Honeybee gut microbiota promotes host weight gain via bacterial metabolism and hormonal signaling Hao Zheng (郑浩)a,1, J. Elijah Powella, Margaret I. Steelea, Carsten Dietrichb, and Nancy A. Morana,1 aDepartment of Integrative Biology, University of Texas at Austin, Austin, TX 78712; and bStrategy and Innovation Technology Center, Siemens Healthcare GmbH, 91052 Erlangen, Germany Edited by Joan E. Strassmann, Washington University in St. Louis, St. Louis, MO, and approved March 24, 2017 (received for review February 1, 2017) Social bees harbor a simple and specialized microbiota that is spa- fermentation products of dietary fiber accumulate in the human tially organized into different gut compartments. Recent results on colon in concentrations up to 80–130 mM (12) and serve as a the potential involvement of bee gut communities in pathogen major energy source for intestinal epithelial cells (13) or as the protection and nutritional function have drawn attention to the im- main respiratory substrate of the host (14). Moreover, microbial pact of the microbiota on bee health. However, the contributions of metabolites can have a profound effect on gut physiology; for gut microbiota to host physiology have yet to be investigated. Here example, their effects on oxygen concentration, pH, and redox we show that the gut microbiota promotes weight gain of both potential can be essential for host health (15). As neuroactive whole body and the gut in individual honey bees. This effect is likely compounds, SCFAs produced by the gut microbiota can affect mediated by changes in host vitellogenin, insulin signaling, and neural and immune pathways of the host and thereby influence gustatory response. We found that microbial metabolism markedly brain function and behavior (16). reduces gut pH and redox potential through the production of short- In the case of honey bees, genome-based investigations showed chain fatty acids and that the bacteria adjacent to the gut wall form that G. apicola strains potentially digest complex carbohydrates an oxygen gradient within the intestine. The short-chain fatty acid (i.e., pectin from pollen cell wall) that are otherwise indigestible profile contributed by dominant gut species was confirmed in vitro. by the host (17). A recent study documents that G. apicola strains Furthermore, metabolomic analyses revealed that the gut commu- can also use several sugars that are harmful to bees (18). How- nity has striking impacts on the metabolic profiles of the gut com- ever, how the bee gut microbiota affects host physiology and the MICROBIOLOGY partments and the hemolymph, suggesting that gut bacteria gut microenvironment has not yet been described. Hence, we degrade plant polymers from pollen and that the resulting me- compared germ-free (GF) honey bees to those with a conven- tabolites contribute to host nutrition. Our results demonstrate tional gut community (CV) to identify how the gut microbiota how microbial metabolism affects bee growth, hormonal signaling, affects weight gain, expression of genes underlying hormonal behavior, and gut physicochemical conditions. These findings in- dicate that the bee gut microbiota has basic roles similar to those pathways, gut physicochemical conditions, and metabolite pools found in some other animals and thus provides a model in studies of in the gut and hemolymph. – host microbe interactions. Results and Discussion Gut Microbiota Promotes Host Body and Gut Weight Gain. honeybee | gut microbiota | short-chain fatty acids | insulin | metabolomics To observe effects of the microbiota on growth of individual hosts, we per- formed serial measurements of whole-body wet weight in the oney bees (Apis mellifera) provide a critical link in global presence and absence of the gut microbiota. GF and CV bees Hfood production as pollinators of agricultural crops (1), and were obtained from pupae that were removed from hives, allowed their economic value is over $15 billion annually in the United to emerge in sterile laboratory conditions, and then fed either States alone (2). Honey bee populations have undergone elevated colony mortality during the last decade in the United States, Significance Canada, and Europe (3). A potential role of gut microbial com- munities in the health of honey bees has recently become more widely appreciated (4). Perturbation of the gut microbiota leads Honey bees are globally important plant pollinators. Guts of to higher mortality within hives and greater susceptibility to a adult workers contain specialized bacteria not found outside bacterial pathogen, suggesting a crucial role of normal microbiota bees. Experimental results show that gut bacteria increase in bee health (5). Honey bees are associated with specific in- weight gain in young adult bees, affect expression of genes testinal microbiota that is simpler than the microbiota found in governing