Monocarboxylate transporters (MCTs) are bi-directional symporters that couple proton movement to a variety of glycolytic metabolites, including lactate and pyruvate. As such, MCTs not only play a crucial role in pH regulation, but could also function in the distribution of substrates between organelles, cells and tissues. This function may be critically important during hypoxia, when tissues depend on glycolytic flux for energy. We have examined the role of MCTs during hypoxia in an estuarine teleost, the common killifish, Fundulus heteroclitus, that routinely encounters environmental hypoxia. We cloned and sequenced a killifish MCT that resembled the MCT-2 isoform of mammals, and examined its expression pattern and regulation during hypoxia using real-time quantitative PCR. Killifish MCT mRNA levels were highest in the intestine (~2x) and lowest in the heart (~0.2x) as compared to the expression in the liver. Exposure to hypoxia (0.5 mg L-1) resulted in a significant increase in plasma lactate and glucose within 4 hours. However pyruvate and glycogen concentration in white muscle did not change significantly during this time. We observed no change in the mRNA levels of this MCT isoform in any tissue during 16h of hypoxia. This could indicate the regulation of this MCT at another level, a secondary role in hypoxia, or a role for this isoform unrelated to hypoxia tolerance. / Thesis (Master, Biology) -- Queen's University, 2009-09-02 15:54:00.643

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Experience of stress and/or cortisol, the end-product of activation of the hypothalamic-pituitary-interrenal (HPI) axis, may serve as a cue to trigger developmental plasticity. In fish, most research in this area has focused on effects of maternal stress or maternal cortisol levels on development, particularly with respect to the HPI axis and stress responses, and little attention has been paid to the effects of an endogenous stress response during early life. In the current study, zebrafish (Danio rerio) at four developmental stages (4, 7, 15 or 35 days post fertilization, dpf) were subjected to an air exposure stressor twice a day for two days. Individuals stressed early in life exhibited decreased survival and growth, increased whole-body Na+ and Ca2+ concentrations, and altered HPI axis activity associated with changes in anxiety-related behaviour at 7 to 35 dpf, with most effects diminishing with increasing age. Stress at 7 dpf was particularly effective at eliciting phenotypic changes, suggesting this age represents a critical window for cortisol to influence development. Finally, stress at 35 dpf induced masculinization, suggesting that cortisol influences sexual differentiation in zebrafish. These findings demonstrate that early-life stress in zebrafish triggers developmental plasticity, with effects on physiology and behaviour mediated by the HPI axis in an age-dependent manner.

In this work, behavioural field studies and metabolic studies in the laboratory were conducted to elucidate the extent of adaptation of the platypus Ornithorhynchus anatinus to its highly specialised semiaquatic lifestyle. Energy requirements of platypuses foraging, resting and walking were measured in a swim tank and on a conventional treadmill using flow-through respirometry. Foraging behaviour and activity pattern of platypuses in the wild were investigated at a sub-alpine Tasmanian lake where individuals were equipped with combined data-logger-transmitter packages measuring foraging activity or dive depth and ambient temperature. Energy requirements while foraging in the laboratory were found to depend on water temperature, body mass and dive duration and averaged 8.48 W kg-1. Mean rate for subsurface swimming was 6.71 W kg-1. Minimum cost of transport for subsurface swimming platypuses was 1.85 J N-1m-1 at a speed of 0.4 m s-1. The metabolic rate of platypuses resting on the water surface was 3.91 W kg-1 while minimal RMR on land was 2.08 W kg-1. The metabolic rate for walking was 8.80 and 10.56 W kg-1 at speeds of 0.2 and 0.3 m s-1, respectively. Minimal cost of transport for walking was predicted to be 2.13 J N-1m-1 at a speed of 1.7 m s-1. A formula was derived, which allows prediction of power requirements of platypuses in the wild from measurements of body mass, dive duration and water temperature. Activity patterns of platypuses in the wild were highly variable. Forty percent of the platypuses studied showed patterns, which deviated considerably from the nocturnal pattern generally reported for the species. Some animals showed diurnal rhythms while others temporarily followed the lunar cycle. Foraging trips lasted for an average of 12.4 h of continuous foraging activity per day (maximum: 29.8 hours). There were significant differences in diving behaviour between sexes and seasons. Activity levels were highest between August and November and lowest in January. While foraging, platypuses followed a model of optimised recovery time, the optimal breathing theory. Mean dive duration was 31.3 seconds with 72 % Energetics and foraging behaviour of the platypus 6 of all dives lasting between 18 and 40 seconds. Mean surface duration was 10.1 seconds. Mean dive depth was 1.28 m with a maximum of 8.77 m. Up to 1600 dives per foraging trip with a mean of 75 dives per hour were performed. Only 15 % of all dives were found to exceed the estimated aerobic dive limit of 40 seconds indicating mainly aerobic diving in the species. Total bottom duration per day was proposed as a useful indicator of foraging efficiency and hence habitat quality in the species. In contrast to observations made earlier in rivers, temporal separation was found to play a vital role for social organisation of platypuses in the lake system that was investigated. It is suggested that high intra-specific competition as well as limited burrow sites and a limited number of at the same time highly productive foraging locations were responsible for this observation. Mean burrow temperature in the wild was 17.5 and 14.2ºC in summer and winter, respectively, and was fairly constant over the platypus's resting period. In the cooler months, burrow temperature was up to 18ºC higher than ambient air temperature. By combining both field and laboratory data, a time-energy budget for the platypus was created. Mean field metabolic rate was 684 kJ kg-1 day-1 and was significantly higher in the winter months. Mean food requirement was 132 g fresh matter kg-1 day-1. Feeding rates were 68 % higher in winter than in summer. While platypuses in the swim tank were found to expend energy at only half the rate of semiaquatic eutherians of comparable body size, cost of transport at optimal speed as well as field metabolic rates were in line with findings for eutherians. These patterns suggest that locomotor efficiency of semiaquatic mammals might have reached a limit for energetic optimisation. The semiaquatic lifestyle seems to pose comparable energetic hurdles for mammals regardless of their phylogenetic origin.

