Molecular Ecology (2011) 20, 619–628 doi: 10.1111/j.1365-294X.2010.04959.x A simple and distinctive microbiota associated with honey bees and bumble bees VINCENT G. MARTINSON,* BRYAN N. DANFORTH,† ROBERT L. MINCKLEY,‡ OLAV RUEPPELL,§ SALIM TINGEK– and NANCY A. MORAN**,1 *Center for Insect Science, University of Arizona, 1007 East Lowell St. Rm 204, Tucson, AZ 85721-0088, USA, †Department of Entomology, Cornell University, Ithaca, NY 14853-5905, USA, ‡Department of Biology, University of Rochester, Rochester, NY 14627-0211, USA, §Department of Biology, University of North Carolina, Greensboro, NC 27402-6170, USA, –Agricultural Research Station, Tenom, Sabah, Malaysia, **Department of Ecology and Evolutionary Biology, University of Arizona, Tucson, AZ 85721-0088, USA Abstract Specialized relationships with bacteria often allow animals to exploit a new diet by providing a novel set of metabolic capabilities. Bees are a monophyletic group of Hymenoptera that transitioned to a completely herbivorous diet from the carnivorous diet of their wasp ancestors. Recent culture-independent studies suggest that a set of distinctive bacterial species inhabits the gut of the honey bee, Apis mellifera. Here we survey the gut microbiotae of diverse bee and wasp species to test whether acquisition of these bacteria was associated with the transition to herbivory in bees generally. We found that most bee species lack phylotypes that are the same or similar to those typical of A. mellifera, rejecting the hypothesis that this dietary transition was symbiont- dependent. The most common bacteria in solitary bee species are a widespread phylotype of Burkholderia and the pervasive insect associate, Wolbachia. In contrast, several social representatives of corbiculate bees do possess distinctive bacterial phylotypes. Samples of A. mellifera harboured the same microbiota as in previous surveys, and closely related bacterial phylotypes were identified in two Asian honey bees (Apis andreniformis and Apis dorsata) and several bumble bee (Bombus) species. Potentially, the sociality of Apis and Bombus species facilitates symbiont transmission and thus is key to the maintenance of a more consistent gut microbiota. Phylogenetic analyses provide a more refined taxonomic placement of the A. mellifera symbionts. Keywords: Apis mellifera, bacterial microbiota, insect symbiosis, microbiology Received 15 July 2010; revision received 15 September 2010; accepted 24 September 2010 & Vasquez 2008). This result is contrary to results of Introduction previous culture-based surveys, which showed a com- Recent non-culture-based 16S rRNA and metagenomic plex and inconsistent microbiota of over 6000 bacterial surveys have revealed that the guts of honey bees (Apis strains in A. mellifera guts (Gilliam 1997). The eight mellifera) from the United States, Australia, South characteristic phylotypes constitute 95% of bacterial Africa, Germany, Sweden, and Switzerland harbour 16S rRNA sequences cloned from A. mellifera abdomens, representatives of the same eight bacterial phylotypes and represent five bacterial classes (Fig. S1, Supporting (Fig. 1) (Jeyaprakash et al. 2003; Mohr & Tebbe 2006; information) (Cox-Foster et al. 2007). The association Babendreier et al. 2007; Cox-Foster et al. 2007; Olofsson between these bacteria and A. mellifera is highly con- served despite environmental, geographic, and subspe- Correspondence: Vincent G. Martinson, Fax: +1 520 621 9190; cies differences of hosts (Jeyaprakash et al. 2003; Mohr E-mail: [email protected] 1Current address: Department of Ecology and Evolutionary & Tebbe 2006; Babendreier et al. 2007; Olofsson & Vas- Biology, Yale University, 21 Sachem Street, New Haven, CT quez 2008). These observations suggest that A. mellifera 06520, USA. has a coevolved symbiotic relationship with some or all Ó 2010 Blackwell Publishing Ltd 620 V. G. MARTINSON ET AL. Phylogeny of bacterial phyla Tebbe 2006). In the current study, we further character- γ Gammaproteobacteria ize the A. mellifera microbiota and determine the distri- “Gamma-1” Mycoplasmas β bution of the same or related lineages in gut Chlamydiales “Gamma-2” α Betaproteobacteria communities from members of all but one bee family, Simonsiella/Alysiella “Beta” the wasp clade sister to all bees, and a distantly related ε Alphaproteobacteria wasp that has independently shifted to an exclusively Spirochetes Bartonella Proteobacteria “Alpha-1” pollen and nectar diet. Acetobacteraceae Green sulfur Cyanobacteria “Alpha-2” Deinococcales Actinobacteria Bifidobacterium Methods Bacteroidetes “Bifido” Firmicutes High G+C Lactobacillus Specimen collection and DNA extraction Gram positives Low G+C “Firm-4” Gram positives Thermotogales “Firm-5” Aquificales Bee and wasp specimens were collected