The aim of environmental enrichment is to alter the environment of a captive animal in a way that results in improved mental and physical welfare. The technique has been utilised effectively for many years for captive mammals in a variety of settings. However, until now it has never been considered as a way of improving the welfare of aquatic animals such as fish. Fish that are used in regulatory toxicology studies are at present maintained solely in barren tank environments. Little is known about how these types of environments affect the well-being of the animals residing there and whether they impact either physiological heath or behavioural repertoire. This thesis aims to address this gap in the knowledge regarding the potential for environmental enrichment to improve the welfare of fish used in regulatory toxicology. More specifically it looks at two types of enrichment and the effects of these on the commonly used model species, the zebrafish (Danio rerio). The first type of enrichment studied was glass rod structures of varying heights provided to increase tank complexity and provide refuge. The glass structures did not produce any quantifiable benefits in unstressed fish and appeared to delay the formation of stable social hierarchies. When fish were stressed by a period of chasing, the presence of the glass rods appeared to reduce the magnitude of the cortisol response. Whilst this could be viewed as a potential benefit, it was felt that it would not outweigh the costs of this type of enrichment. The second type of enrichment studied was provision of airstones. Again, no clear evidence was found that fish in tanks with airstones experienced an improvement in welfare. The main observation was the vast increase in mortality in tanks containing these airstones, in particular, those of a smaller size. Regardless of the physiological cause underlying this result, this can only be viewed as a negative consequence and one that appears to rule out airstones as an effective form of enrichment for this species and strain of fish. It was also observed that both stress and the presence of enrichment influenced the absolute deviation from the mean in several endpoints. Since changes in endpoint variation will have effects both on the number of animals required to statistically measure environmentally relevant effects this is a factor that should be considered when researching methods of environmental enrichment. Finally, results from these studies suggest the possibility that laboratory zebrafish do not require the addition of environmental enrichment to tanks in order to promote maximum welfare. Furthermore, as considerable costs would be involved in implementing many types of enrichment (relating to manufacture, cleaning, incompatibility of results with previous studies etc.) it is likely that observed benefits would have to be both substantial and well established in order for changes in regulatory guidelines to take place. For a species such as zebrafish that are extremely easy to breed and maintain in the laboratory with minimal amounts of disease, social problems or mortalities, it may be that current conditions are satisfactory.

Three new NILT genes were successfully cloned and characterized from rainbow trout, with one NILT alternatively spliced into a long isoform containing two Ig domains and a short isoform containing one Ig domain.  The expression of NILTs was studied in six different tissues and two different cell lines, with expression apparent in immunologically important tissues.  Furthermore, phylogenetic analysis showed that NILTs are more closely related to triggering receptor expressed on myeloid (TREM) cells and Nkp44 from humans than to NITRs from rainbow trout. The genomic organisation and structure of the multigene family of NILTs in Atlantic salmon was investigated using a BAC sequencing approach.  This revealed the presence of six novel NILT genes, which either contained one or two Ig domains and several immunoreceptors tyrosine-based inhibitory motifs (ITIMs) in the cytoplasmic region. By homology search two NILT-like genes in zebrafish (<i>Danio rerio</i>) located on chromosome 1 have been obtained. Chromosome 1 in zebrafish also contains the <i>Dare</i>-ZE genes, which are equivalent to the human MHC class I genes located on chromosome 6.  The distance between the later and the TREM genes on chromosome 6 is similar to the distance between the NILT-like genes and <i>Dare</i>-ZE genes on zebrafish chromosome 1.  In addition, two NILT-like Ig domains were obtained from the green spotted pufferfish (<i>Tetraodon nigroviridis</i>), putatively part of the same receptor. The results will contribute to our knowledge of the immune system in fish and provide useful information for the control of inflammatory processes in salmonids.

