Inanga (Galaxias maculatus) are a culturally and economically important fish species in New Zealand and abroad. However, very little is known about their ability to deal with trace element contamination. As a scaleless fish with the ability to survive in relatively extreme environments, they may not fit toxicity models (such as the biotic ligand model; BLM) based on other fish species. The aim of this study was to determine how this fish responds to elevated trace elements in both the laboratory and field in order to determine the applicability of these toxicity models. In order to determine the impacts of stress on ion transport and subsequent metal toxicity, inanga were exposed to handling stress and measures of ion uptake were collected. Handling stress was shown to result in increased ventilation rates, resulting in stimulated sodium (Na+) efflux. A compensatory increase in Na+ influx was also measured as a result of this stress. Inanga largely recovered from this ionoregulatory stress within 2 hours, with full recovery after 24 hours. This was indicative of a rapid homeostatic response for maintaining ion balance. Enhanced Na+ uptake in response to this stress resulted in increased copper (Cu) uptake in Cu-contaminated water, suggesting stressed fish will accumulate more Cu (and likely other Na+ mimics) than an unstressed fish. These results suggest a heightened vulnerability of inanga to this type of contaminant as a result of exercise stress during migrations. A combination of field and laboratory studies was used in order to measure trace element accumulation in inanga. In situ field studies showed changes to aluminum (Al) and iron (Fe) body burdens when inanga were placed in streams of varying trace element concentrations along the West Coast of the South Island. However, other trace elements measured did not alter over the period of exposure (9-10 days). Biochemical biomarker analysis showed no changes in the activity of Na+/K+-ATPase (NKA), but a marker of lipid peroxidation (thiobarbituric acid reactive substances; TBARS) was elevated in one stream. Analysis suggested that stream pH was the major driver of this effect, whether directly or via changes to metal bioavailability. Subsequent laboratory exposures (96 h) of inanga to 1.2, 2.7, 10.8, and 44 µg L-1 dissolved Fe and 5.6, 23.3, 60.7, and 128.7 µg L-1 dissolved zinc (Zn) showed no difference in whole body trace element accumulation, ammonia excretion, ion influx (Ca2+ and Na+), and TBARS. There were significant differences in oxygen consumption (MO2) after Fe exposures, with increases in the 2.7 and 44 µg L-1 dissolved Fe exposures. Laboratory exposure results suggest inanga are relatively insensitive to short-term Fe and Zn exposures. Both in vivo (whole body partitioning) and in vitro (Ussing chamber) techniques were used to determine the influence of cutaneous ion transport on preventing trace element accumulation. Results suggest inanga use their skin as an additional site of calcium (Ca2+) and Na+ uptake. This is the first study to confirm these ion transport capabilities in inanga, and revealed that up to 48% of Na+ uptake may occur across the skin. Pharmacological inhibition of Ca2+ uptake was achieved by known Ca2+ channel blockers (verapamil and lanthanum). Furthermore Fe and Zn impaired cutaneous Ca2+ transport, indicating that ion transport pathways in the skin modulate in response to these metals.

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.