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

This dissertation characterizes the influences of water chemistry on the acute toxicity of lead (Pb) to two of the long-standing sentinel test organisms commonly employed by the United States Environmental Protection Agency (USEPA), the fathead minnow (Pimephales promelas) and daphnid (Ceriodaphnia dubia), for parameterization of an acute Pb Biotic Ligand Model (BLM). In addition, a toxicogenomic approach was employed to identify genes that might serve as molecular markers of Pb exposure and long-term effects, as well as provide new insights as to the underlying toxic mechanisms of chronic Pb exposure in P. promelas. The endpoints of growth, reproduction, Pb accumulation, prey capture ability, and swimming performance of P. promelas were examined to assess the influences of water chemistry during chronic Pb exposures and to potentially link microarray-identified genes to outcomes of ecological significance. Importantly, this work revealed that calcium does not protect against acute toxicity to C. dubia or chronic Pb accumulation by P. promelas, indicating that current hardness-based regulations are inappropriate and provide further support for the need for alternative approaches to setting environmental regulations for Pb. The findings reported herein should facilitate the arrival of such an approach in the form of a new acute Pb BLM. However, different responses with respect to the influences of water chemistry on the acute toxicity of Pb were exhibited by these species suggesting that development of separate BLMs for P. promelas and C. dubia should be considered to ensure adequate protection for both species. Furthermore, the influences of water chemistry were found to be inconsistent during acute and chronic Pb exposures to P. promelas and thus caution against inferring chronic effects from acute exposures. A number of Pb-responsive genes were identified that exhibited a strong potential for serving as robust indicators of Pb exposure and accumulation in P. promelas. While these genes also provided insight as to the likely toxic mechanisms of Pb, additional work will be necessary to firmly link these genes to chronic outcomes of ecological relevance in the context of ambient water chemistry.

Worldwide, an increasing demand for fish and fisheries products, together with socioeconomic pressure for industry expansion, is placing considerable pressure on wild fish stocks – more than 80% of which are considered by the Food and Agriculture Organisation of the United Nations (FAO) to be either maximally- or over-exploited. Adding value to the existing catch and/or improving the sustainability of current wild capture methods may offer a means of providing industry growth while negating the need for increased landings. In particular, the peri-mortem condition of a fish plays an integral role in the condition of the tissues post-mortem and hence in product quality, with harvesting techniques that result in stress or fatigue yielding a lower quality product. An understanding of the physiology of the target species and its response to harvest is therefore essential to implementing targeted improvements in harvesting technologies. For species harvested using trawl-based technologies, this includes knowledge of their exercise physiology, in particular their swimming capacity, since this is a key determinant of the interaction between fish and trawl gears, and hence of the nature and severity of stress experienced and of the condition of fish at landing. This thesis describes a series of discrete studies relating to the exercise physiology of juvenile snapper, Pagrus auratus, an iconic New Zealand finfish that comprises important recreational and commercial fisheries. In particular, we sought to characterise the capacity of snapper for sustained swimming activity, including how performance may differ between fish of different size or with environmental temperature; to determine the consequences of exhaustive exercise for both subsequent swimming activity, an important determinant of survival in escaping or discarded catch, and for tissue biochemistry, which ultimately determines product quality in harvested fish; to validate the use of laboratory-based simulations for the study of capture-related stress by comparing the response of laboratory-exercised snapper with commercially caught fish; and to determine the tolerance of snapper to environmental hypoxia, and further, the possible consequences of hypoxia for swimming capacity and for recovery in fish retained for subsequent rested-type harvest. The capacity of snapper for sustained swimming activity was characterised through the use of incremental exercise tests to determine critical swimming speeds, Ucrit. Juvenile snapper (94-107 mm length, 16-157 g mass) demonstrated a strong swimming capacity, with