Monoclonal antibodies (mAbs) have been essential tools in the elucidation of the immune system of mammals, and their application to identify surface molecules on leukocytes have allowed important functions of these cell to be identified (such as receptors that bind antigens, ligands involved in cell to cell signaling or in initiating immune response activity). Not only have mAbs been used to discriminate cells during different stages of cell development, but have also assisted in understanding the dynamics of molecules expressed during functional processes. Such molecules detected on human leukocytes are called human leukocyte differentiation antigens or HLDA. In order to group the antibodies that detect similar molecules and have similar patterns of reaction, immunologists have organised the mAbs that bind to these antigens into Clusters of Differentiation (CD). So far, there are about 350 leukocyte surface molecules detected by mAbs with a CD nomenclature for human leukocytes (www.hcdm.org). In fish immunology there is a great need to produce mAbs that are able to differentiate the various components of the fish immune system to assist in the elucidation of the fish immune system. The present study was an endeavour to develop and characterise mAbs that could be accredited to such scheme. A better understanding of the fish immune system is urgently required so that effective strategies of control can be developed for significant diseases during fish farming. Monoclonal antibodies were prepared by immunizing mice with thymic leukocytes from rainbow trout. The leukocytes were activated with the lectin Concanavalin A to promote the activation and proliferation of the target T-cell population. The selection of clones producing antibodies during screening was performed on the basis of the response of the supernatant from hybridomas using three consecutive assays. First, selection was determined by the positive staining of cells from the thymus in a Dot blot assay. Secondary screening was performed by means of flow cytometry (FCM) and the criterion for selection was the preferential detection of leukocytes gated in the lymphocyte region. Finally, the positive supernatants from hybridomes were evaluated to determine their effectiveness in the detection of modifications in the labelled cells during a multiple way activation by detection of foreign histocompatibility complex enhanced with mitogens. Monoclonal antibody TcOm15 was selected from 564 hybridomas produced and then used to stain cells from various Rainbow Trout tissues. It was clear from FCM, microscopy and Western blot analysis that mAb TcOm15 not only reacted with thymic cells but also with cells from other tissues. Differential staining of cells with mAb TcOm15 was observed with 27.1 ±1.4 % of leukocytes from peripheral blood leukocytes (PBL) stained in comparison to 2.0 ±0.2 % from the thymus, 13.8 ±0.4 % from the spleen, and 5.6 ±0.6 % cells stained from head kidney. The labeled cells showed characteristics of lymphocytes and monocytes, presenting a distinctive staining in immunohistochemistry and confocal microscopy. Western blot analysis, using electrophoresed proteins under denaturing conditions with leukocytes from several different tissues, showed that mAb TcOm15 did not detect a single protein. At least three proteins appeared to be identified by the mAb at 105, 160 and 200 kDa. The proteins were identified as α Actinin-4, non-erythroid Spectrin αII chain or Ig-like protein and non-muscle Myosin (MYH10) by MALDI-TOF analysis. Three of these identities are for compositional molecules for the cytoskeleton of different types of cells, and one it is associated to immunoglobulin superfamily. The identification of these proteins by mAb TcOm15 suggests an ability of this mAb to detect a specific function, possibly related with the synchronicity of expression or interaction of cytoskeleton-membrane proteins forming a multiprotein complex. Another possibility is as a carrier role for a protein during interactions. Colocalization of the mAb with F actin from the cytoskeleton was also observed suggesting the possibility that mAb TcOm15 detects a specific site in a multi-protein complex from the cytoskeleton. The molecule detected showed down-regulation in a dose dependant way with Concanavalin A and the expression was almost lost following stimulation of cells with phorbol 12-myristate 13-acetate stimulation. Leukocytes from the PBL and thymus up-regulated the expression of the TcOm15 molecule under mitogenic conditions in vitro, and results from in vivo experiments suggested the possibility of up-regulation on thymic cells. In conclusion, the results obtained in the present study provide information on a potentially useful marker (mAb TcOm15) for a cytoskeleton-membrane antigen that is modulated during stimulation of teleost lymphocytes. Additionally, this may enable insights into the relationship between cytoskeletal proteins and membrane associated immunoglobulin. Future research is necessary in order to explain this relationship and to determine the functional participation of the TcOm15 molecule during the activation of rainbow trout cells.

