Surveys of 16S rDNA sequences from the honey bee, Apis mellifera, have revealed the presence of eight distinctive bacterial phylotypes in intestinal tracts of adult worker bees. Because previous studies have been limited to relatively few sequences from samples pooled from multiple hosts, the extent of variation in this microbiota among individuals within and between colonies and locations has been unclear. We surveyed the gut microbiota of 40 individual workers from two sites, Arizona and Maryland USA, sampling four colonies per site. Universal primers were used to amplify regions of 16S ribosomal RNA genes, and amplicons were sequenced using 454 pyrotag methods, enabling analysis of about 330,000 bacterial reads. Over 99% of these sequences belonged to clusters for which the first blastn hits in GenBank were members of the known bee phylotypes. Four phylotypes, one within Gammaproteobacteria (corresponding to “Candidatus Gilliamella apicola”) one within Betaproteobacteria (“Candidatus Snodgrassella alvi”), and two within Lactobacillus, were present in every bee, though their frequencies varied. The same typical bacterial phylotypes were present in all colonies and at both sites. Community profiles differed significantly among colonies and between sites, mostly due to the presence in some Arizona colonies of two species of Enterobacteriaceae not retrieved previously from bees. Analysis of Sanger sequences of rRNA of the Snodgrassella and Gilliamella phylotypes revealed that single bees contain numerous distinct strains of each phylotype. Strains showed some differentiation between localities, especially for the Snodgrassella phylotype.

Nitrogen acquisition and assimilation is a primary concern of insects feeding on diets largely composed of plant material. Reclaiming nitrogen from waste products provides a rich reserve for this limited resource, provided that recycling mechanisms are in place. Cockroaches, unlike most terrestrial insects, excrete waste nitrogen within their fat bodies as uric acids, postulated to be a supplement when dietary nitrogen is limited. The fat bodies of most cockroaches are inhabited by Blattabacterium, which are vertically transmitted, Gram-negative bacteria that have been hypothesized to participate in uric acid degradation, nitrogen assimilation, and nutrient provisioning. We have sequenced completely the Blattabacterium genome from Periplaneta americana. Genomic analysis confirms that Blattabacterium is a member of the Flavobacteriales (Bacteroidetes), with its closest known relative being the endosymbiont Sulcia muelleri, which is found in many sap-feeding insects. Metabolic reconstruction indicates that it lacks recognizable uricolytic enzymes, but it can recycle nitrogen from urea and ammonia, which are uric acid degradation products, into glutamate, using urease and glutamate dehydrogenase. Subsequently, Blattabacterium can produce all of the essential amino acids, various vitamins, and other required compounds from a limited palette of metabolic substrates. The ancient association with Blattabacterium has allowed cockroaches to subsist successfully on nitrogen-poor diets and to exploit nitrogenous wastes, capabilities that are critical to the ecological range and global distribution of cockroach species.Flavobacteria ͉ insect endosymbiont ͉ nitrogen conservation ͉ uricolysis

Starting in 2003, numerous studies using culture-independent methodologies to characterize the gut microbiota of honey bees have retrieved a consistent and distinctive set of eight bacterial species, based on near identity of the 16S rRNA gene sequences. A recent study [Mattila HR, Rios D, Walker-Sperling VE, Roeselers G, Newton ILG (2012) Characterization of the active microbiotas associated with honey bees reveals healthier and broader communities when colonies are genetically diverse. PLoS ONE 7(3): e32962], using pyrosequencing of the V1–V2 hypervariable region of the 16S rRNA gene, reported finding entirely novel bacterial species in honey bee guts, and used taxonomic assignments from these reads to predict metabolic activities based on known metabolisms of cultivable species. To better understand this discrepancy, we analyzed the Mattila et al. pyrotag dataset. In contrast to the conclusions of Mattila et al., we found that the large majority of pyrotag sequences belonged to clusters for which representative sequences were identical to sequences from previously identified core species of the bee microbiota. On average, they represent 95% of the bacteria in each worker bee in the Mattila et al. dataset, a slightly lower value than that found in other studies. Some colonies contain small proportions of other bacteria, mostly species of Enterobacteriaceae. Reanalysis of the Mattila et al. dataset also did not support a relationship between abundances of Bifidobacterium and of putative pathogens or a significant difference in gut communities between colonies from queens that were singly or multiply mated. Additionally, consistent with previous studies, the dataset supports the occurrence of considerable strain variation within core species, even within single colonies. The roles of these bacteria within bees, or the implications of the strain variation, are not yet clear.

Phytophagous pentatomid insects can negatively impact agricultural productivity and the brown marmorated stink bug (Halyomorpha halys) is an emerging invasive pest responsible for damage to many fruit crops and ornamental plants in North America. Many phytophagous stink bugs, including H. halys, harbor gammaproteobacterial symbionts that likely contribute to host development, and characterization of symbiont transmission/acquisition and their contribution to host fitness may offer alternative strategies for managing pest species. “Candidatus Pantoea carbekii” is the primary occupant of gastric ceca lumina flanking the distal midgut of H. halys insects and it is acquired each generation when nymphs feed on maternal extrachorion secretions following hatching. Insects prevented from symbiont uptake exhibit developmental delays and aberrant behaviors. To infer contributions of Ca. P. carbekii to H. halys, the complete genome was sequenced and annotated from a North American H. halys population. Overall, the Ca. P. carbekii genome is nearly one-fourth (1.2 Mb) that of free-living congenerics, and retains genes encoding many functions that are potentially host-supportive. Gene content reflects patterns of gene loss/retention typical of intracellular mutualists of plant-feeding insects. Electron and fluorescence in situ microscopic imaging of H. halys egg surfaces revealed that maternal extrachorion secretions were populated with Ca. P. carbekii cells. The reported findings detail a transgenerational mode of symbiont transmission distinct from that observed for intracellular insect mutualists and illustrate the potential additive functions contributed by the bacterial symbiont to this important agricultural pest.

Beneficial microbial associations with insects are common and are classified as either one or a few intracellular species that are vertically transmitted and reside intracellularly within specialized organs or as microbial assemblages in the gut. Cockroaches and termites maintain at least one if not both beneficial associations. Blattabacterium is a flavobacterial endosymbiont of nearly all cockroaches and the termite Mastotermes darwiniensis and can use nitrogenous wastes in essential amino acid and vitamin biosynthesis. Key changes during the evolutionary divergence of termites from cockroaches are loss of Blattabacterium, diet shift to wood, acquisition of a specialized hindgut microbiota, and establishment of advanced social behavior. Termite gut microbes collaborate to fix nitrogen, degrade lignocellulose, and produce nutrients, and the absence of Blattabacterium in nearly all termites suggests that its nutrient-provisioning role has been replaced by gut microbes. M. darwiniensis is a basal, extant termite that solely retains Blattabacterium, which would show evidence of relaxed selection if it is being supplanted by the gut microbiome. This termite-associated Blattabacterium genome is ϳ8% smaller than cockroach-associated Blattabacterium genomes and lacks genes underlying vitamin and essential amino acid biosynthesis. Furthermore, the M. darwiniensis gut microbiome membership is more consistent between individuals and includes specialized termite gut-associated bacteria, unlike the more variable membership of cockroach gut microbiomes. The M. darwiniensis Blattabacterium genome may reflect relaxed selection for some of its encoded functions, and the loss of this endosymbiont in all remaining termite genera may result from its replacement by a functionally complementary gut microbiota.