Niche partitioning facilitates coexistence of closely related honey bee gut bacteria
Curation statements for this article:-
Curated by eLife
Evaluation Summary:
Brochet et al. address an outstanding ecological question related to how closely related microbial symbionts co-exist in the gut as stable communities. To tackle this question, the authors use an elegant model relying on honeybees colonized with a defined bacterial community. They provide compelling empirical evidence that a nutritionally complex diet together with microbial metabolic diversity play a key role in enabling co-existence of closely-related honeybee gut microbiota.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #1 agreed to share their name with the authors.)
This article has been Reviewed by the following groups
Listed in
- Evaluated articles (eLife)
- Microbiology and Infectious Disease (eLife)
Abstract
Ecological processes underlying bacterial coexistence in the gut are not well understood. Here, we disentangled the effect of the host and the diet on the coexistence of four closely related Lactobacillus species colonizing the honey bee gut. We serially passaged the four species through gnotobiotic bees and in liquid cultures in the presence of either pollen (bee diet) or simple sugars. Although the four species engaged in negative interactions, they were able to stably coexist, both in vivo and in vitro. However, coexistence was only possible in the presence of pollen, and not in simple sugars, independent of the environment. Using metatranscriptomics and metabolomics, we found that the four species utilize different pollen-derived carbohydrate substrates indicating resource partitioning as the basis of coexistence. Our results show that despite longstanding host association, gut bacterial interactions can be recapitulated in vitro providing insights about bacterial coexistence when combined with in vivo experiments.
Article activity feed
-
Author Response
**Reviewer #1 (Public Review): **
[...] Their studies were complemented by transcriptomics and metabolomics and these results support the general conclusions that pollen contains diverse carbon sources which could be used in complementary ways by the different species, which have diverse metabolic capabilities encoded in their genomes.
Reply: We thank the reviewer for the positive assessment of our manuscript.
One of the points that was not completely explored in the paper is what happens in the simplified diet both in vitro and in the Bee gut. They propose in the discussion that in the presence of few and simple carbon sources (sugars) there is competition for nutrients and competitive exclusion is driving loss of some species. But this is not fully addressed in the paper.
Reply: All four species can colonize the …
Author Response
**Reviewer #1 (Public Review): **
[...] Their studies were complemented by transcriptomics and metabolomics and these results support the general conclusions that pollen contains diverse carbon sources which could be used in complementary ways by the different species, which have diverse metabolic capabilities encoded in their genomes.
Reply: We thank the reviewer for the positive assessment of our manuscript.
One of the points that was not completely explored in the paper is what happens in the simplified diet both in vitro and in the Bee gut. They propose in the discussion that in the presence of few and simple carbon sources (sugars) there is competition for nutrients and competitive exclusion is driving loss of some species. But this is not fully addressed in the paper.
Reply: All four species can colonize the gut individually and grow on their own in axenic cultures when providing the simple sugars or the pollen as the only carbon source. When cultured together, all four strains are stably maintained in the presence of pollen. However, three of the four strains steadily decrease in abundance in the simple sugars. These findings are, in our opinion, consistent with the consumer-resource model (more resources = more species that can coexist) and the competitive exclusion principle which predicts that if two or more strains compete for the same nutrients they will not be able to coexist. We have added a corresponding section on line 423-425.
The system they use (with 4 closely related bacterial species) is a simplified system. Therefore, it is not clear if the same general findings will hold in more complex systems. But the results supporting that nutrient complexity (in diet) and metabolic diversity (from the microbial side) are key factors to enable co-existence and persistence of complex microbiota communities are strong and likely generalizable. Although, it is possible that with other communities and other hosts other factors will also come into play. Nonetheless, the current study is important because it sets a good example for how these questions can be addressed to study more complex systems.
Reply: It is true that bacterial coexistence does not necessarily need to be dependent on the nutrient complexity and that in other communities the host, the structure of the environment, or cross-feeding activities may play a more important role. We have discussed this point in the revised manuscript starting on line 423 and on line 427.
Overall, the study described here is complete, and rigorous, except for a few points that still need to be addressed and clarified. Namely, it would be interesting to understand what drives exclusion of some members of the community in the simplified diet.
Reply: See our reply above.
Importantly, the current study opens the door for new studies (including in vitro studies) on the identification of network interactions that are important for Microbe-Microbe interactions that enable co-existence in other systems. Additionally, this study also highlights the importance of identifying the relevant nutritional (and metabolic) conditions for addressing those questions given the importance of the metabolic context in shaping microbe-microbe interactions.
Reply: Thank you. We agree.
