Genetic drift and host-adaptive features likely underlie cladogenesis of insect-associated Lachnospiraceae.

Phylogenetic and functional group analysis of the genomes of anaerobic bacteria isolated from Periplaneta americana digestive tracts suggest that they represent novel Lachnospiraceae genera. PAL113 and PAL227 isolate genomes encoded short-chain fatty acid biosynthetic pathways and plant fiber and chitin catabolism and other carbohydrate utilization genes common in related Lachnospiraceae species, yet the presence of operons containing flagellar assembly pathways were among several distinguishing features. In general, PAL113 and PAL227 isolates encode an array of gene products that would enable them to thrive in the insect gut environment and potentially play a role in host diet processing. We hypothesize that cladogenesis of these isolates could be due to their oxygen sensitivity, reliance upon the host for dispersal and genetic drift and not necessarily as a result of an ongoing mutualism.

Microbiota Perturbation or Elimination Can Inhibit Normal Development and Elicit a Starvation-Like Response in an Omnivorous Model Invertebrate.

Omnivorous animals, including humans, harbor diverse, species-rich gut communities that impact their growth, development, and homeostasis. Model invertebrates are broadly accessible experimental platforms that enable linking specific species or species groups to host phenotypes, yet often their specialized diets and distinct gut microbiota make them less comparable to human and other mammalian and gut communities. The omnivorous cockroach Periplaneta americana harbors ∼4 × 102 bacterial genera within its digestive tract and is enriched with taxa commonly found in omnivorous mammals (i.e., Proteobacteria, Bacteroidetes, and Firmicutes). These features make P. americana a valuable platform for identifying microbe-mediated host phenotypes with potential translations to mammals. Rearing P. americana insects under germfree conditions resulted in prolonging development time by ∼30% and an up to ∼8% reduction in body size along three dimensions. Germfree rearing resulted in downregulation of gene networks involved in growth, energy homeostasis, and nutrient availability. Reintroduction of a defined microbiota comprised of a subset of P. americana commensals to germfree insects did not recover normal growth and developmental phenotypes or transcriptional profiles observed in conventionally reared insects. These results are in contrast with specialist-feeding model insects (e.g., Drosophila), where introduction of a single endemic bacterial species to germfree condition-reared specimens recovered normal host phenotypes. These data suggest that understanding microbe-mediated host outcomes in animals with species-rich communities should include models that typically maintain similarly diverse microbiomes. The dramatic transcriptional, developmental, and morphological phenotypes linked to gut microbiome status in this study illustrates how microbes are key players in animal growth and evolution. IMPORTANCE Broadly accessible model organisms are essential for illustrating how microbes are engaged in the growth, development, and evolution of animals. We report that germfree rearing of omnivorous Periplaneta americana cockroaches resulted in growth defects and severely disrupted gene networks that regulate development, which highlights the importance of gut microbiota in these host processes. Absence of gut microbiota elicited a starvation-like transcriptional response in which growth and development were inhibited while nutrient scavenging was enhanced. Additionally, reintroduction of a subset of cockroach gut bacterial commensals did not broadly recover normal expression patterns, illustrating that a particular microbiome composition may be necessary for normal host development. Invertebrate microbiota model systems that enable disentangling complex, species-rich communities are essential for linking microbial taxa to specific host phenotypes.

Microbiome reduction prevents lipid accumulation during early diapause in the northern house mosquito, Culex pipiens pipiens.

