No description available.

No description available.

The initial aim of the work described in this Thesis was to investigate the lymphoid organ distribution, phenotype and function of the lymphocyte population identified by candidate anti-Xenopus natural killer (NK) cell monoclonal antibodies (mAb's). Since removal of the thymus gland early in larval life (thymectomy) results in the eradication of T-cells and subsequent increase in the proportion of candidate NK cells, thymectomised (Tx) Xenopus were integral in the study of this subset of lymphocytes. Phenotypic and functional studies respectively demonstrated that mAb-defined candidate NK cells do not belong to the B- or T-cell lineage and display cytotoxic activity towards MHC class-Ia-deficient tumour target cells, strengthening the contention that these cells represent the NK subset in Xenopus. The ontogeny of NK cells was investigated in relation to the emergence of the NK cell inhibitory ligand, MHC class-L Splenic NK cells were found to emerge in 6-7 week-old larvae (stage 56-58), which is ≈5 weeks after T- and B-cells become detectable, and some 2 weeks after MHC-Ia is first detected. However, these cells do not appear to be functionally competent until 6 months of age. The expression and ontogeny of recently cloned β2m (the molecule essential for MHC class-I expression) was also briefly investigated. β2m (both RNA and protein) was detectable in all adult tissues and cell lines, even class-I-deficient tumour cells; β2m transcripts were found in 5 week-old larvae that lack MHC class-I. The emergence of NK antigen on a population of T-cells following in vitro stimulation of splenocytes with PMA and calcium ionophore presented the opportunity to biochemically characterise (through immunoprecipitation) the mAb-defined NK antigen. Proteins precipitated using the anti-NK mAb were either surface labelled with biotin, or metabolically labelled with (^35)S. Both techniques resulted in the detection of a protein 55kDa in size.

No description available.

No description available.

Increased knowledge of the components of the turbot immune system, and the effects of environmental conditions such as water temperature, which influence the appearance of defense mechanisms would allow for the introduction of new strategies to cope with the problem of a high, variable mortality rate observed during the early rearing stages of turbot larvae. Partial sequences for a number of turbot non-specific and specific immune genes have been identified. These include partial sequences for transforming growth factor-beta 1 (TGFb1) (186bp), transferrin (492bp), interleukin-1 beta (IL-1b) (1025bp), recombinase activating gene 1 (RGA-1) (548bp) and immunoglobulin M (IgM) (606bp). Expression studies looking at the dose dependency of lipopolysaccharide (LPS) and polyinosinic:polycytidylic acid (poly I:C) induced gene expression in a turbot cell line (TFC) and turbot leucocytes was studied. The studies using turbot leucocytes show that a number of genes of the turbot immune system are induced in response to stimulation with LPS, these include IgM, lysozyme, transferrin IL-1b and TGFb1. The response to poly I:C was more selective with only the Mx gene and TGFb1 induced in response to stimulation. To determine the effects of the inclusion of exogenous nucleotides in aquaculture diets on the turbot immune system, a feeding study was carried out. When aided to normal fish feed formulations at a combined inclusion level of 0.03% these additional nucleotides were shown to increase the humoral immune response in terms of increased IgM, RAG-1 and TcRa gene expression. RT-PCR expression studies looking at immune gene development in turbot eggs and newly hatched larvae, and the effect of rearing temperature and stimulants was also carried out using the turbot specific primers. The results show that immune gene expression is detectable from early in development. Low rearing temperatures (10-12oC) had a detrimental effect on the development of immune gene expression.

This thesis describes the mRNA expression patterns of the Bone Morphogenetic Proteins (BMPs), downstream members of the BMP signal pathway, BMP antagonists and candidate target genes in the developing inner ear of wild type zebrafish. The crista Bmp expression pattern is conserved between four vertebrate species. However, unlike in chick, mouse and Xenopus laevis none of the hmps examined are macula markers in zebrafish. This thesis identifies sources of Bmp signalling (the cristae, the endolymphatic duct (ED) and the semicircular canals (SCC)) and possible sites of Bmp action (the cristae, posterior macula, SCC and the mesenchyme around the ED). It also provides the first description of the early stages of ED development, a structure only recently described at later stages in the zebrafish (8dpf), and two mRNA markers of this structure (bmp4 and dachA). In analysis of zebrafish mutants with defective cristae, the presence of cristae correlated with the expression of the hmps and msxc, a putative Bmp target. This suggests the Bmps are required to form cristae and express msxc. Gain and loss of function studies have also supported a role for the Bmps in the development of the posterior macula and SCc. Ectopic hBMP4 protein was applied to the otic vesicle via protein-coated beads. This inhibited the development of the posterior macula and SCC. However, these hBMP4 beads were not sufficient to induce the expression of ectopic msxc, generate ectopic cristae or rescue crista development in mutants. Beads coated in a BMP antagonist did not affect the development of endogenous cristae or the expression of endogenous msxc. Rescued swirl (bmp2b) mutant adult zebrafish exhibit a balance defect. Early stages of inner ear development in rescued embryos were found to progress normally up until 7dpf. However, it is not clear when the rescuing mRNA or protein degrades, and work done by others in the lab has shown that Bmp2b is required at later stages to form adult SCc. The ectopic hBMP4 experiments suggest that moderating levels of Bmp signalling may be required for normal development of the SCC at early stages.

