Dissertations / Theses on the topic 'Cherax quadricarinatus'

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.

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.

Memoria para optar al Título Profesional de Médico Veterinario<br>Gran variedad de seres vivos desarrollan sistemas capaces de crear estructuras mineralizadas, que se usan como protección, sostenimiento, alimentación, reservas de minerales, etc. La formación y degradación de estructuras minerales resulta ser un punto crítico en la fisiología de animales invertebrados como los crustáceos y en particular en el modelo animal utilizado en esta memoria de título, la langosta de tenaza roja, Cherax quadricarinatus, especie cuyo desarrollo está en íntima relación con la formación de una reserva mineral llamada gastrolito, que se fabrica en la parte anterior del estómago cardiaco, en una zona especializada de la pared estomacal, el disco del gastrolito, cuyo epitelio presenta distinta estructura según el momento del ciclo de muda y representa un excelente modelo para el estudio de la biomineralización. La matriz extracelular del gastrolito está formada por moléculas cargadas negativamente, entre las que se destacan los proteoglicanos, que son depositados por el epitelio formador. Los proteoglicanos serían los responsables de entregar un orden definido al depósito de carbonato de calcio amorfo que compone al gastrolito. La etapa del ciclo de muda se determinó por observación del exoesqueleto de los individuos, cuando éste era blando se clasificó en la etapa de postmuda y si estaba rígido en cualquiera de las otras dos etapas: intermuda o premuda. Para diferenciarlas se radiografiaron y en intermuda no se observó la imagen compatible con gastrolito y por el contrario, si fue posible observarlo en premuda. Se extrajeron estómagos de las langostas clasificadas, se procesaron de manera rutinaria para realizar cortes histológicos, los que fueron teñidos con las técnicas de hematoxilina-eosina y azul de alciano a pH 2,5; para describir zonas de interés y reconocer la presencia de glicosaminoglicanos, respectivamente. Además se utilizaron reacciones inmunohistoquímicas con el propósito de identificar temporal, espacial y específicamente algunos proteoglicanos presentes en la matriz orgánica del gastrolito. Se logró establecer un orden espacial y temporal para proteoglicanos específicos: Dermatán sulfato y condroitín-4-sulfato están presentes en los tres estados del ciclo de muda, observándose una mayor intensidad de reacción en las etapas de intermuda y premuda principalmente, lo que sugiere, al igual que en otros estudios similares, que serían necesarios para mantener el orden en el tipo de acumulación de carbonato de calcio en la formación de esta biocerámica. Condroitín-6-sulfato se muestra con una reacción más intensa en intermuda y premuda, disminuyendo su intensidad en postmuda, lo que sugiere una participación más activa durante la formación, luego en postmuda está ausente tanto en el epitelio formador como en el gastrolito en degradación y sólo se observa en el tejido conectivo subyacente al lugar de formación del gastrolito. Queratán sulfato presenta una mayor intensidad de reacción en intermuda y premuda principalmente, sugiriendo un rol en el inicio de la calcificación. En el periodo de postmuda se encuentra en el gastrolito principalmente y en el epitelio formador con menor intensidad. Esta distribución temporal de los proteoglicanos sugiere la influencia en el desarrollo de los gastrolitos de la langosta de tenaza roja<br>Financiamiento: Proyecto Fondap 11980002 y Proyecto ECOS-CONICYT C07-B02

