final concentration of the solvent was. 350: For clarity, which workers were isolated to provide faecal samples -the recently eclosed workers or the original 'nurse' workers? 361-366: good practice to isolate and recheck health like this 379-380: those pesky queens! 376: it is not clear if the number of brood inoculated was controlled/standardised between colonies, and if not, was to total number of spores applied to the colony standardised? also was it just the L2/3 brood inoculated? 383: was there cat-litter or similar in the base to absorb faeces or could pools of faeces in the arena facilitate dispersal between foraging workers? 386: Did this caffeine treatment include MeOH as before? 431-432: It would be nice to include these normality plots as supplementary Some minor formatting inconsistencies; In line 520: '10' is placed in bold which is inconsistent with the rest of the formatting. Lines 575-576: a grey background is included, which is inconsistent with other references.

09-Apr-2021
Dear Dr Folly: Your manuscript has now been peer reviewed and the reviews have been assessed by an Associate Editor. The reviewers' comments (not including confidential comments to the Editor) and the comments from the Associate Editor are included at the end of this email for your reference. As you will see, the reviewers and the Associate Editor have raised some concerns with your manuscript and we would like to invite you to revise your manuscript to address them.
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Best wishes, Professor Hans Heesterbeek mailto: proceedingsb@royalsociety.org Associate Editor Board Member: 1 Comments to Author: Both reviewers are convinced by the importance of the study and the strength of the experimental design. Both also ask for some of the arguments in the discussion, e.g. the importance to CRP, extrapolating from the study of "only" one compound, to either be clarified or turned down a bit. I agree with both sentiments. Because the requested changes are editorial and the reviews comments give detailed guidance, I would recommend accepting the manuscripts, but urge the authors to carefully consider the reviewers comments.
Reviewer(s)' Comments to Author: Referee: 1 Comments to the Author(s) The authors report an interesting and important study documenting the importance of phytochemicals in protecting pollinators from pathogens. The study is well designed and the results clearly support the claims by the author on the potential benefits of caffeine. Caffeine as a beneficial phytochemical has been recently researched in the context of pollinator health with specific reference to honeybees and their pathogen Nosema ceranae. Given that the authors here document benefits of caffeine on a different species of Nosema, it strengthens their case to cite these other studies. In addition, phytochemicals benefiting pollinator health is also well documented and authors here fail to cite some important recent publications. The discussion section is rather weak as the authors do not propose any suggestions for mode of action of caffeine but instead just repeat the introduction premise on the importance of pollinator habitat plants. The study is fairly small but the results are clear. Please cite the following publications during revision and improve discussion to include some of the ideas proposed in these publications Lu, Y.-H., et al. (2020). "Identification of Immune Regulatory Genes in Apis mellifera through Caffeine Treatment." Insects 11 (8) Referee: 2 Comments to the Author(s) The authors Folly et al, explore the phytochemicals present in the nectar and pollen of plants planted as part of Agri-environmental schemes (AES) and determine if caffeine, a chemical found in one of the screened plants (Sainfoin), can have a beneficial effect on bumblebees exposed to the parasite Nosema bombi. Manipulative experiments examined the prophylactic and therapeutic effects of caffeine on queen less microcolonies established from commercially sourced Bombus terrestris colonies and exposed to Nosema bombi. In addition, whole colony effects were explored using wild caught queen B.terrestris queens. The results show that caffeine can have both a prophylactic and therapeutic effect on N. bombi infection and in whole colony caffeine can reduce infection intensity particularly in younger bumblebees.
Overall, this was an enjoyable paper to read with well laid out justification for the experimental work undertaken with clearly outlined methods and analysis employed and clearly presented results. net effect. It is for that reason, I also feel the title and general tone of the manuscript is too broadbrush and should be toned down.

Introduction
The introduction is succinct and informative, however, the relevance of N. bombi to bumblebee populations, which is not mentioned until its brief appearance in the discussion could be expanded upon to help provide better context for the importance of the study.

Methods
The methods should better integrate the chemistry work. These are currently as supplementary methods but there is no clear link to them in the main manuscript. The manuscript would benefit from at least an overview of what has been done included in the methods and reference to the supplementary methods.

Results
The 'Phytochemical identification' column, which is differently named in supplementary table 3, despite referring to the same concept, should be renamed to 'Phytochemical identified' for clarity. Additionally, abbreviations are used in both supplementary table 2 and supplementary table 3 without being included in the table legends. In Figure 1, poor resolution and small size make reading the y-label difficult. Additionally, it is not clear why in line 135 there is reference to Figure 3. For Figure 3, the in-figure legend is unnecessary. In line 417, the word 'number' is misspelled.

Discussion
Throughout the report it does not become clear what the relevance of CRP is to the manuscript, as to my understanding, only plants involved in the AES scheme are included. Is it only included because of its similar approach to AES? Clarification needed. While the discussion briefly mentions field applicability, a broader exploration of this could be beneficial. This could involve a brief mention of potential interactions with other phytochemicals, exposure to different doses of caffeine or a further exploration of what is meant by increased potential for nutritional deficiencies. While it is clear that in the experiment food is freely provided, which would not be the case for field conditions, it is not clear why the presence of flowers which have the alkaloid would be more likely to lead to nutritional deficiencies. Perhaps, if such flowers are less nutritious themselves or through influencing foraging choices? In any clarity on this could improve the overall understanding of the topic for the reader.
Line 52: font size changes Line 94: may benefit from some system specific references 217-232: what about possible negative effects of phytochems? 254: clarify its not species specific within Bombus. 271-272: are the planting schemes in the US? 307: what was the concentration of the inoculant? How was the inoculant also deemed clear of other parasites? 308-309: mention the use of UV irradiation to remove microbes, a necessary step for the experiment to ensure that the bees are only inoculated with the pathogen being assessed in the study. However, the sterilisation of different bee pathogens require different doses of irradiationis it possible to include details on this or the supplier of the pollen. 309: was the pollen of a known flower? Was it tested for phytochemicals? 315: nice to see you checked repeatedly for adult parasite presence 329: to clarify, was caffeine placed on pollen at all? 330: though you describe the volume of MeOH added to the sucrose, you haven't noted the volume of sugar water to interpret what the final concentration of the solvent was. 350: For clarity, which workers were isolated to provide faecal samples -the recently eclosed workers or the original 'nurse' workers? 361-366: good practice to isolate and recheck health like this 379-380: those pesky queens! 376: it is not clear if the number of brood inoculated was controlled/standardised between colonies, and if not, was to total number of spores applied to the colony standardised? also was it just the L2/3 brood inoculated? 383: was there cat-litter or similar in the base to absorb faeces or could pools of faeces in the arena facilitate dispersal between foraging workers? 386: Did this caffeine treatment include MeOH as before? 431-432: It would be nice to include these normality plots as supplementary Some minor formatting inconsistencies; In line 520: '10' is placed in bold which is inconsistent with the rest of the formatting. Lines 575-576: a grey background is included, which is inconsistent with other references.

05-May-2021
Dear Dr Folly I am pleased to inform you that your manuscript entitled "Agri-environment scheme nectar chemistry can suppress the social epidemiology of parasites in an important pollinator" has been accepted for publication in Proceedings B.
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Sincerely, Professor Hans Heesterbeek Editor, Proceedings B mailto: proceedingsb@royalsociety.org Associate Editor: Board Member Comments to Author: The authors replied and adopted reviewers comments comprehensively and I am happy to recommend the revised version of the manuscript for publication.