- Email: [email protected]
Two gut community enterotypes recur in diverse bumblebee species
Current Biology
Magazine Correspondence
Two gut community enterotypes recur in diverse bumblebee species Jilian Li1,8, J. Elijah Powell2,8, Jun Guo5,8, Jay D. Evans4, Jie Wu1, Paul Williams6, Qinhui Lin7, Nancy A. Moran2,*, and Zhigang Zhang3,* Pollinating insects are key to the evolutionary and ecological success of flowering plants and enable much of the diversity in the human diet. Gut microbial communities likely impact pollinators in diverse ways, from nutrition to defense against disease [1,2]. Honeybees and bumblebees harbor a simple yet specialized gut microbiota [3] dominated by several newly described bacterial species, including Gilliamella apicola, Frischella perrara, Snodgrassella alvi and specialized species of Lactobacillus. These bacterial groups are absent from the guts of other bees studied to date [3,4]. Although simple, the gut microbiota of honeybees and bumblebees share several similarities to those of mammals. First, both are socially transmitted, and young individuals are colonized through interactions with older individuals [5]. Second, in guts of both humans and honeybees, a particular bacterial species is typically represented by many strains; for example, in honeybees, both G. apicola and S. alvi occur as numerous strains with distinct gene sets [6]. Third, in both humans and social bees, individual hosts vary in the composition of their gut communities [5,7,8]. This variation likely reflects the interplay of symbiont specialization to distinct hosts and opportunity for transfer among hosts. A striking finding for human gut communities has been the occurrence of enterotypes, which are defined as “densely populated areas in a multidimensional space of community composition” [8]. Three enterotypes are found in diverse human populations, a result mirrored in chimpanzees [9]. In both humans and chimpanzees one enterotype shows overrepresentation R652
of Bacteroides, Faecalibacterium and Parabacteroides; a second enterotype shows overrepresentation of Lachnospiraceae; and a third shows overrepresentation of Dialester, Ruminococcus, Subdoligranulum and Collinsella. Bumblebees display high species and ecological diversity and, like mammals, contain specialized gut bacteria that are transmitted socially [3,6]. Thus, bumblebees provide an ideal system for examining the evolution and ecology of gut communities across related host species and varied environments. China has the highest diversity of bumblebees thus observed, harboring about 125 of the 250 recognized species [10]. We surveyed the gut microbial communities of 142 workers from 28 species of Chinese bumblebees (Table S1A in Supplemental Information) using high-throughput 454 pyrosequencing of the V6–V8 region of bacterial 16S rRNA. We processed and filtered sequences, clustered them into operational taxonomic units (OTUs) with 97% minimum identity, and excluded plastids, singletons, and OTUs restricted to single samples. Using the resulting OTU table, we tested for the presence of enterotypes (Table S1B) using described methods [8]. Also, we screened samples for the widespread trypanosomatid parasite Crithidia bombi. Detailed experimental procedures and analysis methods are provided in Supplemental Information. A total of 314,401 sequences from 141 samples (2,214 ± 836 (SD) reads per sample) clustered into 380 OTUs. To test for differences in the presence of different genera, we binned OTUs into 77 genera, based on BLAST searches against the SILVA SSU database. Communities sorted into two robust enterotypes based on the Calinski– Harabasz (CH) index, a test criterion for determining numbers of clusters formed by points in a dataset [8] (Figure 1A). Most samples (73%) sorted into Enterotype 2, distinguished by abundant Gilliamella and Snodgrassella (Figure 1B,C). Lactobacillus was common in both enterotypes, while Enterotype 1 had more Serratia and Hafnia (Figure 1B,C). Enterotype 1 samples had more genera on average (p < 0.0001, Kruskal-Wallis) and lower evenness (p < 0.0001, Kruskal-Wallis). When analyzed at the OTU level,