The structural polysaccharide-degrading enzymes, cellulase and xylanase, in the digestive system of the freshwater redclaw crayfish, Cherax quadricarinatus were investigated in an attempt to elucidate the potential capacity of crayfish to utilise the cell wall components of plant material in their diet. Cellulase, xylanase and lichenase were all found in the gastric juice, hindgut juice and hepatopancreas of the redclaw crayfish. The specific activities of cellulase and xylanase in the crayfish gastric juice were 2788 and 2060 units per ml, respectively. In contrast, cellulase and xylanase activities were only just detectable in the intestinal juice of the omnivorous Sea Garfish examined as a reference. Analysis of the relative activity of cellulase and xylanase in the different parts of the crayfish digestive tract showed that the foregut possessed total cellulase and xylanase activities 91-fold and 83-fold higher than the hindgut, respectively. This indicates that the foregut is the major organ involved in the digestion of dietary cellulose and xylan in the redclaw crayfish. Treatment with antibiotics depressed bacterial populations in the gastric juice to very low levels, but only slightly reduced cellulase and xylanase activities. This result indicates that gastric hydrolases present in the redclaw crayfish are produced from the hepatopancreas. Cellulase from crayfish gastric juice was active across a pH range of 4-9 with a pH optimum of 5. 5. The hepa topancreas cellulase had a similar pH profile to that found in gastric juice. The pH profile of crayfish xylanase was essentially the same as that of the cellulase. The optimum pH values for cellulase and xylanase activities in the redclaw fit well within the observed range of gastric juice pH values in this species. Crayfish cellulase was active across a temperature range of 10°C to 70°C with an optimum between 55°C and 60°C. Similarly, crayfish xylanase was active in a temperature range of 10°C to 65°C with an optimum temperature of 50°C. These results demonstrate the relatively broad stability of these enzymes across a wide range of environmental parameters. Cellulase and xylanase activities in crayfish grown in an earthen pond were significantly lower than that of individuals raised in the laboratory and fed on a commercial pellet diet. Lower levels of cellulase and xylanase activities in the gastric juice of earthen-pond-reared crayfish were accompanied by lower levels of these enzymes in the hepatopancreas. Starvation resulted in a 2- to 3-fold reduction in gastric cellulase and xylanase activities, compared with crayfish fed on a commercial pellet diet. The significant reduction in hepatopancreatic levels of cellulase and xylanase activities was accompanied by a parallel decline in general protein content in starved crayfish, indicating a reduced level of general protein synthesis in the hepatopancreas associated with starvation. Crayfish fed on a diet containing starch and milk (SM) had only slightly lower levels of cellulase and xylanase activities in the gastric juice, compared with a group fed on a commercial diet which contained 7% crude fibre. Cellulase levels in the hepatopancreas were also not significantly different between the two groups, although there was a significant decrease in the hepatopancreatic xylanase activity in the SM group. Cellulase and xylanase activities were significantly lower in the group fed on a starch and milk diet plus 25% fibre (cellulose and xylan) than in the commercial diet-fed crayfish. A marked decrease in the protein content of the hepatopancreas was also observed in this group. The activities of both cellulase and xylanase in juvenile crayfish (body wt. of 20-50 grams) were significantly higher (1.5-3-fold) than those of mature crayfish (body wt of 150-280 grams), indicating that the levels of these digestive enzymes may be positively related to growth rate. A positive relationship between growth rate and enzyme activity was also observed in a study using selected lines with fast growth compared with control lines. In a Gilbert River line selected for fast growth rate both the juvenile and adult crayfish possessed significantly higher cellulase and xylanase activities in gastric juice (1. 4-fold to 1. 9-fold higher) than control individuals. Similarly, higher activities (1.2-fold to 1. 7-fold higher) of both enzymes were seen in the hepatopancreas of the selected line. A parallel result (1. 3-fold to 4. 6-fold higher) was observed in selected vs control lines for Flinders River crayfish stocks. These results suggest that redclaw crayfish produce cellulase and xylanase endogenously in the hepatopancreas, and these enzymes are secreted into the foregut of the animal. Both enzymes were present at very high levels in the gastric juice. Enzyme levels were inflated in lines selected for fast growth rate, but did not increase with increased fibre intake in the diet. The presence of high activities of these fibre-degrading enzymes in the digestive system indicates that the redclaw crayfish has a high capacity to utilise plant cell wall components. As a consequence it is suggested that addition of microbial fibre­degrading enzymes to diets may not be necessary to promote crayfish growth.

