The spotted minnow, Galaxias maculatus has a widespread southern hemisphere and circum-polar distribution including south-western and south-eastern Australia. It was sampled at monthly intervals over 12-18 months, by seine and plankton netting at three localities including a freshwater lake, Moates Lake, and two intermittently flowing, naturally saline rivers, the Jerdacuttup and the Oldfield rivers on the south coast of Western Australia. The resulting data provided an opportunity to describe the biology of G. maculatus in some detail including; environmental variables, life cycle, larval development, diet and parasitism by platyhelminth and nematode worms. Comparisons were made with other studies in south-east Australia, including Tasmania, and New Zealand. The present study confirmed that, at least throughout most of its range in Western Australia, G. maculatus has established a self-sustaining land-locked reproductive strategy. It is hypothesised that the development of land-locked breeding is an adaptive response to changing coastal geomorphology in the Holocene period that restricted ocean access of rivers and their fauna and caused estuaries to become non-tidal. The principal conclusion arising is that the local biology differs largely in degree rather than kind from elsewhere it has been studied; differences in degree are interpreted as local adaptations to an environment that is both variable and unpredictable Field measurement of environmental variables revealed G. maculatus will withstand salinities to approximately 46 ppt and surface water temperatures to 280 c. Very low dissolved oxygen concentrations to <1.0 mg r1 are accommodated by practicing secondary aerial respiration at the water surface. Galaxias maculatus on the south coast of Western Australia were smaller than those reported from populations elsewhere. Overall tota11engths of Western Australian males and females ranged 23-132 mm compared to 38-187 mm length to caudal fork for south-west Victoria, 31-185 mm standard length for Tasmania and 40-152 mm length to caudal fork for New Zealand fish. In the present study, size varied between the lake and one river population that was smaller. It is hypothesised that reduction in size of Western Australian G. maculatus generally is an adaptive response to avoid predation by piscivorous birds in shallow, confined river pools and lakes. There was a well defined, albeit extended, breeding season between autumn and spring with peak spawning in winter. The season was longer in the relatively stable lake situation and shorter ~ the very variable river situation partly due to the influence of river flow, which is continuous into the lake and intermittent and variable in the rivers. A flow dependent upstream spawning migration was part of the reproductive strategy but there was also the capacity, in certain circumstances, of spawning on falling water levels in years of nil or little flow. There was an almost complete cessation of reproductive activity during summer. Fecundity ranged from 296-2 874 eggs with a mean of 912 and was positively correlated with total length. The overall total lengths at which 50% of females and males and attain sexual maturity were estimated at 52 and 49 mm total length, respectively. For 95% of females and males the total lengths were estimated at 74 and 62mm total length, respectively. Ageing by counting annual growth rings was successful for lake inhabiting fish only, the lack of consistency in growth rings in the river environments was attributed to the extreme variability of these environments. The von Bertalanffy growth equation predicted that, on average, at the end of their first, second and third years females were 61, 81 and 88 mm total length respectively. Male predictions were 56, 74 and 80 mm, respectively. Approximately 75% of males and 62% of females attained sexual maturity at the end of their first year. Excluding larval fish, 73.1, 22.7, 4.1 and 0.1 % were 0+, 1+, 2+ and 3+ fish, respectively. The overall sex ratio females:males was 1.09:1.0, the ratio favoured males for very small fish but favoured females as fish aged and grew. Larval development was described in detail for the first time for Australian G. maculatus. The sequence of fin development was the same as that reported for galaxiids elsewhere, i.e. caudal, dorsal, anal, pectoral and pelvic. Adult fin ray counts were; cauda1 16, dorsa1 9, anal 13, pectoral 12 and pelvic 6-7. Myomeres ranged from 45-50. Development of pigmentation and dentition were described; caniniform teeth began to develop during the late postflexion larval stage. Dietary analysis confirmed a previous description of G. maculatus as an euryphagic carnivore. A wide range of invertebrate food groups including copepods, amphipods and ostracods, aquatic insects as well as terrestrial invertebrates (spiders, winged ants and orthopterans) were consumed. Most variation in diet was explained by site, i.e whether fish were from river or lake environments or which river environment. A lack of replicate samples precluded a rigorous statistical analysis of the influence of either fish size or season on diet. However, a provisional analysis suggested these variables have minimal influence. Larval diets comprised copepods, cladocerans and unicellular algae; with the attainment of postflexion larval stage and development of caniniform dentition, a wider range of dietary items were ingested. One cestode, one trematode and two nematode larval worms infected river and lake inhabiting fish. The cestode, Ligula intestinalis, infected 13% of lake inhabiting fish causing gross disfiguration and probably reduced reproductive success, particularly of males. The degree and severity of cestode infection was much less in rivers, perhaps due to their saline waters. The worms' adult hosts in all cases were piscivorous waterfowl particularly the white-faced heron. At present G. maculatus is widespread and abundant throughout its range in Western Australia. As most of its range is in rivers and lakes which are, and will in the future be influenced by clearing for agriculture, it is likely that increased river recharge due to clearing will initially benefit G. maculatus. However long term change, particularly changes to riparian vegetation structure and species composition, are likely eventually to be inimical as the shading value of vegetation and its habitat value for terrestrial invertebrate food are diminished.

