Thesis (Ph.D.)--University of Melbourne, Dept. of Zoology, 2010. / Typescript. Includes journal articles co-written by the author. Includes bibliographical references (leaves: 93-107)

Interactions between non-native yabbies (Cherax albidus) and indigenous marron (Cherax tenuimanus) in the south-west of Western Australia are not well understood. While there is abundant evidence to suggest that invasive freshwater crayfish are detrimental to native species, the nature and degree of impact on marron populations by exotic yabbies remains unclear. Researchers have hypothesized that invasive species make faster and more appropriate use of information about their environment than native species. This greater behavioural plasticity can result in displacement of indigenous species, successful colonisation by invaders, and subsequent disturbance to natural ecosystems and representative biodiversity. / The research presented in this thesis examines the behavioural responses of an indigenous crayfish (C. tenuimanus) and an invasive crayfish (C. albidus) to waterborne odours derived from food, alarm sources and finfish predators. This study was undertaken to assist in the understanding of predatory and competitive interactions between indigenous and non-indigenous crayfish and fish predators, with particular relevance to Western Australia. Predation and competition are major forces influencing community structure in ecosystems; therefore knowledge of competitive and predatory interactions will be of benefit when considering future translocation policies. / Behavioural trials were conducted in two culture systems (54 L aquaria and a 70,000 L mesocosm), where marron and yabbies were exposed to a range of water-borne odours from finfish predators (silver perch and Murray cod), with and without competition from conspecific and heterospecific crayfish. A number of variables likely to influence crayfish behaviour were investigated: strength of chemical odour; crayfish size, gender, diurnal and nocturnal activity patterns; predator size; prior-residence; suitable habitat/shelter; and feed availability. / A key innovation in this research was the high replication in the aquarium-based observation trials using a Latin Cube design, which resulted in greater statistical strength and lower variability. More importantly, this research deviated from the tradition of exclusively using the ‘individual crayfish’ approach for odour-detection experiments and tested these results in a 70,000 L communal observation tank. This was an important development in crayfish behavioural experimentation, particularly as several key findings from the individual crayfish approach were confirmed in a multi-species environment. / Results from this study supported the hypothesis that invasive crayfish species make more appropriate use of a wider range of information about their environment than native crayfish species. Yabbies were found to possess behavioural characteristics not present in marron, such as clearer behavioural modifications to food and heterospecific odour, and cautionary behaviour in the presence of odour from a finfish predator. During simulated daylight conditions, marron displayed behaviours conducive to predation that were not present in yabbies, including less time spent in shelter and more time spent in locomotory activity. However, during specialised night-time observational studies developed during this research, these differences were not evident. This would not seem to be an unusual result, given that crayfish naturally forage at night and become more active; however, it may have important implications for future behavioural studies of crayfish, indicating a bias associated with day-time approaches. Crayfish size also played a role in behavioural modifications to water-borne odours. Larger marron displayed clearer changes in behaviour and were more responsive to heterospecific alarm odour than juveniles. Furthermore, juveniles of both species were more active than adults and sub-adults. / The expansion of the yabby population into Western Australian habitats occupied by marron has been facilitated through translocation for aquaculture, and biological characteristics of the species, some of which are typical of other invasive crayfish species including: tolerance of a variety of conditions; rapid growth; early sexual maturity; burrowing to escape drought and predation; capable of multiple spawns in a growth season; and aggressiveness. Another characteristic of invasive crayfish species also shared by yabbies, as supported by the results of this study, is high behavioural plasticity. / Although marron do not share the same level of behavioural plasticity found in yabbies, their larger body size increases their success in competitive interactions. The comparatively smaller body size of yabbies may be the major factor limiting their population expansion in the presence of marron, especially in water-bodies where shelter is a limited resource. / Marron are an important endemic species in Western Australia, but their conservation is threatened by competition and predation from exotic species. The research presented in this thesis indicates that invasive yabbies are more receptive to chemical stimuli and better equipped to respond to predation risk than marron. This information will be of benefit when considering future translocation policy in Western Australia and highlights the need for a cautious approach to species introductions.