Memoria para optar al Título Profesional de Médico Veterinario<br>La biomimética o mimetismo de lo biológico es una disciplina muy antigua que trata de imitar algo preexistente en la naturaleza, por lo tanto tiene como fuente de inspiración alguna característica o habilidad biológica de los organismos para generar materiales útiles para el ser humano. Debido a la necesidad del ser humano por alcanzar nuevos logros y seguir superando sus limitaciones son que la biomimética ha alcanzado la importancia tecnológica que tiene actualmente. Por tal razón numerosas investigaciones se han realizado con el objeto de dilucidar los diferentes mecanismos moleculares que permitan conocer como se forman estos biomateriales de origen biológico y obtener materiales más complejos con características superiores a las ya existentes. Dado al menor conocimiento que se tiene sobre los materiales amorfos, en la presente memoria de título se estudió un tipo de reserva inorgánico de tipo amorfo del carbonato de calcio (CaCO3) del crustáceo Cherax quadricarinatus llamado gastrolito. Cherax quadricarinatus es un crustáceo que vive en zonas de agua dulce y que presenta un exoesqueleto que muda constantemente. Los gastrolitos son concreciones altamente calcificadas, encontradas en la zona cardiaca del estómago de algunos Decápodos, que actúan como reservorios temporales formados por CaCO3 de tipo amorfo (CCA), material inorgánico crucial para el proceso de muda de su exoesqueleto y un excelente modelo de mineralización de CCA. En estudios de biomineralización se han usado variados tipos de aditivos para evaluar la participación de éstos en ensayos de cristalización in vitro de CaCO3. En esta memoria de título se utilizaron diferentes polímeros acídicos comerciales tales como: ácido fítico (AF), ácido poliaspártico (APS) y ácido poliacrílico (APA) en conjunto con sustratos (esponja y gastrogel) y proteoglicanos (PGS) de fracciones solubles (PG-FS) e insoluble (PG-FI) obtenidos a partir del gastrolitos como aditivos en la cristalización del CaCO3. Los ácidos AF, APS y APA son polímeros comerciales usados por el ser humano en diferentes procesos industriales y que han mostrado tener efectos modificadores sobre la cristalización del CaCO3. 7 El proceso in vitro de cristalización del CaCO3 fue el método de difusión de gases y las técnicas de caracterización de los cristales obtenidos fueron la microscopía electrónica de barrido (SEM) y la difracción de rayos X (XRD), con los cuales se pudo observar la morfología y determinar la estructura cristalina del CaCO3 obtenidos en las diferentes combinaciones ensayadas de polímeros, sustratos (esponja y gastrogel) y proteoglicanos de ambas fracciones (PG-FS; PG-FI) durante la cristalización de CaCO3. Encontramos que los sustratos esponja y gastrogel al ser usados como aditivos únicos en los ensayos de mineralización de CaCO3 mostraron una clara tendencia a la formación de estructuras amorfas y un efecto modulador mecánico del depósito cristalino. Con PG-FS se obtuvieron cristales de CaCO3 con morfología semejantes a los polimorfos de aragonita y calcita y estructuras cristalinas del tipo aragonita, calcita y vaterita, los cuales se determinaron mediante XRD. Sin embargo, con PG-FI se obtuvieron cristales con morfología similar a los polimorfos de aragonita y vaterita y se determinaron estructuras cristalinas del tipo aragonita, calcita y vaterita mediante XRD. Por otro lado, el uso de APA como único aditivo indujo la formación de estructuras cristalinas con morfologías esféricas observadas con SEM y estructuras cristalinas del tipo aragonita y calcita determinadas por XRD. Cuando el APS fue usado se observaron cristales con morfologías espiculadas y estructuras cristalinas del tipo aragonita, calcita y vaterita mediante XRD. Sin embargo, con AF como aditivo se observaron cristales con morfologías irregulares y se determinaron los polimorfos aragonita, calcita y vaterita mediante XRD. Por último, cuando AF se utilizó en presencia de otros aditivos o sustratos se obtuvieron cristales con morfología espiculadas, esféricas, romboédricas, etc. La determinación morfológica de los cristales de CaCO3 in vitro obtenidos mediante el proceso de difusión de gases fueron observados claramente mediante SEM y la determinación cristalina de sus polimorfos mediante XRD confirmando la importancia del efecto nucleador de los diferentes polímeros en combinación con sustratos de origen biológico, creemos, sin embargo, que el uso de nuevas técnicas de caracterización como AFM, XPS y microscopía electrónica de transmisión (TEM) potenciarían enormemente la presente memoria de título contribuyendo a un mejor entendimiento de los procesos moleculares involucrados en los ensayos de cristalización biológica de minerales inorgánicos como el CaCO3. Finalmente concluimos que es importante seguir ahondando en el conocimiento de la formación cristalina de materiales inorgánicos con el objeto de desarrollar nuevos materiales útiles para el ser humano y la creación de nuevas tecnologías de materiales, los cuales podrían llevar al hombre a nuevas fronteras aún no conocidas.