**Reviewer #2 (Public Review): **
[...] Strengths: The use of community profiling, transcriptomics, and metabolomics adds depth, as does the comparison of defined culture conditions to the host environment. The main conclusions drawn by the authors is that the presence of pollen is necessary for gut species to coexist, and that the different species, although closely related, respond in distinct ways to nutrients in pollen and consume different profiles of nutrients from pollen.
Reply: We thank the reviewer for the positive feedback and the many valuable comments which helped us to further strengthen our manuscript.
Weaknesses: The main weakness I see with this work is the choice of in vitro comparison conditions. The strains are cultured either on pollen or sugar water, whereas in vivo bees are fed a diet of pollen and sugar water, or only sugar water. A direct comparison is possible between the strains grown on sugar water in vitro or in vivo, but I think that in several places, the authors may have to reconsider or modify their interpretations comparing in vitro culture on pollen/pollen extract with the in vivo growth of the community on pollen and sugar water. Because there is sugar in the bee diet, differences in assembly dynamics, transcription, or metabolite consumption between pollen-containing culture conditions and the bee gut might stem from the dietary intake of sugar, or from an aspect of the host environment.
Reply: We agree with the reviewer that the nutrient conditions that were used in vitro and in vivo are not identical and may have impacted the relative abundance of some of the community members, the transcriptional profiles, or the metabolite changes. Nevertheless, we believe that our experimental design is valid to test the main hypothesis of our study, i.e. a complex, pollen-based diet facilitates coexistence, while simple sugars lead to the dominance of a single strain independent of the environment (culture tube versus host). An important point to consider here is that bees will pre-digest the consumed pollen, and partially absorb dietary nutrients such as amino acids, glucose, and fructose, before they reach the bacteria in the hindgut. Consequently, the in vivo and in vitro conditions will never be the same even if we would have used the identical nutrients in our treatments. Also, pollen by itself contains glucose, fructose, and sucrose. So, although we have not added glucose to the in vitro pollen condition, this simple sugar was present in the corresponding condition. We have added a corresponding section in the discussion on line 402-422. This said, while we cannot recapitulate the exact same nutritional conditions in vitro, we still think that our main conclusions hold which is that we can recapitulate the pollen-dependent coexistence found in vivo.
Reviewer #3 (Public Review):
[...] Overall, the paper is strong and the arguments and conclusions put forth are well supported by the data. I only have a few suggestions:
Reply: We thank the reviewer for the positive evaluation of our manuscript.
- The study focuses on one strain each of the 4 Firm-5 species; however, there is diversity within each species. This is only briefly mentioned in the paper at the very end, and I think the authors should address this a bit more directly. In particular, they have previously generated a large amount of genomic data from some of these other strains, so it is likely possible to infer or speculate, based on this data, whether they expect different strains within each species to utilize similar nutrients. Also, I'm wondering if the authors can comment on how their findings could extend to the related bumble bee gut microbiome. Such a discussion would help enhance the applicability and importance of this study.
Reply: We agree that the large amount of strain-level diversity within a given species is an important point to consider. However, we would like to not expand this point much further as it would require a relatively complex genomic analysis. Also, considering that many of the strain-specific transcriptional changes are in genes shared with the other species, I am not sure how much such an analysis would reveal. Anyways, we plan to compare the coexistence between strains from the same versus another lineages in a follow-up study.
As for the bumble bees, we currently do not know how many strains or species of Lactobacillus Firm5 can coexist in bumble bees. Therefore, we feel that a discussion extending to bumble bees would be too speculative. However, we included a sentence in the discussion which states that since pollen facilitates coexistence, it follows that dietary differences are likely to influence the diversity of Lactobacillus Firm5 and give the example of the Asian honey bee, which seems to only harbor one species of this phylotype. See line 479-488.
- It is interesting that different species ended up dominating in the in vivo vs. in vitro simple sugar-based communities. What do the authors think may be behind this difference?
Reply: This is indeed an interesting point. We have not used the same sugars in vivo (sucrose) and in vitro (glucose). Moreover, the nutritional and physicochemical conditions in the hindgut are likely different from those found in a culture tube. We have mentioned that these are potential reasons for the observed differences in the relative abundance of different community members between in vivo and in vitro conditions on line 402-422 of the manuscript.
- Since the observed coexistence of these gut microbes is largely due to nutritional niche partitioning, it would be helpful if the authors can comment on the natural variation of key pollen derived metabolites, and if/how we could expect ecological variation in the bee microbiome due to plant pollen availability based on biogeography and seasonality.
Reply: We agree and have included a corresponding sentence in the discussion on line 479. See also our reply to point 1.
