Thesis (Ph. D.)--Auburn University. / Abstract. Vita. Includes bibliographical references (p. 102-115).

Studying species at the molecular level can provide insights into how ecological and biological processes interrelate resulting in the diversity we see today. This information can be applied to conserve species at risk of extinction, or to better manage genetic diversity in species of economic importance. Species that inhabit freshwater riverine systems commonly exhibit population structures that are related to their relative dispersal capability, contemporary stream structure and/or historical stream structure. This thesis examined the populations genetic structure of wild and cultured stocks of the commercially farmed freshwater crayfish, C. quadricarinatus (von Martens), using genetic markers characterized by different modes of inheritance. C. quadricarinatus is distributed naturally in riverine systems in northern Australia, and southern Paupa New Guinea (PNG) and inhabits a variety of freshwater ecosystems ranging from ephemeral to permanent. Life history characteristics of C. quadricarinatus suggest a high level of genetic structuring among wild stocks might exist. However, seasonal flooding coupled with low topography across its distribution in northern Australia may promote sufficient gene flow among rivers to produce genetic homogeneity. Historical gene flow may also influence modern genetic structure as many distinct riverine catchments that C. quadricarinatus inhabits, were once connected at times of lower sea level. Insight into genetic relationships among C. quadricarinatus populations will allow for better management practices of wild populations in the future. The study investigated phylogenetic relationships among C. quadricarinatus representing 17 discrete natural drainages across the natural range in Australia and PNG, using 16s and COI gene sequences. Sequence analysis of both genes resolved two distinct genealogical lineages in Australia and three in PNG. The two divergent Australian lineages concur with original taxonomic descriptions of Reik (1969) based on external morphological differences. The three C. quadricarinatus populations sampled in PNG were all genetically distinct from each other, with one exhibiting a close association with an Australia lineage. The immense physical barriers (rugged mountain ranges) to gene flow in PNG will almost certainly have reduced dispersal capabilities for C. quadricarinatus. During times of lowered sea levels in the past, Australia and southern PNG were a single landmass with terrestrial and freshwater organisms theoretically able to disperse over associated land and via freshwater connections. The close genetic relationship between PNG and Australian C. quadricarinatus support a recent freshwater connection and hence gene flow between northern Australia and PNG C. quadricarinatus populations. Genetic differentiation among some C. quadricarinatus lineages exhibit as much genetic divergence at 16s RNA sequences as taxonomically recognised sub-species in the Cherax genus. Since C. quadricarinatus was originally described as different species based on external morphological differences (Reik, 1969), it is recommended that the taxonomy of C. quadricarinatus in Australia and PNG be re-evaluated. C. quadricarinatus specific microsatellite markers were developed for this study. Five variable loci were employed to investigate the extent of contemporary gene flow among fourteen C. quadricarinatus wild river populations in northern Australia. High FST and genetic distance estimates observed among pair wise comparisons of C. quadricarinatus populations are consistent with limited or no gene flow occurring among drainages. Speculation that C. quadricarinatus may disperse between adjacent or nearby drainages at times of flood, either across floodplains, or via flood plumes therefore seems highly unlikely among the populations examined in the current study. No significant correlation was observed between geographic distance and genetic distance among C. quadricarinatus populations here. C. quadricarinatus populations most closely resemble an island-like model, where gene flow is independent of geographic distance among populations and where genetic divergence occurs to a greater or lesser extent as a result of genetic drift within otherwise isolated populations. A significant number of C. quadricarinatus populations showed deviations from expected Hardy-Weinberg equilibrium (HWE). Samples sizes may not have been sufficiently large to reflect a true representation of genotypic proportions present in the sampled populations due to the highly variable nature of microsatellite loci. Deviations from HWE equilibrium, however, can also result from null alleles. Null allele estimates suggested a large proportion of null alleles were present in the C. quadricarinatus populations analysed. This may be a result of C. quadricarinatus populations confined to discrete drainages experiencing independent evolution, resulting in mutations in primer binding sites. The growing economic potential of C. quadricarinatus culture, both domestically and internationally, prompted expanding the current study to examine genetic diversity levels in commercial C. quadricarinatus stocks. The study employed five microsatellite markers to quantify genetic diversity in four Australian and three C. quadricarinatus culture stocks from overseas. Many C. quadricarinatus culture stocks also showed deviations from HWE expectations. This was not a surprising result given that the wild populations also deviated and domestication can also influence HWE. Relatively high levels of genetic diversity were observed. This probably results from intentional mixing of discrete river strains for production of the first commercial stock. Genetic differentiation estimates among culture stocks and assignment tests indicated that overseas culture stocks are most likely derived from the first commercial culture stock developed in Australia and then disseminated widely (the Hutchings stock). Robin Hutchings was a known supplier of live C. quadricarinatus to many international culture initiatives. Assignment of culture stocks back to their wild origins indicated that all C. quadricarinatus culture stocks sampled possess alleles that originate from the Flinders River (proportions ranged from 33-94%). Domestication of C. quadricarinatus to date has not resulted in significant reductions in levels of genetic diversity (heterozygosity or alleles richness) when compared to wild populations sampled in this study. Comparing culture stocks to wild populations to gauge their 'genetic health' may not be a suitable scale for evaluating genetic diversity in culture stocks. Wild populations are essentially evolving independently, are subjected to harsh seasonal environmental fluctuations resulting in periodic population crashes (genetic bottlenecks), with little or no recruitment from neighbouring drainages (gene flow). This study does however indicate that there is a large amount of genetic diversity distributed among wild populations that has yet to be exploited in culture. Genetic diversity in wild populations provides a resource for future stock improvement programs for C. quadricarinatus culture and thus requires careful conservation and appropriate management.