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.

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.

碩士<br>國立中興大學<br>獸醫學系暨研究所<br>105<br>Cherax quadricarinatus is a common foreign species in Taiwan. It carries with parasite. What the species it is? I use optical microscopy, dissecting microscopy, scanning electron microscopy, and differential interference contrast microscopy to assist the identification of the parasite. In addition, use histopathology to find out whether the parasite causes red claw crayfish lesions? The result shows the species is Craspedella pedum which doesn’t cause lesions in Cherax quadricarinatus. And whether it infects Taiwan&apos;&apos;s common crustaceans: Macrobrachium rosenbergii and Procambarus clarkia? The result shows it doesn’t infect mature Macrobrachium rosenbergii and Procambarus clarkia, but the influence on the young is still unknown. Is it possible to observe the hatching process of the worm eggs inside the carapace of the red claw crayfish to have a more complete understanding of the parasite life history? Due to rapidly deteriorated water quality, I didn’t get more information.

In 2014, a new syndrome was recognized in redclaw crayfish (Cherax quadricarinatus) from farms in northern Queensland. Crayfish started to die with mortality reaching 40% in approximately three weeks after transportation and translocation stress. In trying to identify the cause of the disease, sequence dependent and independent PCRs failed to identify any aetiology. However, using next gen sequencing of RNA, two novel RNA viruses, namely chequa iflavirus and bunya‐like virus were found in the stressed, dying crayfish. Chequa iflavirus is a positive sense, single stranded RNA virus in the order Picornavirales, marginally in the genus Iflavirus. This is the first iflavirus identified from crustracea. The bunya‐like virus found is related to Whenzhou Shrimp Virus 2, a bi‐segmented, unaligned, negative sense, single strand RNA virus. Efficient and sensitive detection methods for these viruses based on RNA dependent RNA polymerase (RdRp) was developed for large scale screening of crayfish on farm. Due to a lack of virus‐free crayfish, house cricket, Acheta domesticus was trialled as a bioassay animal to prove River’s postulates for chequa iflavirus and bunya‐like virus. However, it was unsuccessful as the immune system of cricket might able to destroy and leave no viable virus or enough traces of RNA to be detected by reverse transcription polymerase chain reaction (RT‐PCR). To solve the viral infection problem with chequa iflavirus infection on farm, some possible viral reduction strategies for farmers were trialled. The polyphenol flavonoid quercetin, inhabiting water temperature at 32°C all had statistically significant effects reducing viral copy numbers (P<0.018; P<0.05 respectively). While RNA interference based on RdRp sequence appeared to drop viral copy number 80% at four weeks, it was not statistical significant (P˃0.05). For the crayfish farmers, quercetin is the most favoured treatment as it is abundant, cheap and readily incorporated into the diet and should ameliorate the original, transport‐stress mortality which started this study. Broodstock could be warmed to 32°C in the hatchery to reduce viral dose. Although RNAi treatment was most expensive to implement, it could be delivered with limited amounts of RNAi at the hatchery.

