This thesis focuses on the UK pond newts, the smaller bodied species known as Lissotriton newts and the larger Triturus. The primary aims were to identify and address gaps in the current Tritus/Lissotriton literature; to provide a more complete understanding of this group as many assumptions about morphology and physiology exist untested, for example that larger/fatter females will lay more eggs. Specifically for Lissotriton helveticus, many assumptions are based on the similarly sized Lissotriton vulgaris, potentially missing any species specific differences. A further focus of the work was to provide a clearer view over the whole breeding season; using measures of condition over a season and egg-laying.

Incidence of endocrine disruption in wild fish species has been documented globally and is well characterised in the UK, where the occurrence of intersex in roach (Rutilus rutilus) is widespread. Although this has been associated with concentrations of steroid oestrogens, research indicates that anti-androgenic chemicals may also play a role in inducing these effects. Anti-androgenic activity is commonly detected in wastewater treatment works effluents and some receiving waters, but the chemicals responsible remain largely uncharacterised. This thesis aimed to identify environmental anti-androgens in UK and South Australian catchments and to produce environmentally relevant exposures to assess their potential impacts on sexual disruption in fish, alone and in combination with steroid oestrogens. By using hydrological modelling techniques, pharmaceuticals with an anti-androgenic mode of action were predicted to occur in the ng/L concentration range in UK and South Australian wastewater treatment works effluents and river catchments. This work included analysis of future trends in environmental concentrations of the pharmaceuticals and the steroid oestrogens in these catchments. Modest increases in concentrations by 2050 were predicted in the absence of mitigation, which could increase in the risk posed to fish health by the steroid oestrogens in the future. The effects of the predicted concentrations of two pharmaceuticals, bicalutamide and cyproterone acetate, were then assessed in fathead minnows (Pimephales promelas) and Japanese medaka (Oryzias latipes) based on the UK modelling for the present day. These concentrations did not contribute to endpoints characteristic of sexual disruption, alone or in combination with steroid oestrogens. However, the results did support an environmental role for the steroid oestrogens in intersex induction. Concurrently, effect directed analysis identified some highly potent anti-androgens, such as triclosan and pyrene, in wastewater treatment works effluents from the UK. However, they are likely to make a minor contribution to overall anti-androgenic activity due to their low concentrations. Consequently, more work is required to identify the causes of this activity in the environment and its implications for wild fish health.

No description available.

From all the species that arrive to a novel environment, very few manage to form a viable population. The guppy, a very successful invader, is a highly social species that performs some of its vital tasks (e.g., foraging, avoiding predators) in groups. This thesis aimed to quantify heterospecific association benefits that enhance invasion success. Interactions between invaders and natives could be one of the environmental characteristics of a place that increase its risk of invasion. I evaluated the tendency of an invasive species to associate with native individuals with similar ecological requirements. I tested the hypothesis that invaders gain exploring, acquisition of information and foraging benefits when socializing with natives. In these experiments I used the guppy as the invasive model species and endangered native Mexican topminnows (Poeciliopsis infans, Skiffia bilineata, Ameca splendens, Zoogoneticus tequila, Xenotoca eiseni and Girardinichthys viviparous). I found that guppies shoal with other species in Trinidad (Poecilia picta and Poecilia sphenops), where they are native (Chapter 2) and that this trait remains when they are invasive (Chapter 3). Guppies are equally willing to explore novel environments when accompanied by heterospecifics or conspecifics. Guppies are more willing to explore complex environments than simple ones. Moreover, when exploring simple environments they have a higher association tendency, regardless of the partner' species (Chapter 4), which could lead them to acquire the benefits of grouping behaviour and avoid Allee effects - the disadvantages of being part of a small group. In the contexts in which they were tested guppies gained as much information by associating with heterospecifics as with conspecifics (Chapter 5). Finally, I found that when shoaling in bigger shoals guppies are able to locate food faster and spent more time foraging. The benefits of increased shoal size were maintained when the additional guppies were replaced with heterospecifics. However, they derive more benefits from the species they are more willing to associate with (Chapter 6). These results uncover a mechanism enabling founding individuals to survive during the most vulnerable phase of an invasion and help explain why guppies have established viable populations in many parts of Mexico as well as in every continent except Antarctica.