This thesis documents the annual profiles of the primary reproductive steroids testosterone (T), 17beta-oestradiol (E2) and progesterone (P4), in the reproductive cycles of male and female blue-tongued lizards, Tiliqua nigrolutea. Data collected from a large captive population over three consecutive reproductive seasons are included. Reproductive cycles are discussed in the context of other viviparous squamate reptiles, while a broader comparative approach is used to consider patterns of steroid biosynthesis and peripheral metabolism. The annual patterns of circulating concentrations of T, E2 and P4 have been characterised for both sexes. In males, peak plasma T (10.9 +/- 3.00 ng ml-1) and E2 (778.0 +/- 120.00 pg ml-1) concentrations occur coincident with late spermatogenesis and observations of mating, respectively. Plasma P4 concentrations remain basal (< 1.2 ng ml-1) throughout the annual reproductive cycle. In females, increasing plasma E2 concentrations (275.2 +/- 33.87 pg ml-1 - 715.1 +/- 106.68 pg ml-1) are associated with vitellogenesis and plasma T peaks (6.3 +/- 0.63 ng ml-1) in the mating and peri-ovulatory period. In pregnant females, plasma P4 concentrations are elevated for the first two thirds of gestation, peaking in the second trimester at 12.7 +/- 1.27 ng ml-1 and falling rapidly prior to parturition. Concurrently, plasma P4 concentrations in non-reproductively active adult females remain basal (1 - 2 ng ml-1) throughout the year. There is good circumstantial evidence for a multiennial reproductive cycle in females. Parturition occurs late in the active season, presumably leaving little time for females to store sufficient fat reserves to become vitellogenic in the following spring: reproductive opportunities are effectively missed in at least one year following a reproductive effort. Observed reproductive behaviours, including agonistic male - male interactions, mating, and parturition, are documented. An investigation of gonadal steroid biosynthetic pathways in this viviparous squamate is presented. This compares variation in the relative contributions of the delta-4 and delta-5 steroidogenic pathways according to sex and reproductive condition. The delta-4 pathway predominates in both sexes, aligning this species phylogenetically with other reptiles. However, there are clear differences between sexes and with changing reproductive condition in the patterns of production of pathway intermediates and end-products. Additionally, detection of a possibly novel polar steroid as a major end-product of steroid biosynthesis in both sexes is reported. Peripheral (extragonadal) metabolism of T and E2 in a number of reproductively relevant steroid target tissues is compared at times of year chosen to represent three clearly distinctive reproductive conditions in each sex. There are differences both between sexes, between tissue types and with changing reproductive condition in the relative proportions of steroid conjugates and non-conjugated derivatives produced. Biosynthetic pathway activity and peripheral steroid metabolism both appear to be plastic in response to changing reproductive condition in Tiliqua nigrolutea. With a comprehensive database of information about the reproductive endocrinology and physiology of Tiliqua nigrolutea, this species is now available as a model to further examine selected aspects of the steroid hormone control of reproductive physiology and behaviour in a cool temperate, viviparous reptile.

