Aquaculture represents a sustainable alternative to natural fisheries for provision of high quality, animal protein. Crustaceans make a significant contribution to global aquaculture production, of which decapods are the most economically important group. Among freshwater crayfish, the genus Cherax includes several species that have emerged as important culture species. A suite of favourable biological attributes, including fast growth and an omnivorous feeding habit, have contributed to establishment of successful culture of Cherax quadricarinatus (redclaw) in many countries. Aspects of redclaw production, however, remain relatively undeveloped, in particular feed formulation. To better understand the digestive processes and nutritional requirements of redclaw, this study examined the relationship between diet composition and digestive enzyme activity, growth performance and diet digestibility coefficients. The extent to which redclaw can efficiently utilise complex polysaccharides, such as cellulose, has been speculated on by authors who reported endogenous cellulase activity in this species. I evaluated the use of insoluble α-cellulose by redclaw, demonstrated that high dietary levels (30%) can significantly reduce the specific activity of selected digestive enzymes (amylase and cellulase), while also lowering apparent digestibility coefficients. Inclusion of α-cellulose above 12% also significantly reduced survival rate, specific growth rate and feeding efficiency in this organism which corresponds with low tolerance for insoluble fibre by other decapods. Even though redclaw possess endogenous cellulases, they appear to have only a limited capacity to utilise insoluble fibre in their diets. Further, I assessed the impact of different nutrient profiles on digestive enzyme activity, growth and tail muscle composition in redclaw. Purified diets containing varying levels of dietary protein significantly affected activity of digestive enzymes (protease, amylase and cellulase) and the composition of the tail muscle tissue. Redclaw have a relatively low protein requirement, which was reflected here, as little significant difference was observed in growth rates and the feed conversion ratio was only significantly affected by the lowest protein diet. Manipulation of the non-protein energy component in purified diets (protein to lipid ratio) had no effect on growth performance indices in redclaw. Digestive enzyme activity (protease) was however, strongly influenced by both the amount of protein and lipid in the diet and a significant correlation was observed between protease activity and growth performance indices. The findings here, provide preliminary data for consideration of digestive enzymes such as proteases as potential growth indicators for freshwater crayfish. These enzymes are already recognised as reliable biological indicators for comparison of digestive efficiency and potential growth rate in fish. The relationship between diet composition and digestive enzyme expression observed here, stress the need for further empirical evaluation of specific ingredients in artificial diets for redclaw. A range of single cell, plant and animal-based, agricultural products were assessed for their potential use in diets formulated for redclaw. Analysis of dietary supplements revealed that apparent digestibility of crude protein was generally higher for diets containing plant-based ingredients. A similar outcome was observed for digestibility coefficients of test ingredients. Ingredient type also had a significant effect on digestive enzyme activity. Importantly, a significant correlation was observed for enzyme activity and apparent digestibility coefficients. It appears that redclaw have the capacity to utilise nutrients from a broad range of dietary ingredients successfully including animal, single cell and in particular, plant matter in their diet. Taken together, the results presented here demonstrate that digestive enzyme activities in redclaw are significantly influenced by diet composition. I show clearly that the ability of redclaw to utilise various nutrients (measured as digestibility coefficients) is highly correlated with digestive enzyme activity. Finally, protease activity demonstrated a potential for use as an indicator of redclaw growth performance. The data presented here will contribute to development of better and cheaper feed formulations for use in redclaw aquaculture and have broader applications to freshwater crustacean culture. In particular, the potential for use of plant-based ingredients in aqua-feeds for redclaw will contribute to a more economically and environmentally sustainable redclaw culture.

The most abundant organic compound produced by plants is cellulose, however it has long been accepted that animals do not secrete the hydrolytic enzymes required for its degradation, but rely instead on cellulases produced by symbiotic microbes. The recent discovery of an endogenous cDNA transcript encoding a putative GHF9 endoglucanase in the parastacid crayfish Cherax quadricarinatus (Byrne et al., 1999) suggests that similar cellulase genes may have been inherited by a range of crustacean taxa. In this study, the evolutionary history of the C. quadricarinatus endoglucanase gene and the presence of additional GHF9 genes in other decapod species were investigated. The activity of endoglucanase and endoxylanase enzymes within several cultured decapod species were also compared. The evolutionary history of the C. quadricarinatus endoglucanase gene was assessed by comparing intron/exon structure with that of other invertebrate and plant GHF9 genes. The coding region of the gene was found to be interrupted by eleven introns ranging in size from 102-902 bp, the position of which was largely conserved in both termite and abalone GHF9 genes. These structural similarities suggest GHF9 genes in crustaceans and other invertebrate taxa share a common ancestry. In addition, two introns were observed to share similar positions in plant GHF9 genes, which indicates this enzyme class may have been present in ancient eukaryote organisms. The presence of GHF9 genes in C. quadricarinatus and various other decapod species was then explored via degenerate primer PCR. Two distinct GHF9 gene fragments were determined for C. quadricarinatus and several other Cherax and Euastacus parastacid freshwater crayfish species, and a single GHF9 gene fragment was also determined for the palaemonid freshwater prawn Macrobrachium lar. Phylogenetic analyses of these fragments confirmed the presence of two endoglucanase genes within the Parastacidae, termed EG-1 and EG-2. The duplication event that produced these two genes appears to have occurred prior to the evolution of freshwater crayfish. In addition, EG-2 genes appear to have duplicated more recently within the Cherax lineage. The presence of multiple GHF9 endoglucanase enzymes within the digestive tract of some decapod species may enable more efficient processing of cellulose substrates present in dietary plant material. Endoglucanase and endoxylanase enzyme activities were compared in several parastacid crayfish and penaeid prawn species using dye-linked substrates. Endoglucanase activity levels were higher in crayfish compared with prawn species, which corresponds with the known dietary preferences of these taxa. Endoglucanase temperature and pH profiles were found to be very similar for all species examined, with optimum activity occurring at 60°C and pH 5.0. These results suggest endoglucanase activity in penaeid prawns may also be derived from endogenous sources. Additional in vitro studies further demonstrated crayfish and prawn species liberate comparable amounts of glucose from carboxymethyl-cellulose, which indicates both taxa may utilise cellulose substrates as a source of energy. Endoxylanase temperature and pH profiles were also similar for all crayfish species examined, with optimal activity occurring at 50°C and pH 5.0. These results suggest xylanase activity in crayfish may originate from endogenous enzymes, although it is unclear whether this activity is derived from GHF9 enzymes or a different xylanase enzyme class. In contrast, no endoxylanase activity was detected in the three prawn species examined. Together, these findings suggest a wide range of decapod crustacean species may possess endogenous GHF9 endoglucanase genes and enzymes. Endoglucanases may be secreted by various decapod species in order to digest soluble or amorphous cellulose substrates present in consumed plant material. Further biochemical studies may confirm the presence and functional attributes of additional endoglucanase genes and enzymes in decapods, which may ultimately assist in the design of optimal plant based crustacean aquaculture feeds.