Introduction Glucocorticoids (GCs) are synthesised endogenously in mammals by the hypothalamic pituitary adrenal (HPA) axis in response to stress. These hormones can elicit a number of physiological roles by binding to and activating specific receptors (glucocorticoid or mineralocorticoid receptors- GR or MR). GCs are important in tissue development and maturation and commonly used therapeutically. Mammalian animal studies have suggested that over-exposure to GCs, whether pharmacologically or through induction of maternal stress, is associated with increased cardiovascular disease risk in adult life. The underlying mechanisms underpinning this early life programming are poorly understood, however GC exposure during development may have direct and indirect effects on the structure and function of developing tissues and organs which may predispose to disease in later life. Current mammalian models of programming do not lend themselves well to studying organ development during embryogenesis. The zebrafish provides an ideal model to study this phenomenon due to the transparent nature of developing larvae and the availability of transgenic lines expressing fluorescent markers. Methods GC pathways were comprehensively characterised during zebrafish embryo development using qRT-PCR and steroid ELISAs. The physiological roles of GCs were assessed during early zebrafish development (first 120 hours post fertilisation (hpf)) assessing stress response, swim activity and global development following various genetic and pharmacological manipulations of the GC system. The impact that GC manipulation had on the cardiovascular system was also investigated. Embryos which had been exposed to GC manipulation during early development were then allowed to develop to adulthood in order to assess the long term impact. The same parameters were investigated in the adult as in the embryo. Results The key components of the GC system are present and functional in the developing embryo with de novo cortisol biosynthesis evident from 48hpf. A functioning hypothalamic pituitary inter-renal (HPI) axis is demonstrable from 72hpf. Manipulation of specific components of the GC pathway during early embryonic development influences growth-rate, head-trunk angle, chorion hatch-rate and swim behaviour. Manipulation of GCs during embryogenesis resulted in altered body weight, length and girth in adulthood, with altered stress response and swim behaviour also detected. Embryonic heart development was also affected with a reduction in ventricle cardiomyocyte number, cardiac gene abundance (vhmc) and cardiac function during embryogenesis resulting in structural abnormalities such as fewer trabeculae and increased intra-ventricular space. Embryonic GC manipulation also alters the formation and patterning of intersegmental blood vessels by 120hpf. In adulthood this manifests as a reduced angiogenic capacity. Conclusion The zebrafish embryo represents a valid and physiologically relevant model for GC research. Manipulation of GCs during early development results in altered growth, gene abundance and cardiovascular structure. These findings have significant implications for on-going research addressing GC mediated programming and suggest that the zebrafish is a highly suitable model for GC research.

Methods were developed to quantify variation in gill size and microstructure and applied to three fish species: brown trout, Arctic charr and common carp. Measurements of arch length, number and length of gill rakers, number and length of gill filaments and number, length and spacing of the lamellae were taken for each gill arch and combined by principal component analyses to give length-independent scores of gill size. Levels of fluctuating asymmetry in gill arch length were also examined. Buccal and gill cavity volumes were measured from silicon moulds. Standard histological methods were used to examine gill microstructure. Benthic-feeding charr from a sample collected in Loch Awe, Scotland had relatively larger heads and buccal cavities than did sympatric pelagic-feeding fish Allowing for body size, they also had a more extensive respiratory surface, perhaps reflecting exposure to poorly oxygenated water while feeding on the loch bottom and/or a more active life style. Levels of asymmetry in gill arch length were higher in the pelagic-feeding form, which grow faster than the benthic-feeding form (Chapter 2). Gill size and structure were compared in carp (Chapter 3) and trout (Chapter 4) classified by a standard test as having proactive, reactive or intermediate stress copping styles. Proactive carp and trout had more extensive respiratory surfaces and lower levels of hyperplasia than did reactive fish, intermediate fish lying in between. The opposite was the case for density of mucous cells, which was highest in reactive fish and lowest in proactive ones. These data suggest that maintaining a large respiratory surface may represent an unrecognised cost of a proactive coping style. Common carp were held in mixed groups of proactive and reactive fish in one of 6 combinations of temperature (20oC and 25oC) and dissolved oxygen (3-4, 5-6 and 7-8 mg O2 L-¹) for 10 weeks. At the higher temperature fish had relatively larger heads and longer secondary lamellae, but had fewer mucous cells and a lower percentage of hyperplasia. At the lowest oxygen levels fish had relatively larger heads and a higher degree of hyperplasia than those held in normoxic and hyperoxic conditions. These results suggest that, over weeks, carp are able to “remodel” their respiratory structures in response to their current oxygen requirements. Few clear differences in response were found between proactive and reactive fish (Chapter 5). In semi-extensively farmed carp sampled over their final production year. Shortterm, acute husbandry stressors (grading and crowding) produced striking changes in several potential welfare indicators, including reduced body condition, increased in plasma glucose, lactate and cortisol levels and higher level of body damage. Percentage hyperplasia and secondary lamella number and length also increased. Long-term acute stress (pre-harvest crowding in concrete tanks) was associated with increased levels of skin and fin damage and in hyperplasia and mucus cell number, reflecting high stress levels and/or poor water quality. Glucose, lactate and cortisol levels fell, suggesting either habituation to current conditions or differential mortality by physiological stress status (Chapter 6). The results of Chapters 2-6 are synthesised in a general discussion (Chapter 7) and considered in the context of the existing literature on trophic polymorphism, on stress coping strategies, on the effects of environmental conditions of the welfare of cultured fish and on how gill structure and microstructure relate to other indicators of welfare.

