Mulloway are a large native inshore marine fish that are currently being evaluated by NSW Fisheries for their potential in aquaculture. The current study developed and applied molecular genetic markers to assess the geographical scale at which future hatcheries should be developed for the species. In addition, it evaluated the impact that current breeding practices in NSW have had on genetic diversity in culture cohorts. The study showed that wild Australian populations of this species constitute a single management unit (genetic stock), but that current hatchery practices employed in NSW are eroding natural genetic diversity. Thus a single hatchery could provide cultures stock to the whole Australian industry without compromising wild populations but that hatchery management practices will need to be modified in the future, to minimise levels of inbreeding.

The bluefin tuna have a variety of distinctive anatomical and physiological adaptations that enhance performance. However, our understanding of bluefin tuna physiology is limited by the logistical difficulties of studying these large pelagic fish. This thesis examines some aspects of the metabolic physiology of the southern bluefin tuna. It provides insight into the high-performance, high-energy demand physiology of bluefin. It also examines the metabolic physiology of the mulloway, another important aquaculture species for which physiological information is currently limited. 1. Routine metabolic rate (RMR) of southern bluefin tuna (SBT) (Thunnus maccoyii), the largest tuna specimens studied so far (body mass = 19.6 kg (± 1.9 SE)) was measured in a large (250,000 l) flexible polypropylene respirometer “mesocosm respirometer”. Mean mass-specific RMR was 460 mg kg⁻¹ h⁻¹ (± 34.9) at a mean water temperature of 19°C. When total RMR is added to published values of other tuna species at equivalent swimming speeds, there is a strong allometeric relationship with body mass (654 • Mb ⁰·⁹ ⁵, R ² = 0.97). This demonstrates that interspecific RMR of tuna scale with respect to body mass similar to that of other teleosts, but is approximately 5-fold higher than the standard metabolic rate (SMR) of other active teleost species. 2. This study reports on the first measurements of the metabolic cost of food digestion and assimilation (specific dynamic action, SDA) of a tuna species. Oxygen consumption (MO₂) and swimming velocity of southern bluefin tuna (SBT) (Thunnus maccoyii) were elevated for periods between 20-45 h (longest for the largest rations) post-ingestion of sardines (Sardinops sagax). It is suggested that the purpose of increased swimming velocity was to increase ventilation volume as a response to the enhanced metabolic demand associated with SDA. The magnitude of SDA as a proportion of gross energy ingested (SDA coefficient) averaged 35 ± 2.2 %. This demonstrates that the absolute energetic cost of SDA in SBT is approximately double that recorded in other teleost species. 3. This study examines the effects of sardines (Sardinops sagax) with high- (12.9%) or low- (1.8-4.0%) lipid level on specific dynamic action (SDA) and swimming velocity of southern bluefin tuna (SBT) (Thunnus maccoyii). Fish swam faster during the SDA period with the increase in velocity being greatest for the fish that ingested the high-lipid sardine. Magnitude of SDA was also greater for fish that ingested the high-lipid sardines. However, the energetic cost of SDA as a proportion of ingested energy was not significantly different between fish that ingested the high- (34.3 ± 2.4%) and low-lipid sardines (31.5 ± 2.9%). These results confirm that the high energetic cost of SDA is ecologically relevant. 4. In this study the metabolic and behavioural responses of both fasted and postprandial southern bluefin tuna (Thunnus maccoyii, SBT) to low dissolved oxygen (DO) was examined. In moderate hypoxia (4.44 and 3.23 mg l⁻¹), swimming velocity (U) and routine metabolic rate (RMR) of fasted fish was mildly enhanced. At 2.49 mg l⁻¹, U increase to over double in the normoxic speed, possibly as an escape response. At 1.57 mg l⁻¹, both U and RMR were suppressed and SBT failed to survive the entire 20 h exposure period. This reveals that SBT are remarkably well adapted to low DO. Feeding did not greatly influence their hypoxia tolerance. In a subsequent experiment there were no significant differences in U, RMR and gastric evacuation rates of postprandial SBT in hypoxia (2.84 mg l⁻¹) compared to those in normoxia (7.55 mg l-¹). 