into 95% etha- nol and stored at )80 °C until DNA was extracted Fig. 1 Phylogenetic positions of A. mellifera bacterial gut mic- (Table S1, Supporting information). Specimens were col- robiota associates. Phylogeny based on Brochier et al. (2002). lected randomly as they were foraging at flowers. The two A. mellifera samples were prepared from adults col- lected within a single healthy hive in January 2008 at the of these bacteria, and that the associations are main- Carl Hayden Bee Research Center (Tucson, AZ). For the tained across generations of the host. A. mellifera hive sample a total of eighty adult digestive Many insects (i.e. termites, leaf-cutter ants, aphids, tracts (crop, midgut, ileum, and rectum) were dissected etc.) have evolved non-pathogenic, persistent associa- in 10 mM MgSO4, homogenized with iris dissection scis- tions with microorganisms that provide benefits to both sors followed by a 1.5-mL tube mini-pestle, and passed partners (Hongoh et al. 2008; Moran et al. 2008; Pinto- through an 8-lm filter to remove most host cells. Tomas et al. 2009). Nutritional symbioses represent the Whole abdomens were removed and macerated with majority of known insect-microbe mutualisms, and are sterile dissection scissors for all specimens except larger found in multiple insect lineages that subsist on unusual bees (i.e. Xylocopa sp. and Bombus sp.). For large speci- or low-nutrient diets (e.g. sap, blood, detritus, wood) mens, the abdominal exoskeleton was burdensome to fit (Moran et al. 2008). For example, acquisition of a nutri- into a 1.5-mL tube and prevented complete homogeniza- ent-provisioning bacterial symbiont in a sharpshooter tion of the digestive tract so it was not used in the DNA (Hemiptera: Cicadellidae: Cicadellinae) ancestor coin- extraction. For specimens stored in ethanol, DNA was cided with the transition from a diet of phloem sap extracted using the Gentra PureGene Kit (Qiagen Inc.) to the comparatively nutrient-poor diet of xylem sap preceded by a 30-min lysozyme incubation (outlined in (Moran 2007), illustrating the potential impact microbial the DNeasy kit, Qiagen Inc.) to lyse Gram-positive bacte- symbiosis can have on the ecology of the host insect. rial cells. For specimens stored in lysis buffer (0.1 M Tris– Bees arose from predatory apoid wasps in the early HCl, 0.1 M EDTA, 0.01 M NaCl, 0.005% SDS, 500 lg ⁄ mL to mid-Cretaceous, transitioning from the ancestral car- Proteinase K), DNA was extracted with a standard phe- nivorous life-style to herbivory, depending upon plant nol ⁄ chloroform reaction, followed by ethanol precipita- pollen as their sole protein source (Danforth et al. tion. Resulting DNA was analysed for molecular weight 2006). Pollen cytoplasm is nutrient-rich but is protected and quantity on a 1.0% agarose gel (80 V, 2 h). by a carbohydrate exine that is refractory to most diges- tive systems; nevertheless, diverse insects and verte- 16S rRNA PCR and cloning brates are able to use pollen as food (Roulston & Cane 2000). One hypothesis for the origin of the distinct die- DNA samples for two A. mellifera samples, 11 other bee tary habits and ecology observed in bees is that these species, and three wasp species were sent to the Joint were facilitated by the acquisition of a distinctive micro- Genome Institute (JGI, Walnut Creek, CA). At JGI, par- biota capable of supplementing the host with necessary tial 16S rRNA gene sequences were amplified from nutrients, or assisting in the digestion of pollen. If so, bac- each specimen using universal bacterial primers 27F teria closely related to the lineages found in A. mellifera (5¢-AGAGTTTGATCCTGGCTCAG-3¢) and 1391R (5¢- might be ubiquitous across all bee species, which com- GACGGGCRGTGWGTRCA-3¢) with reaction conditions prise a monophyletic clade within the superfamily listed in the JGI SOP 16S18S rRNA PCR Library Crea- Apoidea. Outside of A. mellifera, few bee species (and tion protocol as in Warnecke et al. (2007). PCR products specimens) have been screened, so the distribution of were screened on a 1% agarose gel (100 V, 1 h) for the these bacterial types in bees has not been clear (Mohr & expected size along with a size ladder and positive Ó 2010 Blackwell Publishing Ltd THE MICROBIOTA OF HONEY BEES AND BUMBLE BEES 621 (known bacterial DNA) and negative (no template alignment program (DeSantis et al. 2006), and potential DNA) controls. Resulting PCR products were cloned chimeric sequences were identified with Bellerophon using the TOPO TA Cloning Kit (Invitrogen), and 384 (Huber et al. 2004). Chimeric sequences were manually clones were chosen for sequencing (forward and verified with the online RDP Chimera Detection pro- reverse). Sequences were automatically edited and gram (Cole et al. 2003) and removed from the data