individual fish attaining critical swimming speeds of up to 7.1 body lengths per second (bl s⁻¹). Swimming performance demonstrated an allometric association, with absolute critical speeds increasing with fish size, whilst relative performance favoured smaller fish. The relation was described by the function Ucrit (m s⁻¹) = 0.003412 [length (mm)] + 0.2669. Critical swimming performance also exhibited variation in response to environmental variables. Thermal performance curves were evident in snapper acclimated to 12, 18 and 24 °C, with the suggestion of optimal performance at acclimation temperatures between 18 and 24 °C. Critical swimming performance was also significantly reduced during exposure to ambient oxygen tensions below 80 mmHg; at 40 mmHg, snapper attained only 21% of the critical swimming speeds observed under normoxic (150 mmHg) conditions. In juvenile snapper (~75 g), exhaustive exercise resulted in severe metabolic, acid-base, haematological and hormonal perturbations, the nature of which were similar to those classically demonstrated in other strong-swimming fish species, especially salmonids. These included the depletion of glycogen from within the white muscle (WM) and the concomitant production of lactate, with a resultant lactacidosis of the plasma; recruitment of erythrocytes from the spleen; and the release of cortisol to the plasma. The recovery of these disturbances required 6 hours under laboratory conditions. As the stresses experienced by fish during commercial capture are often considered to be greater than those which can be induced during laboratory-based simulations, it was necessary to investigate whether the magnitude of the perturbations observed in laboratory-exercised snapper were an appropriate model of those of trawl-caught fish. In trawl-caught snapper (1100 g, 38 cm) obtained under commercially-relevant conditions (tow speed ~3.0 knots; duration 2.25-2.75 hours), the magnitude of the perturbations were greater than for laboratory-exercised fish. While the recovery of some metabolites was evident within the first 18 hours post-capture, their recovery was prolonged relative to laboratory-exercised fish; other metabolites, namely muscle glycogen and plasma cortisol, exhibited no signs of recovery. These observations suggest that the response of snapper to exhaustive exercise within the laboratory may underestimate the severity of the response induced by commercial harvest. This is further suggested by post-capture mortality rates of 14%, whereas no mortality was observed following fatigue at Ucrit. Exhaustive exercise also resulted in the impairment of subsequent critical swimming performance. Immediately following fatigue, snapper (85-160 g) were capable of sustained swimming activity at speeds of up to 60-70% Ucrit; however, critical swimming performance was reduced 30%, presumably due to limitations in WM function. There was no suggestion of the recovery of WM function within the first 30 minutes post-fatigue; thereafter, Ucrit was progressively restored, such that snapper were able to repeat their initial swimming performance in a second Ucrit test performed 2 hours after the conclusion of the first. Snapper were moderately tolerant of hypoxia, oxygen-regulating at reduced oxygen tensions (<100 mmHg) by virtue of increased ventilatory rate and stroke volume, with a distinct bradycardia developing at PO₂ below 60 mmHg. Larger snapper appeared to possess a greater hypoxia tolerance than did smaller fish, with Pcrit resolved to 77 in 20 g fish, and 50 mmHg in 150 and 230 g fish. Exposure to moderate hypoxia (60-80 mmHg) during recovery from an exhaustive exercise event constrained MO₂ max to 78% of that of normoxic fish, however did not appear to impede the return of MO₂ to routine levels. The present study is the first to examine in detail the swimming performance of snapper, and the consequences of exhaustive exercise for physiological condition. By understanding the swimming capacities of snapper, it may be possible to refine harvesting practices (i.e. tow speeds) or utilise technologies (i.e. net design) such that the water velocities through the trawl net are within the range at which the fish can swim sustainably, minimising the extent of stress and fatigue experienced by fish, and hence their effects on both quality and survival. The study also demonstrates that whilst snapper experience significant physiological disturbance during commercial harvesting, including significant mortality, some fish demonstrate the potential for metabolic recovery, which may permit their retention in an on-board tank facility for subsequent rested-type harvest. Finally, the present work highlights a number gaps in our understanding of the link between harvesting conditions and fish condition, and makes a number of suggestions for future studies or directions.