Current Biology 25, R635–R653, August 3, 2015 ©2015 Elsevier Ltd All rights reserved
each enterotype split into two distinct subclusters, based on the CH criterion. Of 28 host species, 15 had individuals with both enterotypes. No differences were evident among Bombus subgenera. Sample numbers were not sufficient for powerful tests of interspecific differences, but three species (B. lepidus, B. trifasciatus and B. waltoni) showed an excess of individuals hosting Enterotype 1 (p < 0.05, Fisher’s exact test). We estimated the average absolute number of bacterial rRNA genes at 1.2 x 108 (± 1.1 x 108) copies or approximately 3 x 107 bacterial cells per gut. Crithidia prevalence was not significantly different (p = 0.41, Fisher’s exact test), at 37% and 28% for Enterotypes 1 and 2, respectively. Similarly Crithidia prevalence did not differ across the OTU-based subclusters (p > 0.6, Fisher’s exact test). Bumblebees and honeybees are hosts to related gut bacterial species, including G. apicola, S. alvi, and specific clusters of Lactobacillus. The occurrence of two very different community types in bumblebees contrasts with gut communities in honeybee workers, which are consistently dominated by strains of G. apicola, S. alvi, and Lactobacillus [3,5]. One hypothesis for this difference is that typical bee-associated bacteria are depleted in some Bombus queens during their overwintering period, an unlikely event for perennial honeybee colonies. Alternatively, bumblebee foragers may sometimes encounter environmental bacteria that invade and largely replace bee-specific bacterial groups. Enterotypes did not differ significantly in the number of 16S rRNA gene copies, parasite loads or obvious host pathologies. However, several lines of evidence suggest that G. apicola and S. alvi, abundant in Enterotype 2, might affect bee health, including pathogen defense [1] and nutrition [2]. By contrast, Hafnia and Serratia, prevalent in Enterotype 1, contain strains of species known to be pathogenic and invasive in insects. Both enterotypes contain abundant Lactobacillus (Figure 1B,C), although the particular species may differ, as Lactobacillus contains both bee-gut-restricted species and environmental species associated with nectar and other substrates [4]. In conclusion, the finding of distinct enterotypes in bumblebees shows a
Current Biology
Magazine scientific and technological talents program (2013HA020). Read data were deposited in GenBank (Bioproject SRP052691). The authors declare no conflict of interest.
A
REFERENCES 2
Relative abundance 0.0 0.4 0.8
Gilliamella ****
Lactobacillus Relative abundance 0.0 0.4 0.8
Hafnia Relative abundance 0.0 0.2 0.4 0.6 0.8
Serratia
****
Enterotype_1 Enterotype_2
Enterotype_1 Enterotype_2
****
Snodgrassella Relative abundance 0.0 0.2 0.4 0.6 0.8
B
Relative abundance 0.0 0.2 0.4 0.6 0.8
1
Enterotype_1 Enterotype_2
****
Enterotype_1 Enterotype_2
C Gilliamella Snodgrassella Hafnia Serratia 0.10.2 0.3 0.4
Lactobacillus
Mean relative Enterotype_1 Enterotype_2 abundance Enterotype_1 Enterotype_2
Figure 1. Gut community composition in Chinese bumblebees. (A) Principle component analysis (PCA) plot of enterotype clusters observed in Chinese Bombus workers (n = 141) belonging to 28 species. Enterotype 1 specimens were dominated by enterobacterial genera, whereas bee-gut specialists S. alvi and G. apicola dominated in Enterotype 2. Lines represent distances of points from centroids; ellipses show areas of highest density. (B) The generic boxplots show differences between the two enterotypes in mean relative abundance of bacterial genera (defined as number of sequences assigned to genus/total number of sequences). All differences are statistically significant (p < 0.0001, Kruskal-Wallis) except for Lactobacillus (p = 0.834, Kruskal-Wallis). Genera were indicated as contributing to enterotypes by SIMPER analysis in R and include: Gilliamella, Snodgrassella, Serratia, Hafnia, and Lactobacillus. (C) Heatmap shows mean relative abundance of genera for both enterotypes. Correspondingly, relative abundances of top six OTUs across 141 worker samples can be seen in Figure S1.
