Insight into the digestive processes of the wood-boring marine crustacean Limnoria quadripunctata
Student thesis: Doctoral Thesis
A better understanding of wood degradation mechanisms used by Limnoria quadripunctata may lead to the development of more specific treatments to control biodeterioration that have fewer side effects than the traditional broad spectrum biocides. Thus, a clear understanding of digestion in wood boring animals is required. This knowledge of the digestive strategies used by L. quadripunctata could also be applied to the degradation of lignocellulose, and provide a novel source of enzymes which could be used to degrade this recalcitrant substrate. Cellulase activity of extracts of L. lignorum were demonstrated in the 1950s (Ray & Julian, 1952) and the possibility of a microbial contribution to this activity was later tested by electron microscope investigations of the digestive gland (hepatopancreas) and hindgut (Boyle & Mitchell 1978, Sleeter et al. 1978) which showed, unlike other isopods (Zimmer et al. 2002), no resident gut microbes. New sequencing data also shows no evidence of symbiont-derived enzyme sequences in messenger RNA harvested from the hepatopancreas of L. quadripunctata (King et al. 2010). Further evidence for an independent digestion mechanism in L. quadripunctata came from an endo-β-1,4glucanase that belongs to the GH7 family of cellulases, also found in termites (Watanabe and Tokuda 2010) being detected in cells of the hepatopancreas of L. quadripunctata by Dymond et al. (2003) using in situ hybridisation experiments. Subsequent detailed examination of the transcriptome of the hepatopancreas of L. quadripunctata revealed large numbers of RNA sequences encoding for a range of enzymes that have been implicated in cellulose degradation in other organisms. Particularly numerous were sequences for glycosyl hydrolase families GH5, 7 and 9 (King et al. 2010). This made L. quadripunctata the first animal reported to possess a GH7 in an animal genome. Most recently Western blots have been used in this study to identify GH5a and GH7a protein in the hepatopancreas and gut of L. quadripunctata as well as successful in situ localisation of GH5a and GH9a RNA in the hepatopancreas. This work confirms the hepatopancreas as the site of secretion of these enzymes and that they are translated into protein which is then found in the gut. Furthermore a carboxymethyl cellulose assay was used in this study and showed positive degradation by hepatopancreas and gut extract which confirms the presence of active amorphous cellulose degrading enzymes in these tissues. In parallel with the efforts to reveal the presence and activity of endogenous enzymes produced by L. quadripunctata, investigations into the chemistry of wood breakdown and utilization by the organism have been undertaken. X-ray diffraction is an effective tool for showing changes in cellulose crystallinity and its use has demonstrated increased crystallinity in wood after digestion, while FTIR has reinforced this observation by showing changes in the hemicelluloses which are the more amorphous components of wood. Furthermore NMR has been used to identify 13C labelled glucose originating from feeding L. quadripunctata with a 13C enriched wheat straw substrate. This glucose was identified in gut extract and labelled 13C trehalose from the hepatopancreas. This provides evidence of digestion products being assimilated into the metabolic pool which has been complemented by evidence for digestion of wood provided by measurements using HPLC to compare, quantify and identify the sugar components of wood lost during digestion. Together this work provides new insight into how wood is affected by digestion in L. quadripunctata and what effect the activity of enzymes involved in this processing is having on the wood.
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