The issue of sustainability has greatest significance in the midst of unilateral bio-socioeconomic degradation resulting from intense and increasing societal pressures placed on the unified global ecology. In such an environment, sustainable development seeks to manage natural resources within a free market economy, aiming to meet the needs of today's population, and to protect and enhance current resource quality and abundance. In this light, techniques of integrated sustainable primary production and wastewater management are the subject matters of this applied research. There are many researchable issues which could be addressed within the subject matter. The first focus in the research scope was driven by the most severe sustainability issue facing Central Queensland (Australia) in 2000: the depletion and degradation of freshwater supplies. Central Queensland (CQ) is an arid sub-tropical region that has suffered from a marked reduction in rainfall and increase in temperature over the last 100 years, {Miles, 2004 #172}, and by the year 2000, conditions had been exacerbated by eight years of severe drought and warmer than average temperatures and resulted in widespread animal and crop failures due to freshwater shortages. Such a problem required a multi-faceted ecological, social, and economic approach. Hence, research centred on investigating the science of integrating regional water-related industries and agribusiness, and biodiverse ecosystems to achieve water and wastewater reuse applications, and associated eco-socioeconomic benefits. Specifically, this research investigates the integration of (a) electrical power station wastewater (b) barramundi culture, (c) red claw culture, (d) constructed wetlands (for water quality management and habitat creation), and (e) hydroponic flower culture. This research produced outcomes of integrated water and wastewater reuse and recycling, marketable agriproducts production (fish, crayfish, and flowers), water and wastewater reuse and conservation, wetland primary production, carbon dioxide sequestration, aquatic pollution control, and biodiversity creation and support. Successful design and management, experimental trialing and evaluation of system components and subjects, and the development of a knowledge base including static and dynamic system models, represent advances in respective research areas, and underpin the emerging discipline of integrated systems approaches to eco-socioeconomic development. Additionally, several gaps in the current body of knowledge regarding integrated systems were filled, and interactive management tools were developed. Apart from this study, the integration of technologies (as described above) has not, to this author's knowledge, been accomplished.

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.