5. In this study, 768 h of simultaneous recordings of metabolic rate (MR, = heat production) and visceral temperature were made in both fasted and postprandial southern bluefin tuna (SBT, Thunnus maccoyii) of two sizes (~10 and 20 kg) and at two water temperatures (~19 and 16°C). Duration and magnitude of specific dynamic action (SDA) were strongly related to duration and magnitude of postprandial visceral warming providing the first empirical evidence of a link between SDA and postprandial visceral warming. Visceral temperature of fasted SBT was also directly related to MR. In this case, source of heat is thought to be metabolic work performed within the red muscles which warmed the viscera through thermal conductance. Visceral excess temperatures were over 1°C warmer in larger than smaller SBT. Better heat retention ability of the larger SBT is likely attributed to improved retia mirabilia development and greater thermal inertia. SBT at 16°C maintained visceral excess temperatures significantly warmer than similarly sized fish at 19°C. This demonstrates some ability of SBT to physiologically regulate visceral warming. 6. In this study, the effect of progressively severe hypoxia levels on the swimming performance and metabolic scope of juvenile mulloway (Argyrosomus japonicus) were investigated. In normoxic conditions (6.85 mg l⁻¹), standard metabolic rate (SMR) and cost of transport were typical for subcarangiform fish species. Mulloway had a moderate scope for aerobic metabolism (5 times the SMR). The critical dissolved oxygen level was 1.80 mg l⁻¹ revealing that mulloway are well adapted to hypoxia. In all levels of hypoxia (5.23, 3.64, and 1.86 mg l⁻¹) the active metabolic rate was reduced however, the critical swimming velocity was reduced only at 3.64, and 1.86 mg l⁻¹. Mulloway metabolic scope was significantly reduced at all hypoxia levels, suggesting that even mild hypoxia may reduce growth productivity. / Thesis (Ph.D.) -- University of Adelaide, School of Earth and Environmental Sciences, 2007

The research conducted for this thesis has produced quantitative data on the biology and population genetic structure of the Mulloway Argyrosomus japonicus in Western Australia, where this sciaenid is recreationally and commercially fished. These data were used 1) to test various hypotheses regarding the relationship between key biological characteristics of A. japonicus and both latitude and environmental variables, 2) to investigate the population structure and genetic characteristics of populations of A. japonicus on the west and south coasts of Western Australia and to assess the relationships between these populations and those of A. japonicus in eastern Australia and South Africa, and 3) to consider the implications of the biological and genetic results for management.

Secondary salinisation has rendered over 100 million hectares of land throughout the world, and over 5 million hectares in Australia, unsuitable for conventional agriculture. The utilization of salinised land and its associated water resources for mariculture is an adaptive approach to this environmental problem with many potential economic, social and environmental benefits. Despite this, inland mariculture is yet to develop into an industrial-scale, rural enterprise. The main aim of this study was therefore to identify and address some of the technical and biological limitations to the development of an inland finfish mariculture industry. Three technical aspects essential to the development of an Australian inland mariculture industry were reviewed; potential sources of water, the species suitable for culture in these water sources and the production systems available to produce them. Based on factors such as their quantity, quality and proximity to infrastructure, the most appropriate water sources were deemed to be groundwater obtained from interception schemes and waters from operational or disused mines. In terms of species, mulloway (Argyrosomus japonicus) were identified as having many positive attributes for inland mariculture, including being temperate and therefore having the ability to be cultured year-round in the regions where the majority of secondary salinity occurs. Seasonal production of barramundi (Lates calcarifer) in ponds in the temperate climatic zones has potential, but may be more appropriate for those salinised water sources located in the warmer parts of the country. Rainbow trout (Oncorhynchus mykiss) were also identified as having excellent potential provided water temperature can be maintained below the upper lethal limit and also have potential for seasonal production, perhaps in rotation with barramundi. In terms of production systems, pond-based culture methods were found to have many advantages specific to inland mariculture. Static ponds enable culture in areas with low groundwater yield and more cost-effective potassium supplementation compared with flow through