The current understanding of lactate metabolism in fish is based almost entirely on interpretation of concentration measurements that cannot be used to infer changes in flux. Moreover, the transporters regulating these fluxes have never been characterized in rainbow trout. My goals were: (1) to quantify lactate fluxes in rainbow trout under normoxic resting conditions, during acute hypoxia, and exercise by continuous infusion of [U-14C] lactate; (2) to determine lactate uptake capacity of trout tissues by infusing exogenous lactate in fish rest and during graded exercise, and (3) to clone monocarboxylate transporters (MCTs) and determine the effects of exhausting exercise on their expression. Such information could prove important to understand the mechanisms underlying the classic “lactate retention” seen in trout white muscle after intense exercise. In normoxic resting fish, the rates of appearance (Ra) and disappearance (Rd) of lactate were always matched (~18 to 13 µmol kg-1 min-1), thereby maintaining a low baseline blood lactate concentration (~0.8 mM). In hypoxic fish, Ra lactate increased from baseline to 36.5 µmol kg-1 min-1, and was accompanied by an unexpected 52% increase in Rd reaching 30.3 µmol kg-1 min-1, accounting for a rise in blood lactate to 8.9 mM. In exercising fish, lactate flux was stimulated > 2.4 body lengths per second (BL s-1). As the fish reached critical swimming speed (Ucrit), Ra lactate was more stimulated (+67% to 40.4 μmol kg-1 min-1) than Rd (+41% to 34.7 μmol kg-1 min-1), causing an increase in blood lactate to 5.1mM. Fish infused with exogenous lactate stimulated Rd lactate by 300% (14 to 56 μmol kg-1 min-1) during graded exercise, whereas the Rd in resting fish increased by only 90% (21 to 40 µmol kg-1 min-1). Four MCT isoforms were partially cloned and characterized in rainbow trout: MCT1b was the most abundant in heart, and red muscle, but poorly expressed in gill and brain where MCT1a and MCT2 were prevalent. MCT4 was more expressed in the heart. Transcript levels of MCT2 (+260%; brain), MCT1a (+90%; heart) and MCT1b (+50%; heart) were stimulated by exhausting exercise. This study shows that: (i) the increase in Rd lactate plays a strategic role in reducing the lactate load imposed on the circulation. Without this response, blood lactate accumulation would double; (ii) a high capacity for lactate disposal in rainbow trout tissues is elicited by the increased blood-to-tissue lactate gradient when extra lactate is administered; and (iii) rainbow trout may be unable to release large lactate loads rapidly from white muscle after exhausting exercise (lactate retention) because they poorly express MCT4 in white muscle and fail to upregulate its expression during exercise.

The current understanding of lactate metabolism in fish is based almost entirely on interpretation of concentration measurements that cannot be used to infer changes in flux. Moreover, the transporters regulating these fluxes have never been characterized in rainbow trout. My goals were: (1) to quantify lactate fluxes in rainbow trout under normoxic resting conditions, during acute hypoxia, and exercise by continuous infusion of [U-14C] lactate; (2) to determine lactate uptake capacity of trout tissues by infusing exogenous lactate in fish rest and during graded exercise, and (3) to clone monocarboxylate transporters (MCTs) and determine the effects of exhausting exercise on their expression. Such information could prove important to understand the mechanisms underlying the classic “lactate retention” seen in trout white muscle after intense exercise. In normoxic resting fish, the rates of appearance (Ra) and disappearance (Rd) of lactate were always matched (~18 to 13 µmol kg-1 min-1), thereby maintaining a low baseline blood lactate concentration (~0.8 mM). In hypoxic fish, Ra lactate increased from baseline to 36.5 µmol kg-1 min-1, and was accompanied by an unexpected 52% increase in Rd reaching 30.3 µmol kg-1 min-1, accounting for a rise in blood lactate to 8.9 mM. In exercising fish, lactate flux was stimulated > 2.4 body lengths per second (BL s-1). As the fish reached critical swimming speed (Ucrit), Ra lactate was more stimulated (+67% to 40.4 μmol kg-1 min-1) than Rd (+41% to 34.7 μmol kg-1 min-1), causing an increase in blood lactate to 5.1mM. Fish infused with exogenous lactate stimulated Rd lactate by 300% (14 to 56 μmol kg-1 min-1) during graded exercise, whereas the Rd in resting fish increased by only 90% (21 to 40 µmol kg-1 min-1). Four MCT isoforms were partially cloned and characterized in rainbow trout: MCT1b was the most abundant in heart, and red muscle, but poorly expressed in gill and brain where MCT1a and MCT2 were prevalent. MCT4 was more expressed in the heart. Transcript levels of MCT2 (+260%; brain), MCT1a (+90%; heart) and MCT1b (+50%; heart) were stimulated by exhausting exercise. This study shows that: (i) the increase in Rd lactate plays a strategic role in reducing the lactate load imposed on the circulation. Without this response, blood lactate accumulation would double; (ii) a high capacity for lactate disposal in rainbow trout tissues is elicited by the increased blood-to-tissue lactate gradient when extra lactate is administered; and (iii) rainbow trout may be unable to release large lactate loads rapidly from white muscle after exhausting exercise (lactate retention) because they poorly express MCT4 in white muscle and fail to upregulate its expression during exercise.