New insights from NMR, FTIR, X-ray diffraction and physical chemistry into digestive processes in the wood-boring marine crustacean Limnoria quadripunctata.
Graham P. MalyonS. LaBarreNelly KervarecPeter D. CaryJ.E. McGeehanXiaoduo XieAndré KlüppelSimon M. Cragg
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Abstract:
This paper summarises preliminary findings of a multi-technique exploration of the degradation of lignocellulose in the marine isopod Limnoria quadripunctata. Scanning electron microscopy revealed connections between the digestive gland and the hindgut that would permit the exchange of fluids between the two organs while the food mass is retained in place within the hindgut. This enables enzymes to be delivered to the substrate and breakdown products to be absorbed. FTIR and X-ray diffraction were used to show changes in wood chemistry during digestion. Cellulose crystallinity appears reduced after digestion, but lignin structure appeared little changed. NMR spectroscopy of animals under various feeding regimes measured the generation of breakdown products and levels of metabolites. Glucose was detected as a direct result of the animals feeding on 13 C-labelled straw. This is the first direct evidence of total cellulose breakdown to the monomer. The importance of oxygen levels during digestion was shown by direct measurement with microelectrodes and indirectly by observing feeding on substrates impregnated with anti-oxidants. The hindgut lumen was found to be relatively anoxic. The antioxidant BHT significantly reduces feeding rates under laboratory conditions. These findings taken together give a picture of the effects of the activity of the recently described suite of digestive enzymes on their substrate.Keywords:
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Shipworms are marine bivalve molluscs, known for their wood boring abilities. They use modified shells to bore into and grind wood, which is then digested. The shipworm’s ability to feed on lignocellulose is dependent on the presence of endosymbiotic bacteria that live in the animal’s gills inside specialized eukaryotic cells called bacteriocytes. These bacteria provide the animal with hydrolytic enzymes for wood digestion, which are translocated from the gills to the caecum, the main site of wood digestion. Unlike other lignocellulose degrading organisms, which harbour symbiotic microbes in their digestive tract, the shipworm caecum hosts only few bacteria but contains a large amount of carbohydrate active enzymes (CAZymes) of both endogenous and bacterial origin.
This study investigates the anatomical, physiological and molecular basis of wood digestion in the shipworm Lyrodus pedicellatus. A combination of meta-transcriptomics, meta-proteomics and microscopic studies of the shipworm digestive organs were used, coupled with recombinant production and characterisation of some of the most expressed lignocellulolytic enzymes.
This multidisciplinary analysis revealed how two structures, the food groove (a mucus stream utilised by filter feeding molluscs to transport food particles from the gills to the digestive system) and the crystalline style (a rotating structure hosted in the stomach involved in extra-cellular digestion) have been co-opted in shipworms to translocate bacteria and their enzymes from the gills to the caecum, to facilitate wood digestion. The transcriptomic and proteomic results indicate that bacterial lignocellulolytic enzymes are expressed in the gills, while the endogenous enzymes are mainly produced by the digestive glands, with complementary CAZy classes being expressed by the bacteria and the shipworms, indicating a subdivision of roles. Five bacterial CAZymes were recombinantly expressed and characterised, showing activity on cellulose, galacto-glucomannan and xylan. This study provides new insights into the mechanisms of wood digestion in shipworms, which may have biotechnological relevance.
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