Early disease investigations in Cherax spp. were observational in nature. Investigators found a variety of pathogens including fungi, ciliates, platyhelminthes, nematodes and viruses. To date, the viral flora identified have been studied using histopathology and transmission electron microscopy. There have been two transmission trials carried out using Cherax spp. infecting viruses, a Reo-like virus and a parvovirus-like virus. Despite multiple detections of viruses in Cherax spp., there has been no molecular characterisation of any of these pathogens. Of the various viral flora identified in Cherax spp., four were classified as parvovirus-like. These include Cherax destructor systemic parvo-like virus, Cherax quadricarinatus gill parvo-like virus, spawner-isolated mortality like virus and Cherax quadricarinatus parvo-like virus (now Cherax quadricarinatus densovirus, CqDV). The focus of this project was to characterise, for the first time, a Cherax spp. infecting virus, CqDV. The isolate was obtained from the staff at the Queensland Government's former Tropical and Aquatic Animal Health Laboratory. Infected tissue was used to recreate the disease at James Cook University. As well as confirming the results of Bowater et al. (2002), we extended these results, characterising the tissue tropism of CqDV using quantitative real-time PCR (qPCR). We revealed that CqDV preferentially targets ectodermal tissues, consistent with histopathology observations, and that infection is systemic. CqDV was detected in the heart and muscle, most likely from infected haemocytes in the haemocoel. The genome of CqDV was sequenced using primer walking. The CqDV genome is 6,334 nucleotides in length (GenBank: KP410261) and has four open reading frames (ORFs), three (non structural proteins, NS3, NS1 and NS2) on the sense strand and one (viral protein, VP) on the anti-sense strand, indicating an ambisense organisation. Bioinformatics analysis of the ORFs identified highly conserved motifs characteristic of the family Parvoviridae, including endonuclease and helicase motifs in the NS1 protein and a phospholipase A2 motif in the VP. Phylogenetic analysis firmly placed CqDV in the subfamily Densovirinae, genus Ambidensovirus, species Decapod ambidensovirus, virus variant Cherax quadricarinatus densovirus. The CqDV isolate grouped with the blattodean-infecting densoviruses but its genome was architecturally similar to the Lipidopteran ambidensovirus 1 group. The CqDV genome shared 75 % amino acid homology across the entire genome with sea star associated densovirus (SSaDV). Although CqDV and SSaDV are phylogenetically similar, they are geographically and environmentally distinct from each other, infecting two different orders, a unique feature not observed in the Densovirinae to date. Sequencing of the transcriptome revealed four introns in the NS ORFs. These introns could produce four new hypothetical proteins. These included two truncated isoforms of NS3, and one truncated isoform each of NS1 and NS2. The new hypothetical proteins were all in frame. Finally CqDV's ability to cause disease in another commercially cultured crayfish, C. destructor was assessed. Although qPCR was able to detect CqDV in C. destructor + CqDV treatment group, no clinical signs of disease were observed. Future work should investigate if CqDV is replicating in C. destructor and whether or not it can be reisolated and is still infectious to C. quadricarinatus. Future work should also focus on characterising the protein expression of CqDV. CqDV's ability to cause disease in echinoderms and SSaDV's ability to cause disease in freshwater crayfish should also be assessed.