striking parallel with mammalian gut communities [8], where enterotypes have been linked to diet, age, and sex, as well as host species. Experimental manipulation of the relatively simple bumblebee gut microbiome will help determine how these microbes affect individual and colony health. SUPPLEMENTAL INFORMATION Supplemental Information including experimental procedures, one figure and one data table,
can be found with this article online at http:// dx.doi.org/10.1016/j.cub.2015.06.031. ACKNOWLEDGEMENTS The work was supported by the Agricultural Science and Technology Innovation Program (CAAS-ASTIP-2015-IAR), the China Agriculture Research System (CARS-45), the Central Public Interest Scientific Institution Basal Research Fund (2015ZL001), the United States National Science Foundation (1415604), the National Natural Science Foundation of China (31471201) and the Yunnan province High-end
1. Koch, H., and Schmid-Hempel, P. (2011). Socially transmitted gut microbiota protect bumble bees against an intestinal parasite. Proc. Natl. Acad. Sci. USA 108, 19288–19292. 2. Kwong, W.K., Engel, P., Koch, H., and Moran, N.A. (2014). Genomics and host specialization of honey bee and bumble bee gut symbionts. Proc. Natl. Acad. Sci. USA 111, 11509–11514. 3. Martinson, V.G., Danforth, B.N., Minckley, R.L., Rueppell, O., Tingek, S., and Moran, N.A. (2011). A simple and distinctive microbiota associated with honey bees and bumble bees. Mol. Ecol. 20, 619–628. 4. McFrederick, Q.S., Cannone, J.J., Gutell, R.R., Kellner, K., Plowes, R.M., and Mueller, U.G. (2013). Specificity between Lactobacilli and hymenopteran hosts is the exception rather than the rule. Appl. Environ. Microbiol. 79, 1803–1812. 5. Powell, J.E., Martinson, V.G., Urban-Mead, K., and Moran, N.A. (2014). Routes of acquisition of the gut microbiota of Apis mellifera. Appl. Environ. Microbiol. 80, 7378–7387. 6. Engel, P., Stepanauskas, R., and Moran, N.A. (2014). Hidden diversity in honey bee gut symbionts detected by single-cell genomics. PLoS Genet. 10, e1004596. 7. Cariveau, D.P., Powell, E.J., Koch, H., Winfree, R., and Moran, N.A. (2014). Variation in gut microbial communities and its association with pathogen infection in wild bumble bees (Bombus). ISME J. 8, 2369–2379. 8. Arumugam, M., Raes, J., Pelletier, E., Le Paslier, D., Yamada, T., Mende, D.R., Fernandes, G.R., Tap, J., Bruis, T. Batto, J.M., et al. (2011). Enterotypes of the human gut microbiome. Nature 473, 174–180. 9. Moeller, A.H., Peeters, M., Ndjango, J.B., Li, Y., Hahn, B.H., and Ochman, H. (2013). Sympatric chimpanzees and gorillas harbor convergent gut microbial communities. Genome Res. 23, 1715–1720. 10. An, J., Huang, J., Shao, Y., Zhang, S., Wang, B., Liu, X., Wu, J., and Williams, P.H. (2014). The bumble bees of North China (Apidae, Bombus Latreille). Zootaxa 3830, 1−89.
