Gut anatomical properties and microbial functional assembly promote lignocellulose deconstruction and colony subsistence of a wood-feeding beetle.

Beneficial microbial associations enhance the fitness of most living organisms, and wood-feeding insects offer some of the most striking examples of this. Odontotaenius disjunctus is a wood-feeding beetle that possesses a digestive tract with four main compartments, each of which contains well-differentiated microbial populations, suggesting that anatomical properties and separation of these compartments may enhance energy extraction from woody biomass. Here, using integrated chemical analyses, we demonstrate that lignocellulose deconstruction and fermentation occur sequentially across compartments, and that selection for microbial groups and their metabolic pathways is facilitated by gut anatomical features. Metaproteogenomics showed that higher oxygen concentration in the midgut drives lignocellulose depolymerization, while a thicker gut wall in the anterior hindgut reduces oxygen diffusion and favours hydrogen accumulation, facilitating fermentation, homoacetogenesis and nitrogen fixation. We demonstrate that depolymerization continues in the posterior hindgut, and that the beetle excretes an energy- and nutrient-rich product on which its offspring subsist and develop. Our results show that the establishment of beneficial microbial partners within a host requires both the acquisition of the microorganisms and the formation of specific habitats within the host to promote key microbial metabolic functions. Together, gut anatomical properties and microbial functional assembly enable lignocellulose deconstruction and colony subsistence on an extremely nutrient-poor diet.

The status and characterization of Enteroramus dimorphus: a xylose-fermenting yeast attached to the gut of beetles.

Enteroramus dimorphus from the gut of the passalid beetle Odontotaenius disjunctus was described originally as a yeast-like fungus of unknown taxonomic affiliation. The fungus can be observed in situ, attached by a specialized cell to the beetle hindgut wall. In a recent study of yeast endosymbionts from a variety of beetles, we discovered that E. dimorphus is a member of the Pichia stipitis (Saccharomycetes) clade, known for xylose fermentation and assimilation. The closest relative of E. dimorphus is the PASS1 isolate, repeatedly acquired from passalid beetles in eastern North America from Pennsylvania to Louisiana. In addition to xylose fermentation and assimilation, these yeasts are characterized by the production of hat-shaped ascospores in culture, assimilation of a wide range of sugars, and synthesis of several vitamins. Enteroramus dimorphus, however, can be distinguished from close relatives by several physiological characteristics and rDNA sequences, which vary slightly from the more widespread PASS1 genotype. We present an amended description of E. dimorphus and discuss its symbiotic phase in association with O. disjunctus, including a holdfast that parallels those of unrelated symbiotic yeasts associated with nematodes.

Compartmentalized microbial composition, oxygen gradients and nitrogen fixation in the gut of Odontotaenius disjunctus.

Coarse woody debris is an important biomass pool in forest ecosystems that numerous groups of insects have evolved to take advantage of. These insects are ecologically important and represent useful natural analogs for biomass to biofuel conversion. Using a range of molecular approaches combined with microelectrode measurements of oxygen, we have characterized the gut microbiome and physiology of Odontotaenius disjunctus, a wood-feeding beetle native to the eastern United States. We hypothesized that morphological and physiological differences among gut regions would correspond to distinct microbial populations and activities. In fact, significantly different communities were found in the foregut (FG), midgut (MG)/posterior hindgut (PHG) and anterior hindgut (AHG), with Actinobacteria and Rhizobiales being more abundant toward the FG and PHG. Conversely, fermentative bacteria such as Bacteroidetes and Clostridia were more abundant in the AHG, and also the sole region where methanogenic Archaea were detected. Although each gut region possessed an anaerobic core, micron-scale profiling identified radial gradients in oxygen concentration in all regions. Nitrogen fixation was confirmed by (15)N2 incorporation, and nitrogenase gene (nifH) expression was greatest in the AHG. Phylogenetic analysis of nifH identified the most abundant transcript as related to Ni-Fe nitrogenase of a Bacteroidetes species, Paludibacter propionicigenes. Overall, we demonstrate not only a compartmentalized microbiome in this beetle digestive tract but also sharp oxygen gradients that may permit aerobic and anaerobic metabolism to occur within the same regions in close proximity. We provide evidence for the microbial fixation of N2 that is important for this beetle to subsist on woody biomass.

Morphological and ecological similarities: wood-boring beetles associated with novel xylose-fermenting yeasts, Spathaspora passalidarum gen. sp. nov. and Candida jeffriesii sp. nov.

Ascomycete yeasts that both ferment and assimilate xylose were reported previously as associates of insects living in woody substrates. Most notable have been reports of Pichia stipitis-like yeasts that are widely associated with the wood-boring beetle, Odontotaenius disjunctus (Coleoptera: Passalidae), in the eastern United States. Our continuing investigation of insect gut yeasts has lead to the discovery of two new xylose-fermenting yeasts that phylogenetic analysis places as sister taxa. The beetle hosts, O. disjunctus and Phrenapates bennetti (Coleoptera: Tenebrionidae), are similar in habitat and appearance, and the presence of similar gut yeasts is an additional common feature between them. Here we describe the new yeast genus Spathaspora, the type species S. passalidarum, and its sister taxon Candida jeffriesii and discuss their natural history, including a comparison with Pichia stipitis, another member of a guild of xylose-fermenting yeasts with similar metabolic traits. In addition a morphologically distinct yeast ascospore type is described for Spathaspora.

Communities of microbes that inhabit the changing hindgut landscape of a subsocial beetle.

Microbes that have adopted endosymbiotic life styles not only have evolved to live in specialized habitats within living organisms, but the living habitats also have evolved to accommodate them. The hindgut of the passalid beetle (Odontotaenius disjunctus) is lined with a cuticle that undergoes dramatic topographic changes during the life cycle of the beetle. This manuscript addresses the changes that have been observed in time and space for the cuticular landscape of the hindgut as well as for the microbial communities within the hindgut. Microbial identity is based on morphology, culture, and extrapolation from previously reported passalid gut inhabitants.

Wood ingestion by passalid beetles in the presence of xylose-fermenting gut yeasts.

During a survey of insect gut micro-organisms, we consistently isolated Pichia stipitis-like yeasts (Fungi: Ascomycota, Saccharomycetes) from the wood-ingesting beetles, Odontotaenius disjunctus and Verres sternbergianus (Coleoptera: Passalidae). The yeasts were isolated from passalid beetles over a wide area, including the eastern and midwestern USA and Panama. Phylogenetic analyses of the nuclear encoded small and large subunit rRNA gene (rDNA) sequences distinguished a well-supported clade consisting of the passalid yeasts and Pichia stipitis, P. segobiensis, Candida shehatae and C. ergatensis. Members of this clade have the ability to ferment and assimilate xylose or to hydrolyse xylan, major components of the polysaccharide, hemicellulose. Sexual reproduction was present in the passalid isolates but was rare among the gut yeasts of other beetles to which they were compared. Minor genetic and phenotypic variation among some of the passalid yeasts was detected using markers from the internal transcribed spacer region of the rDNA repeat unit, morphology, and in vitro metabolic tests. The consistent association of xylose-fermenting yeasts of almost identical genotypes with passalid beetles across a broad geographical distribution, suggests a significant symbiotic association.