ponds. Static ponds also largely overcome the issues associated with the disposal of salt-laden and eutrophied waste water; however yields from static ponds are typically low and limited by the nutrient input into the pond. In response to the yield constraints of static pond culture, a new culture technology known as the Semi-Intensive Floating Tank System (SIFTS) was designed, patented and constructed in collaboration with the aquaculture industry and tested in a static inland saline pond in the wheatbelt of Western Australia. This technology was designed to reduce nutrient input into ponds by the collection of settleable wastes and to provide large volumes of well-oxygenated water to the target species, to ameliorate the loss of fish from low dissolved oxygen during strong microalgal blooms. The three species identified above has having excellent potential for inland mariculture (mulloway, rainbow trout, and barramundi) were grown in SIFTS held within a 0.13 ha static, inland saline water body (salinity 14 ppt) over a period of 292 days, yielding the equivalent of 26 tonnes/ha/year (total for all three species). Rainbow trout were grown with an FCR of 0.97 from 83 to 697 grams over 111 days (SGR, 1.91%/day) between June and September, when average daily water temperatures ranged from 12.3„aC to 18.2„aC. Over the same time period, mulloway grew only from 100 to 116 grams, however, once temperatures increased to approximately 21„aC in October, feed intake increased and mulloway grew to an average size of 384 grams over 174 days with an SGR and FCR of 0.68 %/day and 1.39, respectively. Barramundi stocked in November with an average weight of 40 grams increased to 435 grams in 138 days (SGR 1.73%/day) with an FCR of 0.90. The SIFTS significantly reduced nutrient input into the pond by removing settleable wastes as a thick sludge with a dry matter content of 5 to 10%. The total quantity of dry waste removed over the 292 day culture period was 527 kg (5 tonnes/ha/yr), which was calculated to contain 15 kg of nitrogen (144 kg/ha/yr) and 16 kg of phosphorus (153 kg/ha/yr). The release of soluble nutrients into the pond resulted in blooms of macro- and micro- algae which caused large and potentially lethal diurnal fluctuations in dissolved oxygen within the pond, however, comparatively stable levels of dissolved oxygen were maintained within each SIFT through the use of air lift pumps. It is well documented that saline groundwater is deficient in potassium which, depending on the extent of the deficiency, can negatively impact on the performance of marine species, including fish. The physiological effects of this deficiency on fish, however, have not been previously described. As such, I conducted a bioassay investigating the physiological effects of a hypersaline (45 ppt) groundwater source containing 25% of the potassium found in equivalent salinity seawater (i.e. 25% K-equivalence) on juvenile barramundi. Histopathological examination of moribund fish revealed severe degeneration and necrosis of skeletal muscles, marked hyperplasia of branchial chloride cells and renal tubular necrosis. Clinical chemistry findings included hypernatraemia and hyperchloridaemia of the blood plasma and lowered muscle potassium levels. It was concluded from this study that the principal cause of death of these barramundi was skeletal myopathy induced by unsustainable buffering of blood plasma potassium levels from the muscle. Although such hypokalaemic muscle myopathies have been previously described in mammals and birds, this was the first description of such myopathies in fish. It was hypothesized from the results described above that the physiological effects of potassium deficiency are dependent on salinity and that they would be ameliorated by potassium supplementation. These predictions were tested in a subsequent study which measured the effects of potassium supplementation between 25% and 100% K-equivalence on the growth, survival and physiological response of juvenile barramundi at hyperosmotic (45 ppt), near-isosmotic (15 ppt) and hyposmotic (5 ppt) salinities. Unlike those juvenile barramundi reared at 45 ppt and 25% K-equivalence in the previous study, those reared in 50% K-equivalence water at 45 ppt in this study survived for four weeks but lost weight; whereas at 75% and 100% K-equivalences fish both survived and gained weight. Homeostasis of blood plasma potassium was maintained by buffering from skeletal muscle. Fish reared in 50% K-equivalence at this salinity exhibited muscle dehydration, increased branchial, renal and intestinal (Na+-K+)ATPase activity and elevated blood sodium and chloride, suggesting they were experiencing osmotic stress. At 15 ppt, equal