1
Key Laboratory of Pollinating Insect Biology of the Ministry of Agriculture, Institute of Apicultural Research, Chinese Academy of Agricultural Science, 100093, Beijing, China. 2 Department of Integrative Biology, University of Texas at Austin, 2506 Speedway A5000, Austin, TX 78712. 3State Key Laboratory of Genetic Resources and Evolution, Laboratory of Evolutionary & Functional Genomics, Kunming Institute of Zoology, Chinese Academy of Sciences, Kunming 650223, Yunnan, China. 4United States Department of Agriculture, Agricultural Research Service, Bee Research Laboratory, Beltsville, MD 20705, USA. 5College of Life Science, Kunming University of Science and Technology, Kunming 650500, Yunnan, China. 6Department of Life Sciences, Natural History Museum, London SW7 5BD, UK. 7Computer Network Information Center, Chinese Academy of Sciences, Beijing, China. 8 These authors contributed equally to this work. *E-mail: [email protected] (N.A.M), [email protected] (Z.Z)
Current Biology 25, R635–R653, August 3, 2015 ©2015 Elsevier Ltd All rights reserved
R653
Magazine Correspondence
Two gut community enterotypes recur in diverse bumblebee species Jilian Li1,8, J. Elijah Powell2,8, Jun Guo5,8, Jay D. Evans4, Jie Wu1, Paul Williams6, Qinhui Lin7, Nancy A. Moran2,*, and Zhigang Zhang3,* Pollinating insects are key to the evolutionary and ecological success of flowering plants and enable much of the diversity in the human diet. Gut microbial communities likely impact pollinators in diverse ways, from nutrition to defense against disease [1,2]. Honeybees and bumblebees harbor a simple yet specialized gut microbiota [3] dominated by several newly described bacterial species, including Gilliamella apicola, Frischella perrara, Snodgrassella alvi and specialized species of Lactobacillus. These bacterial groups are absent from the guts of other bees studied to date [3,4]. Although simple, the gut microbiota of honeybees and bumblebees share several similarities to those of mammals. First, both are socially transmitted, and young individuals are colonized through interactions with older individuals [5]. Second, in guts of both humans and honeybees, a particular bacterial species is typically represented by many strains; for example, in honeybees, both G. apicola and S. alvi occur as numerous strains with distinct gene sets [6]. Third, in both humans and social bees, individual hosts vary in the composition of their gut communities [5,7,8]. This variation likely reflects the interplay of symbiont specialization to distinct hosts and opportunity for transfer among hosts. A striking finding for human gut communities has been the occurrence of enterotypes, which are defined as “densely populated areas in a multidimensional space of community composition” [8]. Three enterotypes are found in diverse human populations, a result mirrored in chimpanzees [9]. In both humans and chimpanzees one enterotype shows overrepresentation R652
of Bacteroides, Faecalibacterium and Parabacteroides; a second enterotype shows overrepresentation of Lachnospiraceae; and a third shows overrepresentation of Dialester, Ruminococcus, Subdoligranulum and Collinsella. Bumblebees display high species and ecological diversity and, like mammals, contain specialized gut bacteria that are transmitted socially [3,6]. Thus, bumblebees provide an ideal system for examining the evolution and ecology of gut communities across related host species and varied environments. China has the highest diversity of bumblebees thus observed, harboring about 125 of the 250 recognized species [10]. We surveyed the gut microbial communities of 142 workers from 28 species of Chinese bumblebees (Table S1A in Supplemental Information) using high-throughput 454 pyrosequencing of the V6–V8 region of bacterial 16S rRNA. We processed and filtered sequences, clustered them into operational taxonomic units (OTUs) with 97% minimum identity, and excluded plastids, singletons, and OTUs restricted to single samples. Using the resulting OTU table, we tested for the presence of enterotypes (Table S1B) using described methods [8]. Also, we screened samples for the widespread trypanosomatid parasite Crithidia bombi. Detailed experimental procedures and analysis methods are provided in Supplemental Information. A total of 314,401 sequences from 141 samples (2,214 ± 836 (SD) reads per sample) clustered into 380 OTUs. To test for differences in the presence of different genera, we binned OTUs into 77 genera, based on BLAST searches against the SILVA SSU database. Communities sorted into two robust enterotypes based on the Calinski– Harabasz (CH) index, a test criterion for determining numbers of clusters formed by points in a dataset [8] (Figure 1A). Most samples (73%) sorted into Enterotype 2, distinguished by abundant Gilliamella and Snodgrassella (Figure 1B,C). Lactobacillus was common in both enterotypes, while Enterotype 1 had more Serratia and Hafnia (Figure 1B,C). Enterotype 1 samples had more genera on average (p < 0.0001, Kruskal-Wallis) and lower evenness (p < 0.0001, Kruskal-Wallis). When analyzed at the OTU level,