Photoperiod manipulation is routinely used in the aquaculture industry with the aim to enhance growth by manipulating the timing of reproduction in several commercially important temperate fish species. However, there are clear gaps in our understanding of how photoperiod is perceived by the circadian axis and transmitted to the brain to alter reproduction. Furthermore, due to the wide range of environments inhabited by fish, it is unlikely that one single organization exists. It is therefore believed that comparative studies of temperate species “models” with tropical species such as the African catfish (Clarias gariepinus) that adapted to different environments characterized by weaker light signals can help in such an aim. A number of studies were therefore performed in this PhD project to expand our knowledge on circadian biology and environmental physiological effects in African catfish. The first aim was to characterize the circadian melatonin system in this species (chapter 3). Results clearly showed that the control of melatonin production by the pineal gland was very different in the African catfish as compared to temperate species such as salmon and trout. Indeed, melatonin production appeared to mainly depend on light stimuli perceived by the eyes as opposed to salmonids where light directly perceived by the pineal gland regulates its own melatonin production within photoreceptors. The main evidence was obtained in ophthalmectomised fish that were unable to synthesize and release melatonin into the blood circulation during the dark period. This was the first time that such a decentralized organisation, similar in a way to the mammalian system, was found in any teleost species. In vitro results also supported such findings as African catfish pineal glands in isolation were not able to normally produce melatonin at night as usually seen in all other fish species studied so far. This indirectly suggested that pineal gland photo-sensitivity might be different in this tropical species. Further studies were performed to better determine the amount of light that can be perceived by the African catfish pineal gland depending on light transmittance though the skull (where the pineal gland is located). Surprisingly, it appeared that catfish cranium act as a stronger light filter than in other species resulting in lower light irradiance of the pineal gland. This could explain, although it still needs to be further confirmed, why African catfish photic control of melatonin produced by the pineal would have evolved differently than in temperate species. The work then focused on better characterizing diel melatonin production and endogenous entrainment through exposure to continuous photic regimes (continuous light, LL or darkness, DD) (chapter 4). Daily melatonin profiles of fish exposed to 12L:12D photoperiod (routinely used in indoor systems) confirmed low melatonin production at day (<10 pg/ml) and increase at night (50 pg/ml) as reported in most vertebrate species studied to date. Interestingly, results also showed that melatonin production or suppression can anticipate the change from night to day with basal melatonin levels observed 45 mins prior to the switch on of the light. These observations clearly suggest the involvement of a clock-controlled system of melatonin secretion that is capable of anticipating the next photophase period. Furthermore, when constant light (LL) was applied, day/night melatonin rhythms were abolished as expected due to the constant photic inhibition of AANAT activity (e.g. one of the enzyme responsible for the conversion of serotonin into melatonin). However when fish were exposed to constant darkness (DD), a strong endogenous melatonin rhythm (maintained for at least 4 days and 18 days in catfish and Nile tilapia respectively) was found, demonstrating once again the presence of robust circadian oscillators in this species. The next aim of the doctoral project was then to investigate circadian behaviour of catfish through locomotor activity studies (Chapter 5). African catfish is again a very interesting “model” due to its reported nocturnal activity rhythmicity as compared to most other teleosts species. Locomotor activity is considered as a very useful tool to elucidate the mechanisms of circadian organization in both invertebrates and vertebrates circadian. Results first confirmed the nocturnal activity rhythms in the species. Furthermore, clear circadian endogenous rhythms were observed under constant light (LL) or darkness (DD) during several days before losing rhythmicity. Interestingly, the activity levels varied depending on the stocking density. Finally, the last aim of this project was to test the effects of a range of photoperiodic manipulations on growth performances, sexual development and reproductive performances in African catfish reared from eggs to puberty. Results did not show any differences at the early sages (up to 90 days post hatching) in growth performances nor mortality (high) between control 12L:12D and LL treatments. In contrast, during the juvenile-adult period (from 120 to 360 DPH), significant growth effects were observed, as previously reported in other catfish species, with fish under LL displaying lower growth rate, food consumption and feed conversion efficiency in comparison to most other treatments (12:12, LL, 6:6, 6:18, 12-LL and LL-12) especially 12l:12D. However, no major effects of the photoperiodic treatments were observed with all fish recruited into puberty and developing gonads although differences in the timing of gametogenesis could be observed, especially a delay (circa 2 months) in females exposed to short daylength (6L:18D and 6L:6D). As for egg quality, egg diameter was the only parameter to differ between treatments (slightly larger in egg batch from LL treated females). Overall, none of the photoperiodic regime suppressed maturation in African catfish as opposed to some temperate species. The work carried out during this PhD project clearly advanced our understanding of circadian rhythmicity, light perception and effects of photoperiod on physiology in a tropical species. Future studies are now required to further characterise the circadian system and link it to evolutionary trends within vertebrates.

From Introduction: Rainbow trout (Salmo gairdneri) were introduced into Europe from North America in the latter half of the last century. They can tolerate higher water temperatures and lower oxygen concentrations than the native brown trout (Salmo trutta). Rainbows grow faster than brown trout under similar conditions and are thus particularly attractive for artificial rearing methods. In Denmark there is a thriving rainbow trout farming industry producing about 9,000 metric tons annually which is largely exported for table use (Mills 1971). In Britain production of rainbow trout for food is not on such a large scale but they form the basis of a considerable sport fishery. In Scotland and Northern England although rainbow trout reach sexual maturity they do not generally breed so the population is entirely dependent on restocking with hatchery reared fish, thus although living more or less wild in many British waters , this species is essentially an artificially managed resource upon which man can impose genetic selection (Donaldson and Olson 1957) as well as normal fishery controls. A detailed understanding of the biology and physiology of this species is hence of particular importance.