The critical swimming speed (Ucrit) of rainbow trout parr (Oncorhynchus mykiss) and three life stages of Galaxias maculatus, larval (whitebait), postlarval inanga and adult inanga, were tested at temperatures from 5oC to 25oC. All fish were swum at their acclimation temperature under normoxic conditions to determine the optimal aerobic exercise temperature. To determine whether acclimation affected swimming ability, trout parr acclimated to either 10oC or 20oC were swum at 20oC and 10oC, respectively. The potential effect of mild hypoxia (75% saturation) on trout parr and whitebait was also examined at 10oC, 15oC and 20oC, and also tested separately and in combination were the effects of mild hypoxia and severe anaemia on the prolonged swimming ability of trout smolts at temperatures from 10oC to 20oC. For all trout experiments, blood samples were taken from non-exercised and exercised fish by acute caudal venepuncture to determine haematological responses to both acclimation and exercise. Under normoxic conditions, Ucrit max for trout parr (7.0 0.5 cm fork length) was calculated to be 5.8 body lengths per second (BL s-1) at 15.1oC, but declined at lower and higher temperatures. This result implies that swimming performance was limited by temperature below 15oC, whereas performance at higher temperatures was limited by oxygen availability. In support of this hypothesis, mild hypoxia (75% saturation) had no effect at 10oC or 15oC but caused a significant reduction in Ucrit at 20oC. However, fish acclimated at 20oC showed an adaptive elevation in oxygen carrying capacity due to an increase in mean erythrocyte volume and haemoglobin content. Furthermore, acclimation to 20oC improved warm water swimming performance. Trout parr acclimated to 10oC performed significantly worse than fish acclimated to 20oC when swum at 20oC. However, trout parr acclimated to 20oC performed as well as fish acclimated to 10oC when swum at 10oC. Following exercise, haematocrit was elevated under both normoxic and hypoxic conditions. However, the primary cause of this apparent increase in oxygen carrying capacity was splenic release of erythrocytes under normoxic conditions, whereas stress-induced erythrocytic swelling contributed to the observed increase in hypoxia. This contrasting response was most pronounced at 10oC. Larval whitebait (4.7 - 5.0 cm total length (TL)) also showed a temperature dependence of prolonged swimming ability with Ucrit max calculated to be 5.1 BL s-1 at 17.7oC. Hypoxia significantly reduced Ucrit at 15oC and 20oC, lowering the optimal aerobic temperature to 13.9oC and reducing Ucrit to 4.2 BL s-1. Mild hypoxia therefore had a more pronounced impact on inanga whitebait than trout. Postlarval inanga (3.9 - 4.0 cm TL) performed poorly at higher temperatures with Ucrit max of 5.6 BL s-1 at 9.4oC indicating an ontogenetic change in swimming ability, possibly resulting from a developmental shift in red muscle kinetics or a greater dependence on anaerobic muscle. Adult inanga (5.5 - 6.8 cm TL) prolonged swimming ability showed similar temperature dependence to that of inanga whitebait but lower relative swimming speeds due to their larger size. The dramatic decline in performance exhibited by juveniles at warmer temperatures was not apparent in adults. Ucrit max for adults was 4.0 BL s-1 at 18.3oC. The critical swimming speed of trout smolts, subjected to mild hypoxia (6.8 mg

Inanga (Galaxias maculatus) is an amphidromous fish species that is able to successfully inhabit a variety of salinities. Using an integrated approach this thesis has characterised for the first time the physiological characteristics that facilitate acclimation in inanga. Structural studies using scanning electron microscopy (SEM) and laser scanning confocal microscopy (LSCM) revealed freshwater-acclimated inanga have a high density of apical pits and freshwater-type mitochondria-rich cells (MRCs) that can facilitate ion absorption from the hypo-osmotic environment. In seawater, inanga remodel their gills by increased proliferation of seawater-type MRCs to facilitate ion secretion in the hyper-osmotic environment. Concentration-dependent sodium (Na+) kinetic analysis revealed that at a whole body level, inanga regulate Na+ using a saturable, high affinity, low capacity uptake system which makes them extremely adept at extracting Na+ from very dilute freshwater environments. In fact inanga displayed an uptake affinity (Km) of 52 ± 29 µM, which is one of the lowest ever recorded in freshwater fish. The sodium/potassium ATPase transporter (NKA) is central to Na+ regulation within the gill. In high salinties inanga displayed increased NKA activity (6.42 ± 0.51 µmol ADP mg protein-1 h-1) in an effort to excrete the excess Na+, diffusively gained from the hyper-osmotic environment. This increase in NKA was most likely a reflection of the proliferation of NKA-containing MRCs. The NKA activities seen in freshwater- and 50% seawater-acclimated inanga were similar (2.54 ± 0.19 and 2.07 ± 0.22 µmol ADP mg protein-1 h-1 respectively) to each other suggesting the inanga gill is capable of supporting ion regulation in brackish waters without a significant increase in NKA activities, and the energetically-expensive changes in gill structure and function that accompany such a change. Molecular investigation of NKA isoform expression using quantitative PCR (qPCR) showed that inanga displayed salinity-induced changes in the expression of the three α NKA isoform variants investigated. Isoform α1a exhibited a pattern consistent with an important role in freshwater, confirming results from other fish species. While it is generally accepted that α1b isoform is the predominant NKA isoform in seawater, inanga did not display this pattern with a freshwater dominance seen. None of the salinity-induced changes could quantitatively explain the increased NKA activity in seawater suggesting that different isoforms may convey different activities, that there is also regulation of NKA at a post-transcriptional level, and/or other isoforms or subunits may have a significant role. The importance of the osmoregulatory hormone cortisol and prolactin is widely accepted and inanga were treated with cortisol, prolactin and a combination of the two in an effort to further elucidate their role. NKA activity and NKA isoform expression were assessed but no specific patterns were deduced, except for a decrease in both NKA activity and isoform expression in 100% seawater-acclimated inanga treated with cortisol and prolactin. The reasons for this decrease were not evident, although the impact of stress induced by the injection protocol was likely to be a confounding factor. The development of a new confocal-based technique in this study was able to describe, for the first time, intracellular sodium levels ([Na+]i) as a function of salinity in an intact euryhaline fish gill cell. Using the fluorescent Na+ indicator dye CoroNa Green this study demonstrated the ability of inanga gill cells to maintain [Na+]i in the face of environmental change. Freshwater-acclimated inanga displayed basal [Na+]i of 5.2 ± 1.8 mM, with 12 ± 2.3 mM and 16.2 ± 3.0 mM recorded in 50% seawater- and 100% seawater-acclimated cells, respectively. Low [Na+]i is advantageous in hypo-osmotic environments as it provides a gradient between the cell and the blood which is essential for generating electrochemical gradients cell volume regulation and other cellular homeostatic mechanisms. A slightly elevated [Na+]i seen at the higher sanities would help minimise the diffusive gradient for passive influx from the environment which would be of benefit in hyper-osmotic environments. Upon salinity challenge 50% seawater cells were equally adept at maintaining a constant [Na+]i at any salinity, suggesting these cells are have the necessary constituents to regulate Na+ in both lower and higher salinities. This novel LSCM approach is advantageous relative to existing transport models as it will allow the observation of cellular ion transport in real time, within a native filament structure displaying functional interaction of different cell types. The extreme ion uptake characteristics of the inanga and their amenability to in situ confocal-based studies demonstrated in this study, confirm inanga as a valuable model species for future research.

