Characterisation of the enzyme transport path between shipworms and their bacterial symbionts

Background Shipworms are marine xylophagus bivalve molluscs, which can live on a diet solely of wood due to their ability to produce plant cell wall-degrading enzymes. Bacterial carbohydrate-active enzymes (CAZymes), synthesised by endosymbionts living in specialised shipworm cells called bacteriocytes and located in the animal’s gills, play an important role in wood digestion in shipworms. However, the main site of lignocellulose digestion within these wood-boring molluscs, which contains both endogenous lignocellulolytic enzymes and prokaryotic enzymes, is the caecum, and the mechanism by which bacterial enzymes reach the distant caecum lumen has remained so far mysterious. Here, we provide a characterisation of the path through which bacterial CAZymes produced in the gills of the shipworm Lyrodus pedicellatus reach the distant caecum to contribute to the digestion of wood. Results Through a combination of transcriptomics, proteomics, X-ray microtomography, electron microscopy studies and in vitro biochemical characterisation, we show that wood-digesting enzymes produced by symbiotic bacteria are localised not only in the gills, but also in the lumen of the food groove, a stream of mucus secreted by gill cells that carries food particles trapped by filter feeding to the mouth. Bacterial CAZymes are also present in the crystalline style and in the caecum of their shipworm host, suggesting a unique pathway by which enzymes involved in a symbiotic interaction are transported to their site of action. Finally, we characterise in vitro four new bacterial glycosyl hydrolases and a lytic polysaccharide monooxygenase identified in our transcriptomic and proteomic analyses as some of the major bacterial enzymes involved in this unusual biological system. Conclusion Based on our data, we propose that bacteria and their enzymes are transported from the gills along the food groove to the shipworm’s mouth and digestive tract, where they aid in wood digestion.

Nutritional resources of the yeast symbiont cultivated by the lizard beetle Doubledaya bucculenta in bamboos

Symbiotic fungi of wood-inhabiting insects are often considered to aid wood digestion of host insects when the associated fungi can assimilate wood-associated indigestible materials. In most cases, however, the components of wood that are utilized by fungal symbionts remain poorly understood. The lizard beetle Doubledaya bucculenta (Coleoptera, Erotylidae, Languriinae) farms the symbiotic yeast Wickerhamomyces anomalus inside the cavity of host bamboo internodes, which serves as food for larvae. To determine the carbon sources of the internodes serving as nutritional substrates for W. anomalus, we used ion exchange chromatography measurements to analyze free and structural sugar compositions in fresh pith (FP), yeast-cultured pith (YP), and larva-reared pith (LP) of internodes. Glucose and fructose were the major free sugars in FP and markedly decreased in YP and LP. For structural sugars, no sugar significantly decreased in YP or LP compared with FP. Carbon assimilation tests showed that W. anomalus assimilated glucose, mannose, fructose, and sucrose strongly, xylose and cellobiose moderately, and xylan weakly. Elemental analysis revealed that the compositions of carbon, hydrogen, and nitrogen were not significantly different among tissue types. These results suggest that W. anomalus does not consume bamboo-associated indigestible sugars but most free sugars, mainly glucose and fructose, in the pith. Our findings suggest that a symbiont’s abilities may not always benefit its host in nature.

A rock-boring and rock-ingesting freshwater bivalve (shipworm) from the Philippines

Shipworms are a group of wood-boring and wood-feeding bivalves of extraordinary economic, ecological and historical importance. Known in the literature since the fourth century BC, shipworms are both destructive pests and critical providers of ecosystem services. All previously described shipworms are obligate wood-borers, completing all or part of their life cycle in wood and most are thought to use wood as a primary source of nutrition. Here, we report and describe a new anatomically and morphologically divergent species of shipworm that bores in carbonate limestone rather than in woody substrates and lacks adaptations associated with wood-boring and wood digestion. The species is highly unusual in that it bores by ingesting rock and is among the very few known freshwater rock-boring macrobioeroders. The calcareous burrow linings of this species resemble fossil borings normally associated with bivalve bioerosion of wood substrates (ichnospecies Teredolites longissimus ) in marginal and fully marine settings. The occurrence of this newly recognized shipworm in a lithic substrate has implications for teredinid phylogeny and evolution, and interpreting palaeoenvironmental conditions based on fossil bioerosion features.

