Mechanical and Enzymatic Digestion of Cellulose by Animals

A project undertaken at the School of Life and Environmental Sciences, Deakin University, and supervised by Stuart Linton

Background, Research questions and Methods
Plant material consumed by herbivorous animals contains large quantities of plant fibre. Plant fibre is composed of molecules called cellulose and hemicellulose. Both of these consist of sugar molecules such as glucose joined together by specialised bonds called beta-glycosidic bonds to form large chains. Cellulose molecules are also aligned to form crystalline fibres. Although they are difficult to digest, both cellulose and hemicellulose represent a substantial energy source.

Cellulose and hemicellulose digestion requires two steps. The first step involves mechanical digestion whereby the cellulose fibres are broken up mechanically. Animals possess structures such as cutting mouthparts called mandibles and gastric mills within their stomachs to do this. How this process aids in cellulose digestion is not understood. It is hypothesised that it reduces the size of the cellulose fibres, solubilises individual cellulose molecules and provides more sites for the second digestive step, enzymatic attack. The primary reason why cellulose and hemicellulose are difficult to digest is that they require specialised enzymes (biological catalysts which are able to perform chemical reactions) called cellulases and hemicellulase to break the beta-glycosidic bonds and split the molecules up into their component sugars. Three cellulase enzymes, cellobiohydrolase, endo-beta-1,4-glucanase and beta-1,4-glucanase are thought to be required to completely reduce cellulose to glucose. Recently it was discovered that animals such as crustaceans, insects and molluscs are able to endogenously produce at least one of the cellulases enzymes endo-beta-1,4-glucanase (Wantabe and Tokuda 2001; Davison and Blaxter 2005; Linton et al 2006). This is important since it represents a change in dogma. Previously it was thought that animals lacked this ability and that they utilised micro-organisms within the gut to produce the cellulases. The other cellulases beta-glucosidase and cellobiohydrolase and the hemicellulases laminarinase, lichenase and xylanase remain to be purified and characterised from animals. Also it is not understood if or how these enzymes work together with mechanical digestion in animals to achieve efficient total hydrolysis of cellulose and hemicellulose.

To examine these questions this project will utilise the Christmas Island Red crab, Gecarcoidea natalis and the Christmas Island Blue Crab, Discoplax hirtipes as animal models. Both species are herbivorous and consume mainly leaf litter. Also, they are known to digest cellulose and hemicellulose and to possess cellulase and hemicellulase enzymes within their digestive juice (Greenaway and Linton 1995; Greenaway and Raghaven 1998; Linton and Greenaway 2004; Linton et al 2006).

The first part of this project involves examining the anatomy and function of the gastric mill in these species. The gastric mill is a structure within the stomach which is utilised to grind up food material. It is hypothesised that it will possess tooth like structures which are specifically adapted to grind hard fibrous leaf material. Its structure will be examined using light and electron microscopy. The function of the gastric mill will be investigated by measuring the size of particles produced by the action of the gastric mill on cellulose particles fed to the animal in an artificial diet.

Hemicellulase and cellulase enzymes will be purified from the crabs to determine exactly which enzymes are produced by them. These enzymes will then be characterised by determining their size and their ability to attack cellulose and various hemicelluloses. An artificial system of purified enzymes which will reduce cellulose and hemicellulose to their component sugars will be created to explain how these enzymes are able to digest these molecules within the animal. Also how the cellulase and hemicellulase enzymes work together with mechanical digestion to achieve total cellulose hydrolysis will be investigated by the measuring the release of glucose by the artificial enzyme system from cellulose particles whose size is similar to that produced by the action of the gastric mill.

Anticipated outcomes

This project will provide fundamental biological knowledge which will allow a better understanding of how animals are able to digest cellulose and hemicellulose. It will also allow a greater understanding of how herbivorous crabs such as G. natalis and D. hirtipes are able to efficiently digest leaf litter.

References

Davison, A. and Blaxter, M. (2005). Ancient origin of glycosyl hydrolase family 9 cellulase genes. Molecular Biology and Evolution 22, 1273-1284.

Greenaway, P. and Linton, S. M. (1995). Dietary assimilation and food retention time in the herbivorous terrestrial crab Gecarcoidea natalis. Physiological Zoology 68, 1006-1028.

Greenaway, P. and Raghaven, S. (1998). Digestive strategies in two species of leaf-eating land crabs (Brachyura: Gecarcinidae) in a rain forest. Physiological Zoology 71, 36-44.

Linton, S. M. and Greenaway, P. (2004). Presence and properties of cellulase and hemicellulase enzymes of the gecarcinid land crabs Gecarcoidea natalis and Discoplax hirtipes. Journal of Experimental Biology 207, 4095-4104.

Linton, S. M., Greenaway, P. and Towle, D. W. (2006). Endogenous production of endo--1,4-glucanase by decapod crustaceans. Journal of Comparative Physiology B 176, 339-348.
Watanabe, H. and Tokuda, G. (2001). Animal Cellulases. Cellular and Molecular Life Sciences 58, 1167-1178.

 
Fig. 1. Christmas Island Red crab, Gecarcoidea natalis (Click on image to enlarge)

Fig. 2. Christmas Island Blue Crab, Discoplax hirtipes (Click on image to enlarge)