In order for animals to experience pain and to suffer from it, they have to be capable of conscious perception. Recent evidence suggests that the fetus is maintained in a sleeplike unconscious state and that conscious perception therefore only occurs after birth. The timing of the onset of conscious perception depends on the maturation of underlying neurological processes and is anticipated to be species dependent. Painspecific electroencephalographic (EEG) responses of lightly anaesthetised young of three species born at different levels of neurological development were investigated. The results of the present thesis are in agreement with published data on general neurological, EEG and behavioural development. This information, in addition to the present results, has been used to estimate the approximate time of the onset of conscious perception in tammar wallaby joeys, rat pups and newborn lambs. In wallaby joeys (extremely immature at birth), the EEG remained isoelectric until about 100-120 days of in-pouch age and became continuous by about 150-160 days, with electroencephalographic and behavioural signs of conscious perception apparent by about 160-180 days. In rat pups (immature at birth), the absence of a differentiated EEG suggests that the ability for conscious perception in pups younger than 10-12 days is doubtful. The marginal EEG responses to noxious stimulation in 12-14 day-old pups and the pronounced EEG responses in pups 18-20 days suggest that rats may be capable of conscious perception from 12-14 days onwards. In lambs (mature at birth), full conscious perception is probably not apparent before 5 minutes after birth and may take up to several hours or days to become fully established. Its modulation by the residual neuroinhibitor allopregnanolone, if that occurs, would be highest over the first 12 hours after birth. Overall, the onset of conscious perception does not seem to follow an “on-off phenomenon”, but seems to develop gradually, even in species born neurologically mature. Although conscious perception, and hence pain experience, may be qualitatively different in younger animals, on the basis of the precautionary principle, when significantly invasive procedures are planned, pain relief should be provided from those postnatal ages when pain may first be perceived – i.e. from about 120 days in the tammar wallaby joey, about 10 days in the rat pup and from soon after birth in the lamb.

Chronic physiological stress impacts animal fitness by catabolizing metabolic stores and suppressing reproduction and immunity. This can be especially deleterious for capital breeding carnivores, such as marine mammals, which rely on lipid stores accrued during intensive foraging to sustain prolonged periods of fasting associated with reproduction. Therefore, chronic stress may cause a decrease in fitness in these animals, leading to population declines and potentially detrimental shifts in food web dynamics as a result. However, the impacts and indicators of chronic stress in animals are currently poorly understood. To identify downstream mediators of repeated stress responses in marine mammals, adrenocorticotropic hormone (ACTH) was administered once daily for four days to free-ranging juvenile northern elephant seals (Mirounga angustirostris) to stimulate endogenous corticosteroid release. I then compared blubber tissue transcriptome responses to the first and fourth ACTH administrations to determine the effects of acute and chronic endocrine stress, respectively. Gene expression profiles showed differences in responses to single and repeated ACTH administration, despite similarities in circulating cortisol profiles. We identified 61 and 12 differentially expressed genes (DEGs) in response to the first ACTH and fourth administrations, respectively, 24 DEGs between the first and fourth pre-ACTH samples, and 12 DEGs between ACTH response samples from the first and fourth days. Annotated DEGs were associated with functions in redox and lipid homeostasis, suggesting potential negative impacts of repeated stress on marine mammals. In addition, protein expression profiles were discrete between single and repeated ACTH administrations, and identified changes in expression of extracellular proteins that were not detected at the transcriptome level. We identified 8 and 7 differentially expressed proteins (DEPs) in response to the first and fourth ACTH administrations, respectively, including 5 DEPs in the overall ACTH response, 1 DEP between the first and fourth pre-ACTH samples, and 10 DEPs between ACTH response samples from the first and fourth days. Differentially expressed proteins in response to repeated ACTH administrations were associated with extracellular matrix (ECM) remodeling and suggest a link between glucocorticoid-induced adipogenesis and ECM remodeling in blubber. Other differentially expressed proteins were associated with increased lipid metabolism and decreased immunity, consistent with transcriptome data. Together, the use of transcriptomics and proteomics to detect responses to repeated stress provides more comprehensive insight into the marine mammal stress response and highlights the importance of using multiple discovery-driven approaches for understanding stress physiology. The gene and protein markers identified in this study may be used to identify stressed animals and discriminate between acutely and chronically stressed individuals with higher sensitivity than hormone measurements alone.