insulin and vitellogenin levels, and increase sucrose mammals but shares some features, including host specificity and sensitivity. Gut bacteria also shape the physicochemical condi- social transmission (6) and a shared evolutionary history of bac- tions within the gut, lowering pH and oxygen levels. Peripheral terial and host lineages (7, 8). The bee gut is dominated by eight resident bacteria consume oxygen, thus maintaining anoxia, as core bacterial species that are spatially organized within specific required for microbial activity. Additionally, gut bacteria pro- duce short-chain fatty acids, with acetate and propionate as the gut regions. Few bacteria colonize the crop and midgut; the major metabolites, as in guts of human and other animals. This hindgut (ileum and rectum) harbors the greatest abundance of study demonstrates how bacteria in the honey bee gut affect bacteria. The ileum, a narrow tube with six longitudinal folds, is host weight gain and improves our understanding of how gut dominated by two Gram-negative bacterial species: Snodgrassella symbionts influence host health. alvi, a nonsugar fermenter forms a layer directly on the gut wall, together with Gilliamella apicola, a sugar fermenter that resides Author contributions: H.Z. and N.A.M. designed research; H.Z. and J.E.P. performed re- more toward the center of the lumen (9). The distal rectum is search; H.Z., M.I.S., and C.D. analyzed data; and H.Z. and N.A.M. wrote the paper. dominated by the Gram-positive Lactobacillus spp (10). The authors declare no conflict of interest. In both insects and mammals, the gut microbiota can possess a This article is a PNAS Direct Submission. large repertoire of metabolic capabilities and can contribute sub- 1To whom correspondence may be addressed. Email: [email protected] or nancy. stantively to dietary carbohydrate digestion within the intestinal [email protected]. ecosystem (11). Short-chain fatty acids (SCFAs), namely acetate, This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. propionate, and butyrate, produced by the gut microbiota as main 1073/pnas.1701819114/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1701819114 PNAS Early Edition | 1of6 Downloaded by guest on September 28, 2021 GF NSNS 6 GF GF A CV *** ***y BC)%( *** CV CV 140 5 30 *** NS *** HV Fig. 1. Gut microbiota increases honey bee whole- a d r d 4 body weight, gut weight, hormonal signaling, and 120 e 20 p na p sucrose sensitivity. (A) Whole-body wet weight 3 Inoculation growth curves of GF (n = 45) and CV bees (n = 49) i g thg g 2 100 10 originated from four different hives (the colony i *** NS eW 1 of origin was not statistically significant). (B) Daily initial body weight (%) weight body initial Body weight relative to relative weight Body weight gains of GF and CV bees after feeding 80 0 Gut compartment weight (mg) 0 0 4 8 12 16 GF CV Midgut Ileum Rectum with sterilized food or food supplemented with Days after emergence from pupae hindgut samples of hive nurse bees (day 1 to day 15). (C) Weights of different gut compartments of GF, D 8 *** E CV, and hive nurse bees (n = 25). Bars indicate mean 100 *** – 6 values of 10 15 pooled guts of bees from different = = GF hives (GF, n 4; CV, n 4) and hive bees from dif- 4 CV = Head ** 10 ferent hives (n 3). (D) Differential expression of ilp, 2 inR, and Vg genes in the head or abdomen of GF and CV bees originating from different hives. n = 3. Relative expression 0 ilp1 ilp2 1 (E) Distribution of sucrose-response thresholds of GF 8 *** (n = 27) and CV (n = 41) bees shown as a violin plot. 6 Sucrose concentration (%) The colony of origin was not statistically signif- Abdomen 0.1 0 icance. Each circle indicates a bee response to the 4 GF CV provided concentration of sucrose. In A–D,**P < ** NS 2 0.01, ***P < 0.001 (Mann–Whitney u test for the Relative expression 0 indicated comparisons); error bars indicate SD. In E, inR1 inR2 Vg ***P < 0.001 (χ2 test). NS, not significant. sterilized food or sterilized food inoculated with the gut content of of GF bees (Fig. 1B). After 15 d, the wet weights of both midgut conventional adult nurse bees. This procedure yields GF bees and ileum were also larger in CV bees than in GF bees and were with <105 bacteria per gut consisting of an erratic mix of bacterial similar to those of nurse bees collected from hives (Fig. 1C). In species versus CV bees with >108 bacteria per gut and consisting contrast, rectum weights were not significantly different and were primarily of core bee gut species, as in naturally inoculated hive more variable, probably because of variable levels of pollen and bees (19, 20). Although GF and CV bees showed similar survi- waste accumulation in this gut compartment (Fig. 1C). These re- vorship in laboratory experiments, CV bees attained greater body sults indicate that the presence of conventional gut microbiota is weight (Fig.