碩士<br>國立臺灣大學<br>生物科技研究所<br>106<br>White spot syndrome virus（WSSV）is a major pathogen of aquacultured shrimp. The infection of WSSV occurs not only in shrimp but also in other marine and freshwater crustaceans, including crabs and crayfishes. WSSV has caused large economic losses and there is still no adequate treatment. Unlike vertebrate immunity which is composed of both innate and adaptive responses, invertebrates rely on multiple innate defense reactions to combat infections. However, probiotics have been used in aquaculture for the function of immune stimulation. In this study, three probiotics were used respectively as feed additive: Lactobacillus reuteri Pg4, Bacillus subtilis AC and B. licheniformis CK, all of which have been proved to increase the immune responses in vitro or in vivo experiments. In immune test, both of phenoloxidase（PO）and superoxide dismutase（SOD）activities in probiotic treated groups were significantly higher than normal feed group. In WSSV challenge test, the two Bacillus strains caused the highest survival rate compared to other groups, and the higher PO and SOD activities were also found in the survival shrimps in B. subtilis group. On the other hand, the results of intestinal microbiota analysis proved that WSSV infection could significantly influence the intestinal microbiota, while the L. reuteri Pg4 and B. subtilis AC could decrease some bacteria genera raised by WSSV infection. In conclusion, B. subtilis is the most effective probiotic among three probiotics to improve the survival rate against WSSV infection.

碩士<br>國立高雄海洋科技大學<br>海洋生物技術研究所<br>100<br>Abstract Prophenoloxidase (proPO) and cytosolic manganese superoxide dismutase (cMnSOD) play crucial roles in crustacean innate immunity. In the present study, both of above genes were cloned from hemocytes of the red claw crayfish Cherax quadricarinatus using reverse transcription- polymerase chain reaction (RT-PCR) and rapid amplification of cDNA ends (RACE) method, and then total hemocyte counts (THCs), gene expression and enzyme activities were characterized after injection of white spot syndrome virus (WSSV) or Aeromonas hydrophila. The full length cDNA of proPO is 2930 bp and encodes 665 amino acids, wherease the cMnSOD cDNA is 1096 bp in length and encodes 287 amino acids. The calculated molecular mass of translated protein of proPO and cMnSOD is 75.8 kDa and 31.3 kDa with an estimated pI of 6.08 and 5.82, respectively. Phylogenetic analysis of amino acid sequences showed that C. quadricarinatus proPO and cMnSOD are more closely related to the proPO and cMnSOD of other crayfish than to that of penaeids, crabs, lobsters, or freshwater prawns. Tissue distributation analysis revealed that the proPO is primarily expressed in hemocytes, gill and heart, though the higher expression levels of cMnSOD showed in hemocytes, gill and hepatopancrease. The crayfish artificially infected with WSSV all died within 4 days generally. However, there was no mortality in crayfish when infected with A. hydrophila. THCs significantly decreased in crayfish at 48 h and 72 h after the infection of WSSV compared to the PBS group. In contrast, THCs of crayfish after A. hydrophila challenge were recovered at 48 h and 72 h from the significantly lower level at 24 h. The expression level of proPO and cMnSOD transcripts in hemocytes varied greatly after the challenge of WSSV or A. hydrophila. After WSSV challenge, the expression of both genes were significantly up-regulated at 24 h, but down-regraulated at other time points including 6 h, 12 h, 48 h and 72 h. While after the infection of A. hydrophila, both genes transcripts only significantly increased at 12 h and 24 h compared with the PBS group. There were similiar responses for the enzyme activity toward WSSV and A. hydrophila infection. The PO activities per hemocyte significantly increased from 24 h to 72 h compared to the PBS group, and cMnSOD exhibited higher level later at 48 h and 72 h post virus and bacteria infection.

Damian Rigg studied juvenile redclaw crayfish in aquaculture and examined the factors for a nursery phase to increase survivorship through grow-out. Diet, temperature and culture parameters were recommended as protocols for industry, adoption of these will assist in growing the industry through more surety over survivorship.