- The supplementary information is nicely documented and accessible, but I think it would be even more useful if genome-wide data for the RNA-seq results, not just for select genes, are made available. Furthermore, I suggest including descriptive titles and labels within the supplementary Excel files, as there are many separate sheets and it is not always clear what each one shows.
Reply: This has been included in the revised manuscript.
-
-
-
-
Evaluation Summary:
Brochet et al. address an outstanding ecological question related to how closely related microbial symbionts co-exist in the gut as stable communities. To tackle this question, the authors use an elegant model relying on honeybees colonized with a defined bacterial community. They provide compelling empirical evidence that a nutritionally complex diet together with microbial metabolic diversity play a key role in enabling co-existence of closely-related honeybee gut microbiota.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #1 agreed to share their name with the authors.)
-
Reviewer #1 (Public Review):
In the manuscript "Niche partitioning facilitates coexistence of closely related gut bacteria" by Brochet et. al., the authors work on the identification of the mechanisms that enable co-existence and persistence of multiple bacterial species in the gut.
The authors rely on a gnoto-biotic approach with Bees colonized with a defined bacterial community composed of 4 species. They studied the effect of diet, the host, and microbial interactions in enabling co-existence of these 4 species.
They followed gut colonization of these different species in mono-colonized animals and in co-culture under two different diets (simple sugar, or pollen). They observed that pollen could sustain persistence of these species, unlike the simpler diet where the community was dominated by a single species.
To disentangle between …
Reviewer #1 (Public Review):
In the manuscript "Niche partitioning facilitates coexistence of closely related gut bacteria" by Brochet et. al., the authors work on the identification of the mechanisms that enable co-existence and persistence of multiple bacterial species in the gut.
The authors rely on a gnoto-biotic approach with Bees colonized with a defined bacterial community composed of 4 species. They studied the effect of diet, the host, and microbial interactions in enabling co-existence of these 4 species.
They followed gut colonization of these different species in mono-colonized animals and in co-culture under two different diets (simple sugar, or pollen). They observed that pollen could sustain persistence of these species, unlike the simpler diet where the community was dominated by a single species.
To disentangle between the role of the host and microbe-microbe interactions in this process they performed similar experiments in laboratory cultures. In laboratory in vitro cultures they also observed co-existence and persistence in the pollen diet, but one-species domination was observed when glucose was the main carbon source. Therefore, they concluded that a complex diet (and not the host) was key for enabling persistence, as the results were similar in the laboratory cultures.
Their studies were complemented by transcriptomics and metabolomics and these results support the general conclusions that pollen contains diverse carbon sources which could be used in complementary ways by the different species, which have diverse metabolic capabilities encoded in their genomes.
One of the points that was not completely explored in the paper is what happens in the simplified diet both in vitro and in the Bee gut. They propose in the discussion that in the presence of few and simple carbon sources (sugars) there is competition for nutrients and competitive exclusion is driving loss of some species. But this is not fully addressed in the paper.
The system they use (with 4 closely related bacterial species) is a simplified system. Therefore, it is not clear if the same general findings will hold in more complex systems. But the results supporting that nutrient complexity (in diet) and metabolic diversity (from the microbial side) are key factors to enable co-existence and persistence of complex microbiota communities are strong and likely generalizable. Although, it is possible that with other communities and other hosts other factors will also come into play. Nonetheless, the current study is important because it sets a good example for how these questions can be addressed to study more complex systems.
Overall, the study described here is complete, and rigorous, except for a few points that still need to be addressed and clarified. Namely, it would be interesting to understand what drives exclusion of some members of the community in the simplified diet.
Importantly, the current study opens the door for new studies (including in vitro studies) on the identification of network interactions that are important for Microbe-Microbe interactions that enable co-existence in other systems. Additionally, this study also highlights the importance of identifying the relevant nutritional (and metabolic) conditions for addressing those questions given the importance of the metabolic context in shaping microbe-microbe interactions.