The fornical tract, a major input-output pathway of the hippocampus, of the primate brain makes crucial contributions to visual memory, as effects after surgical or aetiological lesions of this tract are widely documented in the monkey and human literature. Here, a series of experiments sought to further elucidate the functions of this structure with a battery of novel tasks in macaque monkeys, conducted either on a touchscreen or in an ambulatory chamber, so as to offer a more global view of the mnemonic role accomplished by it. After receiving bilateral transection of the fornix, monkeys are impeded in the 'fast learning' phase of a large number of new visuospatial conditional problems, with major impairments seen in eliminating non-perseverative errors. These fornix transected monkeys are however facilitated in the initial acquisition of a visuovisual conditional task, with facilitation seen in their improved ability in eliminating perseverative errors. It is also demonstrated in an ambulatory apparatus, in comparison to control monkeys, these monkeys are impaired in the new learning of visuospatial context of environments, albeit still displaying intact locomotor and exploratory behaviour patterns. Contrary to the relatively clear role in new learning, the involvement of the fornix in memory retention over the very long-term is unknown. It is shown here that once some visuospatial information is learnt; the fornix is no longer implicated in the retention of the material. The effects of fornix transection are also found to be detrimental on a spatial recognition task, with impairments observed in acquisition of the more demanding stages of the task. The overall results covered in this thesis support previous work suggesting that the fornix mediates the new learning of visual information, and I further propose that this fornical involvement lies primarily in the learning of spatio-temporal contexts, particularly during 'fast learning', as well as in task-sets acquisition. I also argue for dissociation in the contributions of the fornix and hippocampus to some memory processes in the macaque.

Scottish red deer (Cervus elaphus scoticus) are smaller in size and mature later than many continental subspecies. Previous investigations at the Rowett Institute have shown that when Scottish red deer are fed well they grow almost as large as European deer, so the differences between subspecies appear principally determined by the environment. To test this, one group of 6 stag calves was fed to appetite while a second group of 6 was offered 70% as much (on a metabolic body weight basis) during the winter and both groups fed to appetite during the following summer. After the first winter the restricted group were lighter and skeletally smaller than the unrestricted group. Despite compensatory growth during the summer significant differences in weight and size remained at the end of the period of feeding to appetite. This pattern was repeated during the second and third years of the study, Plane of nutrition bad little effect on size and composition of antlers but had profound effects on the timing of growth of the first antlers. Although the restricted stags were less fat than the unrestricted stags both groups showed the same relationship of fat content of the body to empty body weight. The restricted plane of nutrition delayed the growth and moult of wool and hair but did not affect length of either. The restricted plane of nutrition exerted only slight effects on levels of testosterone, oestradiol 17 beta,prolaotin and thyroriue and no effect on the timing of their cycles. A field study on the effect of winter undernutrition on stag calves at Glensaugh Deer Farm gave similar results. Cycles of body weight and food intake were shown to be of smalleramplitude in captive hinds than stage. Superior cervical ganglioneotomy of Soay rams diminished the amplitude of cycles of food intake and growth as much as cycles of reproduction but failed to abolish either.

For the largely immature population of crabs studied no difference in length weight relationships could be detected between males and females unless fresh weight was used for comparison. Females and immature males showed isometric growth throughout their life while mature males showed allometric growth. The use of length, fresh weight, dry weight and ash-free dry weight for the basis of comparative metabolic studies is discussed. Oxygen consumption rate was found to be related to the 0.799 power of body dry weight or ash-free dry weight for both males and females and resting and active rates. Scope for activity was constant at 3.7 across the entire body weight range. A marked diurnal rhythm of oxygen consumption rate was found, the exact form of which depended on season being bi-modal in short day length and uni-modal in long day length. The beat rates of heart and scaphognathite were found to show a high degree of correlation throughout all phases of the daily cycle. A typical short period rhythm of beat rates is shown during the resting phase. Ventilation volume showed a linear relationship with scaphognathite beat rate up to active rates of pumping. Above this ventilation efficiency was impaired. The short period rhythm had a period of almost exactly twenty minutes, and was divided into three phases, the high-rate, the low-rate and the changing phase. In order to conserve metabolic stores, the duration of the low-rate phase is increased, at the expense of the level and duration of the high-rate phase, with increasing starvation. Percentage utilisation of oxygen was at its highest during the high-rate phase (40 - 42%) and it appears that efficient oxygen exchange is limited to the rates of pumping of the active phase. Possible functions for the short period rhythm are discussed in the light of this observation. Haemocyanin was more or less absent from the local population of Cancer but not Carcinus and Portunus. Reasons for this are discussed. A new method for measurement of haemocyanin oxygen capacity was used. Effectiveness of oxygen uptake by the blood was low due to the lack of haemocyanin, but effectiveness of oxygen removal from the ventilatory water was high due to a decrease in ventilation:perfusion ratio. Ventilation:perfusion ratio did not change greatly with increased starvation. The physiological significance of these measurements is discussed.