The mosquito microbiome is critical to multiple facets of their biology, including larval development and disease transmission. For mosquitoes that reside in temperate regions, periods of diapause are critical to overwintering survival, but how the microbiome impacts this state is unknown. In this study, we compared the midgut microbial communities of diapausing and non-diapausing Culex pipiens and assessed how a reduced midgut microbiome influences diapause preparation. High community variability was found within and between non-diapausing and diapausing individuals, but no specific diapause-based microbiome was noted. Emergence of adult, diapausing mosquitoes under sterile conditions generated low bacterial load (LBL) lines with nearly a 1000-fold reduction in bacteria levels. This reduction in bacterial content resulted in significantly lower survival of diapausing females after two weeks, indicating acquisition of the microbiome in adult females is critical for survival throughout diapause. LBL diapausing females had high carbohydrate levels, but did not accumulate lipid reserves, suggesting an inability to process ingested sugars necessary for diapause-associated lipid accumulation. Expression patterns of select genes associated with mosquito lipid metabolism during diapause showed no significant differences between LBL and control lines, suggesting transcriptional changes may not underlie impaired lipid accumulation. Overall, a diverse, adult-acquired microbiome is critical for diapause in C. pipiens to process sugar reserves and accumulate lipids that are necessary to survive prolonged overwintering.

Microbial colonization promotes model cockroach gut tissue growth and development.

BACKGROUND: Digestive tissues are essential for diet processing and nutrient accessibility, especially in omnivores, and these functions occur despite and in collaboration with dynamic microbial communities that reside within and upon these tissues. Prolonged host development and reduced digestive tissue sizes have been observed in germ-free animals, and normal host phenotypes were recovered following the re-introduction of typical gut microbiomes via coprophagy. RESULTS: High-resolution histological analyses of Periplaneta americana cockroach digestive tissues revealed that total prevention of microbial colonization of the gut had severe impacts on the growth and development of gut tissues, especially the posterior midgut and anterior hindgut subcompartments that are expected to be colonized and inhabited by the greatest number of bacteria. Juveniles that were briefly exposed to normal gut microbiota exhibited a partial gut morphological recovery, suggesting that a single inoculation was insufficient. These data highlight gut microbiota as integral to normal growth and development of tissues they are in direct contact with and, more broadly, the organism in which they reside. CONCLUSIONS: We draw on these data, host life history traits (i.e. multigenerational cohousing, molting, and filial coprophagy and exuvia feeding), and previous studies to suggest a host developmental model in which gut tissues reflect a conflict-collaboration dynamic where 1) nutrient-absorptive anterior midgut tissues are in competition with transient and resident bacteria for easily assimilable dietary nutrients and whose growth is least-affected by the presence of gut bacteria and 2) posterior midgut, anterior hindgut, and to a lesser degree, posterior hindgut tissues are significantly impacted by gut bacterial presence because they are occupied by the greatest number of bacteria and the host is relying upon, and thus collaborating with, them to assist with complex polysaccharide catabolism processing and nutrient provisioning (i.e. short-chain fatty acids).

Multiple concurrent and convergent stages of genome reduction in bacterial symbionts across a stink bug family.

Nutritional symbioses between bacteria and insects are prevalent and diverse, allowing insects to expand their feeding strategies and niches. A common consequence of long-term associations is a considerable reduction in symbiont genome size likely influenced by the radical shift in selective pressures as a result of the less variable environment within the host. While several of these cases can be found across distinct insect species, most examples provide a limited view of a single or few stages of the process of genome reduction. Stink bugs (Pentatomidae) contain inherited gamma-proteobacterial symbionts in a modified organ in their midgut and are an example of a long-term nutritional symbiosis, but multiple cases of new symbiont acquisition throughout the history of the family have been described. We sequenced the genomes of 11 symbionts of stink bugs with sizes that ranged from equal to those of their free-living relatives to less than 20%. Comparative genomics of these and previously sequenced symbionts revealed initial stages of genome reduction including an initial pseudogenization before genome reduction, followed by multiple stages of progressive degeneration of existing metabolic pathways likely to impact host interactions such as cell wall component biosynthesis. Amino acid biosynthesis pathways were retained in a similar manner as in other nutritional symbionts. Stink bug symbionts display convergent genome reduction events showing progressive changes from a free-living bacterium to a host-dependent symbiont. This system can therefore be used to study convergent genome evolution of symbiosis at a scale not previously available.