The Notch signalling pathway is pivotal in ensuring that the processes of arterial specification, angiogenic sprouting and haematopoietic stem cell (HSC) specification are correctly carried out in the dorsal aorta (DA), a primary arterial blood vessel in developing vertebrate embryos. Using the zebrafish as a model organism, and additional experimental observations from mouse and cell line models to guide mathematical modelling, this thesis aims to better understand the mechanisms involved in the establishment of a healthy vasculature in the growing embryo. We begin by studying arterial and HSC specification in the zebrafish DA. Mathematical models are used to analyse the dose response of arterial and HSC genes to an input Notch signal. The models determine how distinct levels of Notch signalling may be required to establish arterial and HSC identity. Furthermore, we explore how Delta-Notch coupling, which generates salt-and-pepper patterns, may drive the average gene expression levels higher than their homogeneous levels. The models considered here can qualitatively reproduce experimental observations. Using laboratory experiments, I was able to isolate DA cells from transgenic zebrafish embryos and generate temporal gene expression data using qPCR. We show that it is possible to fit ODE models to such data but more reliable data and a greater number of replicates at each time point is required to make further progress. The same VEGF-Delta-Notch signalling pathway is involved in tip cell selection in angiogenic sprouting. Using an ODE model, we rigourously study the dynamics of a VEGF-Delta-Notch feedback loop which is capable of amplifying differences betwen cells to form period-2 spatial patterns of alternating tip and stalk cells. The analysis predicts that the feeback strengths of Delta ligand and VEGFR-2 production dictate the onset of patterning in the same way, irrespective of the parameter values used. This model is extended to incorporate feedback from filopodia, growing in a gradient of extracellular VEGF, which are capable of facilitating tip cell selection by amplifying the resulting patterns. Lastly, we develop a PDE model which is able to properly account for VEGF receptor distributions in the cell membrane and filopodia. Receptors can diffuse and be advected due to domain growth, defined by a constitutive law, in this model. Our analysis and simulations predict that when receptor diffusivity is large, the ODE model for filopodia growth is an excellent approximation to the PDE model, but that for smaller diffusivity, the PDE model provides valuable insight into the pattern forming potential of the system.