A polymerase chain reaction (PCR)-based cDNA subtraction technique termed suppression subtractive hybridization (SSH) was used to investigate the possible viral aetiology of cellular changes in decapod crustacea. These included hypertrophied nuclei with marginated chromatin (signet rings)but without Cowdry type A (CA) intranuclear inclusion bodies in the gills of freshwater redclaw crayfish Cherax quadricarinatus (Chapter 4), spheroid cells in the lymphoid organ of banana prawn Penaeus merguiensis (Chapter 6) and to identify differential gene expression which were associated with these two cellular changes. In both cases, viral genomes were not detected in SSH cDNA libraries, but multiple-transcripts were identified being induced in the hypertrophied nuclei population of redclaw crayfish and the hatchery population of banana prawn. These transcripts represented protein related to immunity, proteases and inhibitors, synthesis, processing and regulation-related proteins, structural and cytoskeletal related proteins, energy and metabolism factors, and ribosomal proteins, which are all known to be involved in biological process and defence mechanisms against infectious pathogens, in particular viral diseases. To investigate the probability of the viral aetiology of the lesions in C. quadricarinatus (Chapter 5) and P. merguiensis (Chapter 6) due to virus with no poly(A) tail, reverse transcriptase (RT)-PCR using primers designed from Bunyaviridae were performed. However, the results showed those were not the case. The PCR amplification using HPV140F/HPV140R primers revealed the absence of Penaeus merguiensis densovirus (PmergDNV) or related sequences in the redclaw populations (Chapter 5), but suggested the presence of PmergDNV in the hatchery population, while it was undetected in the wild population (Chapter 6). This suggested that these spheroid cells may be formed as defensive response against the viral infection. Further studies were conducted to uncover the cause of the signet ring changes in the gills of redclaw crayfish using several parvovirus primers for PCR amplifications (Chapter 5). Instead of identifying the exogenous viral sequences, the presence of endogenous Brevidensovirus-like elements (EBreVEs) were detected for the first time in C. quadricarinatus. Nine fragments that can be assembled into four consensus sequences were found from different sources of crayfish suggesting the widespread nature of these elements in C. quadricarinatus populations in northern Queensland, Australia. The most remarkable feature of these elements is that they are located in the same region relative to IHNNV sequences and most likely have originated from the non-structural protein of ancestral virus. Even though definitive insertion sites could not be determined, probably most of these elements, if not all, are randomly inserted within the mobile elements (microsatellites) of the host genomes. In addition, the presence of these endogenous virus-like elements may have immunological function for the host through RNAi pathway against infection of the more closely related exogenous viruses. Taken together, these studies have provided an insight into the host-viral interaction at the molecular level. This knowledge may contribute to future research on crustacean immunity into establishing a holistic approach to combat the devastating impact of infectious diseases, in particular viral pathogens, in order to maintain production in crustacean aquaculture.

碩士<br>國立嘉義大學<br>水生生物科學系研究所<br>106<br>A 2*3 factorial experiment was conducted to evaluate the effects of dietary cholesterol and phospholipids levels on the growth, survival, fatty acids composition, tissue triglycerol, cholesterol and phospholipids concentration. Basal diets supplemented with 0 or 0.5% cholesterol plus 0 or 2 or 4% phospholipids were fed to 120 individually reared Australian red claw crayfish (Cherax quadricarinatus) with initial weight of 12.0 g for 12 weeks. The results showed that survivals of all groups were higher than 85% and no difference was observed. There was no significant difference in weight gain among all groups (p >0.05) although the shrimps fed with combination of 0.5% cholesterol and 0% phospholipids had the highest WG. For the composition of the muscle, the amount of crude protein decreased as lecithin increased. Furthermore, for crayfish fed with cholesterol unsupplemented diet, there was a significant difference between in crude protein among group C0L4, and groups C0L0, and C0L2. There were no significant difference in ash, moisture, and crude lipid. Furthermore, no significant difference was observed in hepatopancreas composition. The fatty acids of eighteen carbons were the major composition in red claw crayfish hepatopancreas and muscle, respectively accounted for 64.8% and 48.7%. C18:2 fatty acid increased with lecithin supplemented, but no difference with cholesterol supplemented. There was no significant difference in muscle (p>0.05). When the diets phospholipids content was 8.1179 mg/kg, the C24:1 fatty acid in the muscle was significantly higher than the other groups. Moreover, cholesterol supplemented groups were significantly higher than no cholesterol supplemented groups (p<0.05).