Nutritional symbiosis and ecology of host-gut microbe systems in the Blattodea.

Cockroaches and termites (Order: Blattodea) have been the subject of substantial research attention for over a century due, in part, to a subset of them having a strong propensity to cohabitate with humans and their structures. Recent research has led to numerous insights into their behavior, physiology, and ecology, as well as their ability to harbor taxonomically diverse microbial communities within their digestive systems, which include taxa that contribute to host growth and development. Further, recent investigations into the physiological and behavioral adaptations that enable recalcitrant polysaccharide digestion and the maintenance of microbial symbionts in cockroaches and termites suggests that symbionts contribute significantly to nutrient provisioning and processing.

Cultivable, Host-Specific Bacteroidetes Symbionts Exhibit Diverse Polysaccharolytic Strategies.

Beneficial gut microbes can facilitate insect growth on diverse diets. The omnivorous American cockroach, Periplaneta americana (Insecta: Blattodea), thrives on a diet rich in plant polysaccharides and harbors a species-rich gut microbiota responsive to host diet. Bacteroidetes are among the most abundant taxa in P. americana and other cockroaches, based on cultivation-independent gut community profiling, and these potentially polysaccharolytic bacteria may contribute to host diet processing. Eleven Bacteroidetes isolates were cultivated from P. americana digestive tracts, and phylogenomic analyses suggest that they were new Bacteroides, Dysgonomonas, Paludibacter, and Parabacteroides species distinct from those previously isolated from other insects, humans, and environmental sources. In addition, complete genomes were generated for each isolate, and polysaccharide utilization loci (PULs) and several non-PUL-associated carbohydrate-active enzyme (CAZyme)-coding genes that putatively target starch, pectin, and/or cellulose were annotated in each of the isolate genomes. Type IX secretion system (T9SS)- and CAZyme-coding genes tagged with the corresponding T9SS recognition and export C-terminal domain were observed in some isolates, suggesting that these CAZymes were deployed via non-PUL outer membrane translocons. Additionally, single-substrate growth and enzymatic assays confirmed genomic predictions that a subset of the Bacteroides and Dysgonomonas isolates could degrade starch, pectin, and/or cellulose and grow in the presence of these substrates as a single sugar source. Plant polysaccharides enrich P. americana diets, and many of these gut isolates are well equipped to exploit host dietary inputs and potentially contribute to gut community and host nutrient accessibility.IMPORTANCE Gut microbes are increasingly being recognized as critical contributors to nutrient accessibility in animals. The globally distributed omnivorous American cockroach (Periplaneta americana) harbors many bacterial phyla (e.g., Bacteroidetes) that are abundant in vertebrates. P. americana thrives on a highly diverse plant-enriched diet, making this insect a rich potential source of uncharacterized polysaccharolytic bacteria. We have cultivated, completely sequenced, and functionally characterized several novel Bacteroidetes species that are endemic to the P. americana gut, and many of these isolates can degrade simple and complex polysaccharides. Cultivation and genomic characterization of these Bacteroidetes isolates further enable deeper insight into how these taxa participate in polysaccharide metabolism and, more broadly, how they affect animal health and development.

Conspecific coprophagy stimulates normal development in a germ-free model invertebrate.

Microbial assemblages residing within and on animal gastric tissues contribute to various host beneficial processes that include diet accessibility and nutrient provisioning, and we sought to examine the degree to which intergenerational and community-acquired gut bacteria impact development in a tractable germ-free (GF) invertebrate model system. Coprophagy is a common behavior in cockroaches and termites that provides access to both nutrients and the primary means by which juveniles are inoculated with beneficial gut bacteria. This hypothesis was tested in the American cockroach (Periplaneta americana) by interfering with this means of acquiring gut bacteria, which resulted in GF insects that exhibited prolonged growth rates and gut tissue dysmorphias relative to wild-type (WT) P. americana. Conventionalization of GF P. americana via consumption of frass (feces) from conspecifics and siblings reared under non-sterile conditions resulted in colonization of P. americana gut tissues by a diverse microbial community and a significant (p < 0.05) recovery of WT level growth and hindgut tissue development phenotypes. These data suggest that coprophagy is essential for normal gut tissue and organismal development by introducing beneficial gut bacteria to P. americana, and that the GF P. americana model system is a useful system for examining how gut bacteria impact host outcomes.