Lampreys (Petromyzontiformes) are an ancient vertebrate group, comprising 40 currently recognised species that range throughout the Northern and Southern Hemispheres. Despite a conserved morphology, lampreys nevertheless express a diverse range of life history strategies. Unusually for vertebrates larval lampreys are filter-feeding organisms prior to undergoing an extensive anatomical reorganisation, and the adoption of either a parasitic or a non-parasitic adult life. Parasitic lampreys consume the flesh and blood of actinopterygian fishes, either in marine or freshwater environments, while non-parasitic lampreys do not feed following their metamorphosis from the larval form. Morphological and genetic similarities between pairs of parasitic and non-parasitic lampreys have led to taxonomic confusion regarding the specific status of many non-parasitic forms, and the suggestion that the loss of the trophic adult phenotype is the result of a single species capable of producing alternative life history strategies. In this thesis it is argued that at least some paired species of lampreys do not comprise two distinct evolutionary lineages; rather, that non-parasitic lampreys represent one extreme in a continuum of life history variation expressed by a parasitic species. Some lamprey species, such as the European river lamprey Lampetra fluviatilis, are morphologically variable, exhibiting divergent phenotypes in response to ecological pressures, such as alternative foraging environments. Loch Lomond, Scotland contains a population of L. fluviatilis that feeds exclusively in the lake and exhibits a reduced body size and an overall morphology distinct from the typical anadromous form. Its foraging strategy indicates that it may be capable of switching hosts in the face of declining numbers of a presumed favoured and formerly abundant host, suggesting a certain amount of plasticity in its trophic ecology that may have ensured its survival in this freshwater lake. This freshwater-resident form, as well as anadromous L. fluviatilis and the non-parasitic species L. planeri, were found to spawn in a single river system within the Loch Lomond basin, and this site is crucial for the continued presence of this life history variant in Loch Lomond. The appearance of sexually mature specimens of three discrete phenotypes in this river, each representing an alternative life history strategy that may, or may not, belong to a single species, provides a crucial opportunity to test the strength of assortative mating between lamprey species pairs. Within this system the strength of assortative mating was found to be weak, and points to the possibility that freshwater-resident L. fluviatilis are mitigating gene flow between large anadromous parasitic L. fluviatilis, and small, non-parasitic L. planeri. As well as weak behavioural isolation, inter-specific sneak male mating tactics were documented among these populations, and represents the first time this phenomenon has been observed between paired lamprey species. Such behaviour indicates a lack of species-specific cues acting between L. fluviatilis and L. planeri, and suggests that hybrid offspring could be common in some systems. Testing hybrid viability (survivorship) between Loch Lomond’s two L. fluviatilis life history strategies and the sympatric L. planeri revealed no post-zygotic barriers to gene flow, at least in the form of gamete incompatibility. Perhaps more convincingly though, when comparing traditional morphometrics and body shape variation, as well as mitochondrial DNA sequences, between L. fluviatilis expressing different foraging strategies with populations of L. planeri, no robust species specific differentiation was observed. In fact, species delimitation between L. fluviatilis and L. planeri appears to be related solely to overall body size, which is itself a function of life history strategy. However, life history strategy was not correlated with current species designation as relationships among mtDNA haplotypes indicate non-parasitic populations have evolved independently multiple times throughout the geographic range of L. fluviatilis in Europe. Therefore, L. planeri should not be considered as a distinct species, either morphologically or genetically. Instead, L. fluviatilis appears capable of expressing a range of life history strategies; from parasitic anadromous populations through to non-parasitic stream-resident populations. The overall research approach employed in this thesis, i.e., the combination of ecological, behavioural, taxonomic and molecular studies, could be used to robustly examine the evolutionary ecology of parasitic and non-parasitic lampreys elsewhere.

The proteome is in constant flux and therefore it is important to understand the contribution of protein dynamics to the function of an organism. The zebrafish (Danio rerio) is a recognised model organism that is widely used to investigate physiological processes. The focus of this thesis was to develop a method to calculate the rates of synthesis of heart proteins of zebrafish on a proteome-wide scale. The initial stage of the project involved the optimisation of a method to characterise the protein complement of individual zebrafish hearts. It was concluded that for the rapid screening of proteins 1-dimensional gel electrophoresis in conjunction with high resolution liquid chromatography-tandem mass spectrometry (LC-MS/MS) was the most appropriate experimental approach. In order to determine the rates of protein synthesis, zebrafish were administered with a stable isotope-labelled amino acid ([2H7] L-leucine) via the diet and its incorporation into heart proteins was monitored over an 8 week time course. Using this method it was possible to calculate the synthesis rates of over 600 proteins. The experimental strategy was then applied to define the changes in protein synthesis rates in hearts from zebrafish that were subjected to chronic unpredictable stress (CUS). A variety of stressors on zebrafish comprising air emersion, net chasing or net confinement were employed to model CUS. These approaches were validated by a parallel behavioural analysis. The results revealed that glycolytic and gluconeogenic enzymes as well as proteins involved in hypoxia had significantly altered synthesis rates in response to induced stress. This thesis describes for the first time a proteomics approach to determine the rates of synthesis of individual proteins in the zebrafish and its application to investigate the effects of stress conditions on heart proteome dynamics.

No description available.