Zooplankton are the major primary consumers in pelagic ecosystems, providing the principal pathway for energy transfer from primary production to higher trophic levels. The marine copepod Calanus finmarchicus is an important component of the pelagic food web in the North Atlantic and peripheral ecosystems, and forms an essential dietary component of a number of commercially important fish. As part of its life cycle, many C. finmarchicus overwinter in a diapause phase (a dormant overwintering phase where development is suppressed in adaptation to the seasonal food supply) at depths of 500 to 2000 m, but little is known about the triggers that initiate and terminate diapause, or the internal processes associated with these triggers. Understanding these processes is important, given that subtle changes in the environmental conditions which may affect diapause could have consequences for the entire Calanus-based ecosystem. In this study I took advantage of relatively easy access to a deep (> 100 m), isolated population of C. finmarchicus in Loch Etive (a sea loch on the west coast of Scotland) to sample Calanus finmarchicus monthly between April 2006 and June 2007 and measure lipid dynamics and gene expression associated with diapause. Chapter 1 of this thesis provides a general introduction to diapause and Calanus finmarchicus, Chapter 2 reports on the population of C. finmarchicus in Loch Etive, Chapter 3 reports changes in the lipid class and fatty acid composition of individual copepods, Chapter 4 reports on differential gene expression between diapausing and active C. finmarchicus and Chapter 5 provides a general discussion and puts this research into context. This study provides some initial insight into possible gene expression patterns, but further work is needed to attribute specific gene expression patterns with initiation and termination of diapause.

This work presents an advanced modelling procedure, which applies both structural modelling and kinetic modelling approaches to the trypanothione metabolic network in the bloodstream form of Trypanosoma brucei, the parasite responsible for African Sleeping sickness. Trypanothione has previously been identified as an essential compound for parasitic protozoa, however the underlying metabolic processes are poorly understood. Structural modelling allows the study of the network metabolism in the absence of sufficient quantitative information of target enzymes. Using this approach we examine the essential features associated with the control and regulation of intracellular trypanothione level. The first detailed kinetic model of the trypanothione metabolic network is developed, based on a critical review of the relevant scientific papers. Kinetic modelling of the network focuses on understanding the effect of anti-trypanosomal drug DFMO and examining other enzymes as potential targets for anti-trypanosomal chemotherapy. We also consider the inverse problem of parameter estimation when the system is defined with non-linear differential equations. The performance of a recently developed population-based PSwarm algorithm that has not yet been widely applied to biological problems is investigated and the problem of parameter estimation under conditions such as experimental noise and lack of information content is illustrated using the ERK signalling pathway. We propose a novel multi-objective optimization algorithm (MoPSwarm) for the validation of perturbation-based models of biological systems, and perform a comparative study to determine the factors crucial to the performance of the algorithm. By simultaneously taking several, possibly conflicting aspects into account, the problem of parameter estimation arising from non-informative experimental measurements can be successfully overcome. The reliability and efficiency of MoPSwarm is also tested using the ERK signalling pathway and demonstrated in model validation of the polyamine biosynthetic pathway of the trypanothione network. It is frequently a problem that models of biological systems are based on a relatively small amount of experimental information and that extensive in vivo observations are rarely available. To address this problem, we propose a new and generic methodological framework guided by the principles of Systems Biology. The proposed methodology integrates concepts from mathematical modelling and system identification to enable physical insights about the system to be accounted for in the modelling procedure. The framework takes advantage of module-based representation and employs PSwarm and our proposed multi-objective optimization algorithm as the core of this framework. The methodological framework is employed in the study of the trypanothione metabolic network, specifically, the validation of the model of the polyamine biosynthetic pathway. Good agreements with several existing data sets are obtained and new predictions about enzyme kinetics and regulatory mechanisms are generated, which could be tested by in vivo approaches.