The gill-associated symbiont microbiome is a main source of woody-plant polysaccharide hydrolase genes and secondary metabolite gene clusters inNeoteredo reynei, a unique shipworm from south Atlantic mangroves

Teredinidae is a family of highly adapted wood-feeding and wood-boring bivalves, commonly known as shipworms, whose evolution is linked to the acquisition of cellulolytic gammaproteobacterial symbionts harbored in bacteriocytes within the gills. In the present work we applied metagenomics to characterize microbiomes of the gills and digestive tract ofNeoteredo reynei, a mangrove-adapted shipworm species found over a large range of the Brazilian coast. Comparative metagenomics grouped the symbiotic gammaproteobacterial community of gills of differentN. reyneispecimens, indicating closely related bacterial types are shared, while intestine and digestive glands presented related, and more diverse microbiomes that did not overlap with gills. Annotation of assembled metagenomic contigs revealed that the symbiotic community ofN. reyneigills was a hotspot of woody-polysaccharides degrading hydrolase genes, and Biosynthetic Gene Clusters (BGCs), while in contrast, the digestive tract microbiomes seems to play little role in wood digestion and secondary metabolites biosynthesis. Metagenome binning recovered the nearly complete genome sequences of two symbioticTeredinibacterstrains from the gills, a representative ofTeredinibacter turnerae“clade I” strain, and a yet to be cultivatedTeredinibactersp. type. TheseTeredinibactergenomes, as well as unbinned gill-derived gammaproteobacteria contigs, code for novelty including an endo-β-1,4-xylanase/acetylxylan esterase multi-catalytic carbohydrate-active enzyme, and a trans-acyltransferase polyketide synthase (trans-AT PKS) gene cluster with the gene cassette for generating β-branching on complex polyketides. Multivariate analyzes have shown that the secondary metabolome encoded on the genomes ofTeredinibacterrepresentatives, including the genomes binned fromN. reyneigill’s metagenomes, stand out within the Cellvibrionaceae family by size, and enrichments for polyketide, nonribosomal peptide and hybrid BGCs. Results grouped here add to the growing characterization of shipworm symbiotic microbiomes and indicate that theN. reyneigill gammaproteobacterial community is a prolific source of biotechnologically relevant enzymes for wood-digestion and bioactive compounds production.

Discovery of chemoautotrophic symbiosis in the giant shipwormKuphus polythalamia(Bivalvia: Teredinidae) extends wooden-steps theory

The “wooden-steps” hypothesis [Distel DL, et al. (2000)Nature403:725–726] proposed that large chemosynthetic mussels found at deep-sea hydrothermal vents descend from much smaller species associated with sunken wood and other organic deposits, and that the endosymbionts of these progenitors made use of hydrogen sulfide from biogenic sources (e.g., decaying wood) rather than from vent fluids. Here, we show that wood has served not only as a stepping stone between habitats but also as a bridge between heterotrophic and chemoautotrophic symbiosis for the giant mud-boring bivalveKuphus polythalamia. This rare and enigmatic species, which achieves the greatest length of any extant bivalve, is the only described member of the wood-boring bivalve family Teredinidae (shipworms) that burrows in marine sediments rather than wood. We show thatK. polythalamiaharbors sulfur-oxidizing chemoautotrophic (thioautotrophic) bacteria instead of the cellulolytic symbionts that allow other shipworm species to consume wood as food. The characteristics of its symbionts, its phylogenetic position within Teredinidae, the reduction of its digestive system by comparison with other family members, and the loss of morphological features associated with wood digestion indicate thatK. polythalamiais a chemoautotrophic bivalve descended from wood-feeding (xylotrophic) ancestors. This is an example in which a chemoautotrophic endosymbiosis arose by displacement of an ancestral heterotrophic symbiosis and a report of pure culture of a thioautotrophic endosymbiont.

Influence of Input Variables over the Wood Digestion in a Sulfite Pulp Mill for Biorefinery Purposes

Eucalyptus globulus is the most profitable specie in Europe for pulping. However, in recent years some diseases and pests have caused its defoliation, affecting the available quantity of wood. In this sense, the mills are studying how to avoid the loss of pulp yield as well as the optimization of byproducts in order to take advantage of every component present in the wood using biorefinery processes. One of the possible options is to complete the consumption of E. globulus with other species such as Eucalyptus nitens.The objective of this paper is to study the behavior of both species related to the dissolving pulp manufacturing process as well as the characteristics of the resulting products. Parameters that can be very useful for the evaluation of the raw material, such as the growing data or density of both species, have been compared. Major attention has been focused on the composition of both species and how it affects the characteristics of the possible final products.E. nitens presents good results of forestry characteristics, being better than E. globulus in terms of growing and resistance to frosts. The cellulose content of both species is similar, however the digestion process in the case of E. nitens needs to be improved in order to optimize the involved energy and the obtained products.