Salinity and oxygen availability have long been recognised as important factors influencing animal physiology and therefore species distribution. The maintenance of appropriate cellular ion levels is critical for many essential physiological processes, but at the same time is energetically expensive. Since hypoxia is likely to impose aerobic limitations for ATP generation, the maintenance of salt and water homeostasis could be at risk during hypoxia. The amphidromous inanga (Galaxias maculatus) is well known for its salinity tolerance and its life cycle that involves several salinity related migrations. During these migrations inanga also frequently encounters hypoxic waters, and therefore must maintain energy homeostasis when aerobic metabolism may be compromised. The present study has investigated behavioural, physiological, biochemical and molecular mechanisms by which inanga tolerate changes in salinity and hypoxia. After 14 days of acclimation to salinities ranging from freshwater to 43‰, inanga showed physiological acclimation. This was evident by no changes in metabolic rates or energy expenditures through this salinity range. Energy balance seemed to be tightly and efficiently controlled by changes in the proportion of protein and lipids used as energy substrate. No mortalities and only minor changes in plasma osmolality also indicated salinity acclimation. The remarkable osmoregulatory capacity of inanga was also evidenced after a seawater challenge. The osmotic balance of inanga was only disrupted during the first 24 hours after the challenge, evidenced by an increase in plasma osmolality and plasma Na+, and a decrease in muscle water content. These physiological changes were correlated with changes at the molecular level. Different isoforms of the catalytic subunit of the Na+,K+-ATPase (NKA) were isolated, partially sequenced and identified in inanga. Phylogenetic analysis grouped inanga isoforms (α-1a, α-1b, α-1c) with their respective homologues from salmonids. Patterns of mRNA expression were also similar to salmonids, with α-1a being downregulated and α-1b being up-regulated following seawater challenge. Previous to this study, NKA isoform switching was reported to occur only in salmonids and cichlids. The presence of NKA subunits that change with environmnetal salinity in inanga indicates that this isoform switching phenomenon is much more widespread among teleost lineages than previously thought. Aiming to elucidate the hypoxia tolerance of inanga, oxygen consumption rate as a function of decreasing external PO2 was evaluated. At no point did inanga regulate oxygen consumption, suggesting that this species is an oxyconformer. This is the first robust demonstration of the existence of oxyconforming in fish. Evaluation of the scaling relationship between oxygen consumption and fish size in normoxia, showed that the exponent of this relationship fell within the range previously reported for fish. However, in hypoxic conditions the scaling relationship was less clear suggesting different size-related mechanisms for tolerating hypoxia. Analysis of the aerobic and anaerobic metabolism of small and large fish, showed that smaller inanga were able to sustain aerobic metabolism for longer than larger inanga, which instead relied on anaerobic metabolism for extending their survival. This knowledge is likely to be of value for the conservation of this iconic fish species, by incorporating these size related differences in hypoxia tolerance in streams management. In light of the unusual oxyconforming response of inanga, a study examining the behavioural responses of this species to declining dissolved oxygen was performed. Inanga did not display a behaviour that might reduce energy expenditure during oxygen limitation; instead swimming activity and speed were elevated relative to normoxia. As hypoxia deepened inanga leaped out of the water, emersing themselves on a floating platform. Once emersed, fish exhibited an enhanced oxygen consumption rate compared to fish that remained in hypoxic water. Although this emersion behaviour was hypothesised to be of physiological advantage, both aquatic hypoxia and emersion resulted in similar physiological and biochemical consequences in inanga. While in hypoxic water oxygen availability seemed to be the limiting factor, in air failure of the circulatory system was hypothesised to be the cause of a similar metabolic signature to that found in aquatic hypoxia. Overall, inanga seemed to be not particularly well adapted to tolerate aquatic hypoxia. In light of the increasing likelihood of anthropogenic-induced hypoxia in inanga habitats, this is likely to have negative consequences for the future of inanga populations in the wild. Although this study provides the mechanisms behind the exceptional salinity tolerance of inanga, its susceptibility to hypoxia is likely to impose further constraints for the osmoregulatory processes that guarantee inanga survival during life cycle migrations. The results of the present study are relevant for understanding and managing the fishery of this economically- and culturally important fish species.