-
Reviewer #2 (Public Review):
This paper investigates the mechanisms that allow closely related species to coexist in a gut community. A simple expectation is that more closely related species will overlap more in their ecological niche, and thus tend to compete. However, factors that add complexity and heterogeneity, such as diet, immune response, gut morphology and bacteriophage may cause the realized niche of species to overlap less in the gut environment. The honeybee gut is a beautiful model and is also a good choice to test the competition-relatedness hypothesis, because the core microbiota of bees is made up of distinct phylotypes each containing closely related species. The authors select a single phylotype and compare the community assembly in a gnotobiotic colonization model and in defined culture conditions based on the bee …
Reviewer #2 (Public Review):
This paper investigates the mechanisms that allow closely related species to coexist in a gut community. A simple expectation is that more closely related species will overlap more in their ecological niche, and thus tend to compete. However, factors that add complexity and heterogeneity, such as diet, immune response, gut morphology and bacteriophage may cause the realized niche of species to overlap less in the gut environment. The honeybee gut is a beautiful model and is also a good choice to test the competition-relatedness hypothesis, because the core microbiota of bees is made up of distinct phylotypes each containing closely related species. The authors select a single phylotype and compare the community assembly in a gnotobiotic colonization model and in defined culture conditions based on the bee diet. I believe that all of my concerns are easily addressable, and I think that this manuscript will be a very nice contribution to this active area of research.
Strengths: The use of community profiling, transcriptomics, and metabolomics adds depth, as does the comparison of defined culture conditions to the host environment. The main conclusions drawn by the authors is that the presence of pollen is necessary for gut species to coexist, and that the different species, although closely related, respond in distinct ways to nutrients in pollen and consume different profiles of nutrients from pollen.
Weaknesses: The main weakness I see with this work is the choice of in vitro comparison conditions. The strains are cultured either on pollen or sugar water, whereas in vivo bees are fed a diet of pollen and sugar water, or only sugar water. A direct comparison is possible between the strains grown on sugar water in vitro or in vivo, but I think that in several places, the authors may have to reconsider or modify their interpretations comparing in vitro culture on pollen/pollen extract with the in vivo growth of the community on pollen and sugar water. Because there is sugar in the bee diet, differences in assembly dynamics, transcription, or metabolite consumption between pollen-containing culture conditions and the bee gut might stem from the dietary intake of sugar, or from an aspect of the host environment.
-
Reviewer #3 (Public Review):
Brochet et al. find that four species of the Lactobacillus Firm-5 lineage, one of the core bacterial lineages of the honey bee microbiome, are able to coexist because they utilize different pollen-derived flavonoids and sugars. They demonstrated this both in vivo, in gnotobiotic bees, and in vitro with laboratory co-cultures. Simple yet robust experiments involving diet or growth media with just simple sugars resulted in loss of diversity, whereas diets and media supplemented with pollen allowed the persistence of all four Firm-5 species over multiple serial passages. The authors then proceeded to examine the genes that were differentially expressed in response to different nutrient growth conditions, as well as the presence of metabolites to infer utilization of pollen-derived nutrients. The results paint a …
Reviewer #3 (Public Review):
Brochet et al. find that four species of the Lactobacillus Firm-5 lineage, one of the core bacterial lineages of the honey bee microbiome, are able to coexist because they utilize different pollen-derived flavonoids and sugars. They demonstrated this both in vivo, in gnotobiotic bees, and in vitro with laboratory co-cultures. Simple yet robust experiments involving diet or growth media with just simple sugars resulted in loss of diversity, whereas diets and media supplemented with pollen allowed the persistence of all four Firm-5 species over multiple serial passages. The authors then proceeded to examine the genes that were differentially expressed in response to different nutrient growth conditions, as well as the presence of metabolites to infer utilization of pollen-derived nutrients. The results paint a convincing picture of niche partitioning via differentiation in both encoded metabolic capabilities and in the differential expression of commonly encoded genes among co-resident bacterial species.
Overall, the paper is strong and the arguments and conclusions put forth are well supported by the data. I only have a few suggestions:
The study focuses on one strain each of the 4 Firm-5 species; however, there is diversity within each species. This is only briefly mentioned in the paper at the very end, and I think the authors should address this a bit more directly. In particular, they have previously generated a large amount of genomic data from some of these other strains, so it is likely possible to infer or speculate, based on this data, whether they expect different strains within each species to utilize similar nutrients. Also, I'm wondering if the authors can comment on how their findings could extend to the related bumble bee gut microbiome. Such a discussion would help enhance the applicability and importance of this study.
It is interesting that different species ended up dominating in the in vivo vs. in vitro simple sugar-based communities. What do the authors think may be behind this difference?
Since the observed coexistence of these gut microbes is largely due to nutritional niche partitioning, it would be helpful if the authors can comment on the natural variation of key pollen derived metabolites, and if/how we could expect ecological variation in the bee microbiome due to plant pollen availability based on biogeography and seasonality.
The supplementary information is nicely documented and accessible, but I think it would be even more useful if genome-wide data for the RNA-seq results, not just for select genes, are made available. Furthermore, I suggest including descriptive titles and labels within the supplementary Excel files, as there are many separate sheets and it is not always clear what each one shows.
-