Current Biology 25, R635–R653, August 3, 2015 ©2015 Elsevier Ltd All rights reserved
each enterotype split into two distinct subclusters, based on the CH criterion. Of 28 host species, 15 had individuals with both enterotypes. No differences were evident among Bombus subgenera. Sample numbers were not sufficient for powerful tests of interspecific differences, but three species (B. lepidus, B. trifasciatus and B. waltoni) showed an excess of individuals hosting Enterotype 1 (p < 0.05, Fisher’s exact test). We estimated the average absolute number of bacterial rRNA genes at 1.2 x 108 (± 1.1 x 108) copies or approximately 3 x 107 bacterial cells per gut. Crithidia prevalence was not significantly different (p = 0.41, Fisher’s exact test), at 37% and 28% for Enterotypes 1 and 2, respectively. Similarly Crithidia prevalence did not differ across the OTU-based subclusters (p > 0.6, Fisher’s exact test). Bumblebees and honeybees are hosts to related gut bacterial species, including G. apicola, S. alvi, and specific clusters of Lactobacillus. The occurrence of two very different community types in bumblebees contrasts with gut communities in honeybee workers, which are consistently dominated by strains of G. apicola, S. alvi, and Lactobacillus [3,5]. One hypothesis for this difference is that typical bee-associated bacteria are depleted in some Bombus queens during their overwintering period, an unlikely event for perennial honeybee colonies. Alternatively, bumblebee foragers may sometimes encounter environmental bacteria that invade and largely replace bee-specific bacterial groups. Enterotypes did not differ significantly in the number of 16S rRNA gene copies, parasite loads or obvious host pathologies. However, several lines of evidence suggest that G. apicola and S. alvi, abundant in Enterotype 2, might affect bee health, including pathogen defense [1] and nutrition [2]. By contrast, Hafnia and Serratia, prevalent in Enterotype 1, contain strains of species known to be pathogenic and invasive in insects. Both enterotypes contain abundant Lactobacillus (Figure 1B,C), although the particular species may differ, as Lactobacillus contains both bee-gut-restricted species and environmental species associated with nectar and other substrates [4]. In conclusion, the finding of distinct enterotypes in bumblebees shows a
Current Biology
Magazine scientific and technological talents program (2013HA020). Read data were deposited in GenBank (Bioproject SRP052691). The authors declare no conflict of interest.
A
REFERENCES 2
Relative abundance 0.0 0.4 0.8
Gilliamella ****
Lactobacillus Relative abundance 0.0 0.4 0.8
Hafnia Relative abundance 0.0 0.2 0.4 0.6 0.8
Serratia
****
Enterotype_1 Enterotype_2
Enterotype_1 Enterotype_2
****
Snodgrassella Relative abundance 0.0 0.2 0.4 0.6 0.8
B
Relative abundance 0.0 0.2 0.4 0.6 0.8
1
Enterotype_1 Enterotype_2
****
Enterotype_1 Enterotype_2
C Gilliamella Snodgrassella Hafnia Serratia 0.10.2 0.3 0.4
Lactobacillus
Mean relative Enterotype_1 Enterotype_2 abundance Enterotype_1 Enterotype_2
Figure 1. Gut community composition in Chinese bumblebees. (A) Principle component analysis (PCA) plot of enterotype clusters observed in Chinese Bombus workers (n = 141) belonging to 28 species. Enterotype 1 specimens were dominated by enterobacterial genera, whereas bee-gut specialists S. alvi and G. apicola dominated in Enterotype 2. Lines represent distances of points from centroids; ellipses show areas of highest density. (B) The generic boxplots show differences between the two enterotypes in mean relative abundance of bacterial genera (defined as number of sequences assigned to genus/total number of sequences). All differences are statistically significant (p < 0.0001, Kruskal-Wallis) except for Lactobacillus (p = 0.834, Kruskal-Wallis). Genera were indicated as contributing to enterotypes by SIMPER analysis in R and include: Gilliamella, Snodgrassella, Serratia, Hafnia, and Lactobacillus. (C) Heatmap shows mean relative abundance of genera for both enterotypes. Correspondingly, relative abundances of top six OTUs across 141 worker samples can be seen in Figure S1.