A study under controlled conditions of ovarian development and rematuration in the yabby (Cherax destructot) was undertaken. The purpose of the study was to improve fundamental understanding of the reproductive biology of the species and provide a basis for application to hatchery management in culture. A review was made of the current status of yabby culture in Australia and the present understanding of reproductive biology of decapod Crustacea. The review emphasised factors controlling several aspects of ovarian development, in particular the processes of vitellogenesis. The subsequent study was designed within the context of current hatchery practice and was based on existing knowledge of decapod reproduction, The sexual differentiation of the yabby after hatching was investigated by serial histological sections, and experiments were carried out to investigate the possibility of sex reversal of males. Most of this Investigation was concerned with removing the influence of the androgenic gland in directing male development, with the intent of observing the development of the elementary gonadal tissue into ovary. It was found that in contrast to other crustacean species, the sex of the yabby becomes fixed before the development of external secondary sexual characteristics, and before the androgenic gland can be discerned. Ovarian tissue developed in females at less than 8 weeks after hatching. A preliminary examination was undertaken for feminising parasites in gonadal tissue of a hermaphrodite yabby. Investigation of the ovary after spawning demonstrated that whilst the female was held under constant conditions of temperature and photoperiod, little rematuration occurred. Except for generation of previtellogenic oocytes during the first two days, the gonaciosomatic index remained low for up to 5 months after spawning. If the temperature of the female was reduced to 10°C and maintained constant, the previtellogenic oocytes were partially resorbed over a three week period. Rematuration then commenced, albeit at a low rate because of the reduced temperature, A method for standardising gonadosomatic indices was developed which took into account differences in hepatopancreatic nutrient reserves of individuals and loss of one or more appendages. This part of the study also considered constraints to rematuration and developed a method of accounting for differences in the ability of females to remature after spawning. Experiments were carried out to investigate the effect of crowding and temperature manipulation on initiating ovarian rematuration and to determine the rate of rematuration at 22°C once initiated. The duration of low temperature had no effect on rematuration; an overnight cooling was sufficient to initiate the process, Rematuration to the end of stage 2 vltellogenesis was substantially complete within 10 days. Crowding of females suppressed rematuration, but less than ideal water quality was not found to have any effect. The presence of a male initiated rematuration at a similar rate, but also led to stage 3 vitetlogenesis and spawning. A study was made of the pheromonal influence of the male through water borne factors without success. Rematuration could not be induced in ovigerous females. The literature review indicated that ovarian rematuration was under the control of an ovary stimulating hormone produced by the thoracic nerve ganglia. Attempts were therefore made to stimulate ovarian rematuration by incorporating the thoracic nerve into the diet of females. Attempts were also made to induce the release of ovary stimulating hormone from the thoracic nerve with 5-hydroxytryptamine, and also with octopamine. No effects were found, but a significant difference between the neurophysiology of the yabby and northern hemisphere crayfish was observed, and the implications of this finding are discussed. The study did not produce any conclusive evidence of an ovary stimulating hormone for the yabby. A model of ovarian rematuration which collects the findings of the experimental investigations was developed, and was used to suggest a hatchery broodstock management protocol. This model differs from existing models in that rematuration triggers and nutritional status are considered.

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.

The evaluation of the sub-chronic exposure to atrazine on crayfish The aim of this study is to evaluate the sub-chronic effect of atrazin on a behaviour, oxidative stress, antioxidant enzyme aktivities and biochemical profile of haemolymph in. These complex data should help to appraise the impact of this substance in the environment. The total test duration was 28 days and was divided into two periods. The first 14 days the crayfish were exposed to two concentrations of atrazine: 6.86 micrograms per liter (ATRenv = environmental concentration in the water in the Czech Republic) and 1.21 milligrams per liter (ATR10% = is coincident to 10% LC50). After the atrazine treatments the depuration 2 weeks phases in water without any chemicals followed. The results indicate that sub-chronic effect of atrazine influenced neither the behaviour of the crayfish nor the level of oxidative stress (measured by TBARS), whereas the changes of superoxiddismutase (SOD) were observed in all tissues (muscles, gills and hepatopancreas). The changes of enzyme activity were observed in catalase (CAT; hepatopancreas and the muscle tissue), glutathione S-transferase (hepatopancreas and the gills tissue), glutathione reductase (GR; the hepatopancreas tissue) and reduced glutathione (the muscle tissue). The influence of ATRenv on the biochemical profile of haemolymph at the following parameters was estimated only for lactate and alkaline, however phosphatase changes made by ATR10% were significant for glucose, ammonia, lactate and alkaline phosphatase measurements. The sub-chronical effect changed the activity of all antioxidant enzymes in hepatopancreas, muscles and the gills tissue of the observed crayfish. The presented results in this study are giving compact information of impact of atrazine on the crayfish and the whole water environment. The suggestion of using the crayfish for tests of toxicity looks like an ideal supplement for triazine herbicide estimations.

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.