Cladogenesis and Genomic Streamlining in Extracellular Endosymbionts of Tropical Stink Bugs.

Phytophagous stink bugs are globally distributed and many harbor vertically inherited bacterial symbionts that are extracellular, yet little is known about how the symbiont's genomes have evolved under this transmission strategy. Genome reduction is common in insect intracellular symbionts but limited genome sampling of the extracellular symbionts of distantly related stink bugs has precluded inferring patterns of extracellular symbiont genome evolution. To address this knowledge gap, we completely sequenced the genomes of the uncultivable bacterial symbionts of four neotropical stink bugs of the Edessa genus. Phylogenetic and comparative analyses indicated that the symbionts form a clade within the Pantoea genus and their genomes are highly reduced (∼0.8 Mb). Furthermore, genome synteny analysis and a jackknife approach for phylogenetic reconstruction, which corrected for long branch attraction artifacts, indicated that the Edessa symbionts were the result of a single symbiotic event that was distinct from the symbiosis event giving rise to Candidatus "Pantoea carbekii," the extracellular symbiont of the invasive pentatomid stink bug, Halyomorpha halys. Metabolic functions inferred from the Edessa symbiont genomes suggests a shift in genomic composition characteristic of its lifestyle in that they retained many host-supportive functions while undergoing dramatic gene loss and establishing a stable relationship with their host insects. Given the undersampled nature of extracellular insect symbionts, this study is the first comparative analysis of these symbiont genomes from four distinct Edessa stink bug species. Finally, we propose the candidate name "Candidatus Pantoea edessiphila" for the species of these symbionts with strain designations according to their host species.

Compositional differences among female-associated and embryo-associated microbiota of the viviparous Pacific Beetle cockroach, Diploptera punctata.

All cockroach species, except one, harbor the endosymbiont Blattabacterium, transmitted from females to embryos. Adult cockroaches acquire non-Blattabacterium bacteria as part of their gut microbiota over time, but our knowledge of the possible transmission of these non-Blattabacterium bacteria from females to embryos is rudimentary. We characterized the gut microbiota of gravid viviparous Diploptera punctata females and the non-Blattabacterium microbiota of associated developing embryos, as well as the gut microbiota of non-gravid females, and the microbiota of orphan embryos (females not included), following high-throughput sequencing of the 16S rRNA gene to assess bacterial transference. We determined significant differences in community composition between gravid females and associated embryos and overall greater similarity in community composition among embryos than adult females. Results suggest various routes of transference of bacteria from females or the environment to embryos. The bacterial families Halomonadaceae and Shewanellaceae were more abundant in embryos than in gravid females. The functional relevance of these families remains to be elucidated, but provisioning of amino acids deficient in the brood sac secretion is a possibility. Overall, our results highlight the need for further studies investigating the uptake and selective screening of microbes by D. punctata embryos, as well as their functions.

Can (13)C stable isotope analysis uncover essential amino acid provisioning by termite-associated gut microbes?