striking parallel with mammalian gut communities [8], where enterotypes have been linked to diet, age, and sex, as well as host species. Experimental manipulation of the relatively simple bumblebee gut microbiome will help determine how these microbes affect individual and colony health. SUPPLEMENTAL INFORMATION Supplemental Information including experimental procedures, one figure and one data table,
can be found with this article online at http:// dx.doi.org/10.1016/j.cub.2015.06.031. ACKNOWLEDGEMENTS The work was supported by the Agricultural Science and Technology Innovation Program (CAAS-ASTIP-2015-IAR), the China Agriculture Research System (CARS-45), the Central Public Interest Scientific Institution Basal Research Fund (2015ZL001), the United States National Science Foundation (1415604), the National Natural Science Foundation of China (31471201) and the Yunnan province High-end
1. Koch, H., and Schmid-Hempel, P. (2011). Socially transmitted gut microbiota protect bumble bees against an intestinal parasite. Proc. Natl. Acad. Sci. USA 108, 19288–19292. 2. Kwong, W.K., Engel, P., Koch, H., and Moran, N.A. (2014). Genomics and host specialization of honey bee and bumble bee gut symbionts. Proc. Natl. Acad. Sci. USA 111, 11509–11514. 3. Martinson, V.G., Danforth, B.N., Minckley, R.L., Rueppell, O., Tingek, S., and Moran, N.A. (2011). A simple and distinctive microbiota associated with honey bees and bumble bees. Mol. Ecol. 20, 619–628. 4. McFrederick, Q.S., Cannone, J.J., Gutell, R.R., Kellner, K., Plowes, R.M., and Mueller, U.G. (2013). Specificity between Lactobacilli and hymenopteran hosts is the exception rather than the rule. Appl. Environ. Microbiol. 79, 1803–1812. 5. Powell, J.E., Martinson, V.G., Urban-Mead, K., and Moran, N.A. (2014). Routes of acquisition of the gut microbiota of Apis mellifera. Appl. Environ. Microbiol. 80, 7378–7387. 6. Engel, P., Stepanauskas, R., and Moran, N.A. (2014). Hidden diversity in honey bee gut symbionts detected by single-cell genomics. PLoS Genet. 10, e1004596. 7. Cariveau, D.P., Powell, E.J., Koch, H., Winfree, R., and Moran, N.A. (2014). Variation in gut microbial communities and its association with pathogen infection in wild bumble bees (Bombus). ISME J. 8, 2369–2379. 8. Arumugam, M., Raes, J., Pelletier, E., Le Paslier, D., Yamada, T., Mende, D.R., Fernandes, G.R., Tap, J., Bruis, T. Batto, J.M., et al. (2011). Enterotypes of the human gut microbiome. Nature 473, 174–180. 9. Moeller, A.H., Peeters, M., Ndjango, J.B., Li, Y., Hahn, B.H., and Ochman, H. (2013). Sympatric chimpanzees and gorillas harbor convergent gut microbial communities. Genome Res. 23, 1715–1720. 10. An, J., Huang, J., Shao, Y., Zhang, S., Wang, B., Liu, X., Wu, J., and Williams, P.H. (2014). The bumble bees of North China (Apidae, Bombus Latreille). Zootaxa 3830, 1−89.
1
Key Laboratory of Pollinating Insect Biology of the Ministry of Agriculture, Institute of Apicultural Research, Chinese Academy of Agricultural Science, 100093, Beijing, China. 2 Department of Integrative Biology, University of Texas at Austin, 2506 Speedway A5000, Austin, TX 78712. 3State Key Laboratory of Genetic Resources and Evolution, Laboratory of Evolutionary & Functional Genomics, Kunming Institute of Zoology, Chinese Academy of Sciences, Kunming 650223, Yunnan, China. 4United States Department of Agriculture, Agricultural Research Service, Bee Research Laboratory, Beltsville, MD 20705, USA. 5College of Life Science, Kunming University of Science and Technology, Kunming 650500, Yunnan, China. 6Department of Life Sciences, Natural History Museum, London SW7 5BD, UK. 7Computer Network Information Center, Chinese Academy of Sciences, Beijing, China. 8 These authors contributed equally to this work. *E-mail: [email protected] (N.A.M), [email protected] (Z.Z)
Current Biology 25, R635–R653, August 3, 2015 ©2015 Elsevier Ltd All rights reserved
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