Melissa Joyce assessed nutritional requirements of Redclaw. She determined the dietary requirement for Lysine, digestibly of alternative protein sources, and diet effects on redclaw health. She found that redclaw can digest a variety of ingredients, but dietary impact on health needs to be considered when formulating feed for redclaw.

碩士<br>國立高雄海洋科技大學<br>水產養殖研究所<br>102<br>The cultivation of Cherax quadricarinatus, an Australian freshwater crustacean, has progressively developed in various locations; however, the species is cannibalistic, especially during the larval stage. green water culture has been developed for a long period; it is characterized by stable water quality and reduced visibility, which provide a suitable environment for culturing biological organisms. This study investigated the influence of green water environments on the survival rate and growth of Cherax quadricarinatus. In the experiment, the animals used were crayfish juveniles which just left from mother (juvenile III) and adult crayfish. Each tank has 100 juveniles crayfish and 10 adult crayfish in three replicates of four treatments (clean water, A; green water, B; clean water + shelter, C; green water + shelter, D) were carried out for 30 days. Following the experiments, the specific growth rates and growth Increment of Groups B and D were considerably higher than those of Groups A and C; the survival rate obtained from Group D (72%) was significantly higher than those of the other groups. The results from the clear water group samples showed stressed physiological responses, whereas the green water did not influence the health of the Cherax quadricarinatus. Thus, the introduction of algae and shields facilitated the cultivation of the Cherax quadricarinatus larvae.

The demand for fish meal in aquaculture is an expensive part of aquaculture operations and this has generated interest in alternate food sources and use of natural live food. Use of traditional live food such as microalgae and zooplankton in aquaculture is reviewed. The current taxonomy of the redclaw Cherax quadricarinatus (von Martens, 1868), silver perch Bidyanus bidyanus (Mitchell, 1838), and the oligochaetes; Aulophorus sp. (Schmarda, 1861), Branchiura sowerbyi (Beddard, 1892), Tubifex tubifex (Muller, 1774) and the terrestial worm Dendrodrilus rubidus (Savigny, 1826) was elucidated. Culture requirements for worm species and the nutritional and culture requirements for the aquaculture target species were determined as follows: The best substrate for mass production of Aulophorus was 100% cowdung, at a water temperature of 15°C. There was a significant correlation between oxygen concentration and reproduction in Aulophorus sp. with production levels of 3194.5 mg of T. tubifex per cm2 reached within 60 days and 4242.5 mg per cm2 of B. sowerbyi. Both were cultured in a mixture of 75% cowdung, 20% fine sand and 5% mud. An alternate substrate/food (ground peanut/shell, wheat pollard, rolled oats and lucerne pellets) was utilised for the mass propagation of T. tubifex and B. sowerbyi. Production of 2352 mg T. tubifex per cm2 and 2339.34 mg per cm2 of B. sowerbyi was obtained after 60 days. The population density was significantly influenced by substrate type, with highest population densities in the cowdung based substrate. Experimental culture of the terrestial worm D. rubidus was also carried out. Percentage increase in number and mean weight increase of the inoculated worms after a month was 532.2% and 0.50 g and an average of 2797 juveniles weighing 839.1 g was collected from each culture container after four weeks. Trials of oligochaete species as food for C. quadricarinatus juveniles, were conducted, single species and combinations of worm species being used. The nutritive values of T. tubifex and B. sowerbyi, for redclaw stocks from the Gregory, Mitchell, Gilbert, Flinders and Leichhardt river systems of North Queensland were compared with a commercial pellet (Aquafeed) as a reference diet and a lucerne pellet. The nutritive values of Aulophorus sp. and Dendrodrilus rubidus were compared with Aquafeed. Digestibility experiments were performed using Aulophorus sp. and B. sowerbyi.

Research techniques of integrated sustainable primary production and wastewater 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), 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. -- abstract