rates of growth were obtained between all K-equivalence treatments. Buffering of plasma potassium by muscle also occurred but appeared to be in a state of equilibrium. Barramundi at 5 ppt displayed equal growth among treatments. At this salinity, buffering of plasma potassium from muscle did not occur and at 25% K-equivalence blood potassium was significantly lower than at all other K-equivalence treatments but with no apparent effect on growth, survival or (Na+-K+)ATPase activities. These data confirmed the hypothesis that proportionally more potassium is required at hyperosmotic salinities compared to iso- and hypo- osmotic salinities and also demonstrated that barramundi have a lower requirement for potassium than other marine and estuarine species being investigated for culture in inland saline groundwater. In addition to ongrowing fish, saline groundwater has potential for hatchery production. Specific advantages include the vertical integration of inland saline farms and the production of disease-free certified stock through isolation from the pathogens and parasites found naturally in coastal water. To determine the potential of utilizing inland saline groundwater for hatchery production, barramundi larvae were reared from 2 to 25 days post hatch in 14 ppt saline groundwater with either no potassium supplementation (38% K-equivalence) or full potassium supplementation (100% K-equivalence). Growth, survival and swimbladder inflation of these larvae were compared against those grown in control treatments of seawater (32 ppt) and seawater diluted to 14 ppt. Those reared in saline groundwater with 38% K-equivalence exhibited complete mortality within 2 days, whilst those held in groundwater with full supplementation survived at a rate equal to both control treatments (pooled average 51.1 ¡Ó 0.5%). At 25 days post hatch, there was no significant difference in larval length or dry weight between those grown in the 14 ppt control treatment and those in the saline groundwater with full potassium supplementation. There were no significant differences in swim bladder inflation between any of the surviving treatments (average 93.3 ¡Ó 2.5%). This is the first description of rearing barramundi larvae both in low salinity seawater and in saline groundwater, and demonstrates that the requirement for potassium by larval barramundi is higher than for juveniles of the same species. In addition to a deficiency in potassium, saline groundwater in Western Australia often contains an elevated concentration of manganese relative to seawater as a result of anaerobic reduction of manganese oxides or the pedogenic weathering of manganese-bearing rock. The effects of elevated manganese on marine or estuarine fish have not been described and a study was therefore conducted to determine if manganese, at a concentration typical of that found in saline groundwater, has any impact on fish. The effects of 5 mg/L of dissolved manganese on juvenile mulloway at salinities of 5, 15 and 45 ppt were determined by comparing the survival, growth and blood and organ chemistry with those grown at the same salinities without manganese addition. Survival of mulloway at 45 ppt in the presence of 5 mg/L of manganese (73 ¡Ó 13%) was significantly lower than all other treatments, which achieved 100% survival. Those fish grown in seawater without manganese exhibited rapid growth, which was not affected by salinity (SGR = 4.05 ¡Ó 0.29%/day). Those fish grown at 5 ppt and 45 ppt in the presence of manganese lost weight over the two week trial (SGR 0.17 ¡Ó 0.42 and -0.44 ¡Ó 0.83%/day, respectively), whilst those at 15 ppt gained only a small amount of weight (SGR 1.70 ¡Ó 0.20%/day). Growth was therefore affected by manganese and by the interaction of manganese and salinity, but not salinity alone. Manganese was found to accumulate in the gills, liver and muscle of the fish. No gill epithelial damage or other significant histological findings were found, however, significant differences in blood chemistry were observed. Blood sodium and chloride of manganese exposed fish were significantly elevated in hyperosmotic salinity (45 ppt) and depressed at hyposmotic salinity (5 ppt) compared with unexposed fish; consistent with manganese causing apoptosis or necrosis to chloride cells. Blood potassium was significantly elevated and liver potassium significantly reduced at all salinities in the presence of manganese. These findings are consistent with manganese interfering with carbohydrate metabolism. There were no differences in blood sodium, chloride or potassium across salinities in fish not exposed to manganese, demonstrating mulloway are capable of efficient osmoregulation across this salinity range.