Gut-associated microbes of insects are postulated to provide a variety of nutritional functions including provisioning essential amino acids (EAAs). Demonstrations of EAA provisioning in insect-gut microbial systems, nonetheless, are scant. In this study, we investigated whether the eastern subterranean termite Reticulitermes flavipes sourced EAAs from its gut-associated microbiota. δ (13)CEAA data from termite carcass, termite gut filtrate and dietary (wood) samples were determined following (13)C stable isotope analysis. Termite carcass samples (-27.0 ± 0.4‰, mean ± s.e.) were significantly different from termite gut filtrate samples (-27.53 ± 0.5‰), but not the wood diet (-26.0 ± 0.5‰) (F (2,64) = 6, P < 0.0052). δ (13)CEAA-offsets between termite samples and diet suggested possible non-dietary EAA input. Predictive modeling identified gut-associated bacteria and fungi, respectively as potential major and minor sources of EAAs in both termite carcass and gut filtrate samples, based on δ (13)CEAA data of four and three EAAs from representative bacteria, fungi and plant data. The wood diet, however, was classified as fungal rather than plant in origin by the model. This is attributed to fungal infestation of the wood diet in the termite colony. This lowers the confidence with which gut microbes (bacteria and fungi) can be attributed with being the source of EAA input to the termite host. Despite this limitation, this study provides tentative data in support of hypothesized EAA provisioning by gut microbes, and also a baseline/framework upon which further work can be carried out to definitively verify this function.

Habitat visualization and genomic analysis of "Candidatus Pantoea carbekii," the primary symbiont of the brown marmorated stink bug.

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.

The crypt-dwelling primary bacterial symbiont of the polyphagous pentatomid pest Halyomorpha halys (Hemiptera: Pentatomidae).

A recent invader of North America, the brown marmorated stink bug (Halyomorpha halys Stål) is a polyphagous pentatomid that harbors a gammaproteobacterial mutualist in the crypts of specialized midgut gastric caeca (region V4). Histological analyses revealed a single rod-shaped morphology abundant in distal V4 midgut caecal crypts. A strong fluorescence signal was detected when thin sections of these tissues were hybridized with a fluorescently-labeled, Enterobacteriaceae-specific oligonucleotide probe. A single operational taxonomic unit (OTU) assigned to the Pantoea genus represented >99% of 3,454 16S rDNA amplicons obtained from midgut V4 tissues and egg samples. Detection of H. halys primary symbiont in DNA extracted from eggs suggested vertical maternal inheritance as the mode of intergenerational transmission. Consistent detection of the bacterial symbiont in geographically distinct H. halys populations strongly supports an intimate association between these two organisms. An inferred phylogeny of gammaproteobacterial symbionts of pentatomids placed the Pantoea-assigned OTU from H. halys within a clade distinct from primary bacterial symbionts of related stink bugs, Nezara viridula (L.) and Eurydema rugosa Motschulsky. Given these data, Candidatus "Pantoea carbekii" is proposed as the name of the primary bacterial symbiont of H. halys.

Host-specific assemblages typify gut microbial communities of related insect species.

Mutualisms between microbes and insects are ubiquitous and facilitate exploitation of various trophic niches by host insects. Dictyopterans (mantids, cockroaches and termites) exhibit trophisms that range from omnivory to strict wood-feeding and maintain beneficial symbioses with the obligate endosymbiont, Blattabacterium, and/or diverse gut microbiomes that include cellulolytic and diazotrophic microbes. While Blattabacterium in omnivorous Periplaneta is fully capable of provisioning essential amino acids, in wood-feeding dictyopterans it has lost many genes for their biosynthesis (Mastotermes and Cryptocercus) or is completely absent (Heterotermes). The conspicuous functional degradation and absence of Blattabacterium in most strict wood-feeding dictyopteran insects suggest that alternative means of acquiring nutrients limited in their diet are being employed. A 16S rRNA gene amplicon resequencing approach was used to deeply sample the composition and diversity of gut communities in related dictyopteran insects to explore the possibility of shifts in symbiont allegiances during termite and cockroach evolution. The gut microbiome of Periplaneta, which has a fully functional Blattabacterium, exhibited the greatest within-sample operational taxonomic unit (OTU) diversity and abundance variability than those of Mastotermes and Cryptocercus, whose Blattabacterium have shrunken genomes and reduced nutrient provisioning capabilities. Heterotermes lacks Blattabacterium and a single OTU that was 95% identical to a Bacteroidia-assigned diazotrophic endosymbiont of an anaerobic cellulolytic protist termite gut inhabitant samples consistently dominates its gut microbiome. Many host-specific OTUs were identified in all host genera, some of which had not been previously detected, indicating that deep sampling by pyrotag sequencing has revealed new taxa that remain to be functionally characterized. Further analysis is required to uncover how consistently detected taxa in the cockroach and termite gut microbiomes, as well as the total community, contribute to host diet choice and impact the fate of Blattabacterium in dictyopterans.

Obligate insect endosymbionts exhibit increased ortholog length variation and loss of large accessory proteins concurrent with genome shrinkage.

Extreme genome reduction has been observed in obligate intracellular insect mutualists and is an assumed consequence of fixed, long-term host isolation. Rapid accumulation of mutations and pseudogenization of genes no longer vital for an intracellular lifestyle, followed by deletion of many genes, are factors that lead to genome reduction. Size reductions in individual genes due to small-scale deletions have also been implicated in contributing to overall genome shrinkage. Conserved protein functional domains are expected to exhibit low tolerance for mutations and therefore remain relatively unchanged throughout protein length reduction while nondomain regions, presumably under less selective pressures, would shorten. This hypothesis was tested using orthologous protein sets from the Flavobacteriaceae (phylum: Bacteroidetes) and Enterobacteriaceae (subphylum: Gammaproteobacteria) families, each of which includes some of the smallest known genomes. Upon examination of protein, functional domain, and nondomain region lengths, we found that proteins were not uniformly shrinking with genome reduction, but instead increased length variability and variability was observed in both the functional domain and nondomain regions. Additionally, as complete gene loss also contributes to overall genome shrinkage, we found that the largest proteins in the proteomes of nonhost-restricted bacteroidetial and gammaproteobacterial species often were inferred to be involved in secondary metabolic processes, extracellular sensing, or of unknown function. These proteins were absent in the proteomes of obligate insect endosymbionts. Therefore, loss of genes encoding large proteins not required for host-restricted lifestyles in obligate endosymbiont proteomes likely contributes to extreme genome reduction to a greater degree than gene shrinkage.

The nutrient supplying capabilities of Uzinura, an endosymbiont of armoured scale insects.

An emerging common physiological feature of plant sap-feeding insects is the presence of bacterial endosymbionts capable of providing essential nutrients to their host. These microbial partners are inviable outside of specialized host tissues, and therefore a cultivation-independent approach, namely high-throughput next-generation genome sequencing, can be used to characterize their gene content and metabolic potential. To this end, we sequenced the first complete genome of the obligate endosymbiont, Candidatus 'Uzinura diaspidicola', of armoured scale insects. At 263 431 bp, Uzinura has an extremely reduced genome that is composed largely of genes encoding enzymes involved in translation and amino acid biosynthesis. The tiny size of the Uzinura genome parallels that observed in some other insect endosymbionts. Despite this extreme genome reduction, the absence of a known obligate partner bacterial symbiont suggests that Uzinura alone can supply sufficient nutrients to its host.

Genome sequence of Blattabacterium sp. strain BGIGA, endosymbiont of the Blaberus giganteus cockroach.

Cockroaches harbor the obligate flavobacterial endosymbiont Blattabacterium sp., which resides within the host's bacteriocytes and can recycle ammonia and urea nitrogenous wastes into amino acids for the host. We report the complete genome sequence of the Blattabacterium sp. associated with the giant roach Blaberus giganteus.

Independent studies using deep sequencing resolve the same set of core bacterial species dominating gut communities of honey bees.

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.

Distinctive gut microbiota of honey bees assessed using deep sampling from individual worker bees.

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.

Genome shrinkage and loss of nutrient-providing potential in the obligate symbiont of the primitive termite Mastotermes darwiniensis.

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 ∼8% 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.