The integumentary pattern of the plaice Pleuronectes platessa is divisible into the dark patches, the dark zones, the pale zone, the pale flecks, the white spots, orange and black spots and the black dots. These colour pattern-units differ in size and density and in the arrangement of their chromatophores. The ultrastructure of the chromatophores has been observed. The complex phenomenon of "pattern-matching" in the plaice is largely attributable to the differential responses of the upper dermal melan-ophores in the different pattern-units. The plaice responds to very small changes in the visual composition of its background: ; reflectivity; type of pattern; size of the figures comprising the pattern; proportion of black and white in the under-lying substratum; distance of the components of the pattern from both eyes. An analysis of these differential responses has been proposed. "Pattern-matching" in the plaice appears to be predominantly neurally initiated and is later assisted by its hormonal system. The chromatic responses of long white- and long black-adapted fishes are strikingly different from each other and from those of the fishes adapted to a middle tint. An account is given of the neurally controlled responses of the long white- and black-adapted fishes towards patterned backgrounds after their background reversal and their subsequent return to the original backgrounds. The effect of length of background adaptation on the neurally controlled responses of the plaice has been described. It has been suggested that in relation with its nervous activity, the plaice adapts to a new background in three phases: Phase I, II and III. The chromatic activity of intact and unilaterally enucleated fishes suggests the existence of a central integrating mechanism for most of the retinal information. Some retinal stimuli are capable of bringing about melanosomal movements'contralaterally.' Melanization of the unpigmented 'ventral' side of the plaice can be caused under experimental conditions. The pattern thus produced does not correspond with that of the 'dorsal' side. The neurally controlled responses of the 'ventral' melanophores suggest a mode of innervation somewhat different from that of the 'dorsal' melanophores. The differential responses of the melanophores in different pattern-units can be observed not only by a differential retinal stimulation, but also by the administration of some adrenergic drugs. An adrenergic innervation of the melanophores and a differential distribution of adrenoceptor-populations have been suggested to account for the differential responsiveness of these cells. Integumentary transformations in the plaice during background pattern adaptation may be brought about by the selective stimulation of melanophores in various pattern-units through nerve-fibres originating from a central integrating mechanism.

Recently, substantial attention has focused on the role of immunostimulants as prophylactic disease control agents in aquaculture. The purpose of this study was to investigate the effects of a range of putative immunostimulants on immune parameters and disease resisting capabilities in the rainbow trout, Oncorhynchus mykiss. Two plant derived immunostimulants, Ergosan and Chevimmun, were investigated of the modulatory effects on localised and systemic innate immune responses following intraperitoneal administration. With respect to localised effects, injection of ³1mg ml-1 Ergosan or 75% Chevimmun resulted in enhanced leucocyte migration, phagocytosis and Nitroblue Tetrazolium (NBT) reduction. In addition, Ergosan stimulated cytokine (IL-1b, IL-8 and TNF-a(2)) gene expression in peritoneal leucocytes. Of the humoral parameters studied, complement activity was augment following 1mg ml-1 Ergosan administration, whilst 10mg ml -1 stimulated antiprotease activity. Interestingly, Ergosan, but not Chevimmun, enhanced the resistance of trout to Aeromonas salmonicida and Viral Haemorrhagic Septicaemia Virus (VHSV). A third immunostimulant, recombinant trout IL-1b (rIL-1b) enhanced leucocyte migration, phagocytosis and bactericidal activity in vitro at concentrations ³10ng ml-1. In vivo studies demonstrated that intraperitoneally administered rIL-1b (typically 1mg/fish) could attract peritoneal leucocytes, particularly neutrophils, as well as enhancing the phagocytic activity of elicited cells. Finally, the two isoforms of trout rTNF-a, referred to as rTNF-a(1) and rTNF-a(2), were shown to function as chemoattractants in vivo, in addition to modulating the phagocytic response of head kidney leucocytes. The second isoform was the more potent inducer of these two important parameters. Conversely, rTNF-a(1), but not rTNF-a(2), increased bactericidal activity of trout macrophages exposed to an avirulent strain of A. salmonicida.

A variety of different functions have been attributed to the secretions of copepod exocrine glands. Such secretions have been suggested to possess, amongst other properties, hydrodynamic, predator deterrent, cuticle hardening and antifoulant activities. The nature of the secretion of a selected group of exocrine glands of the caligid copepod Lepeophtheirus salmonis was examined. Firstly, the number and pattern of distribution of the defined glands of L. salmon is were identified in all life-stages. In comparison, several species of parasitic, commensal and free-living copepods and other crustaceans were examined to determine the extent of the distribution of such glands within the Crustacea. Histochemical techniques and ultrastructural analysis of glandular tissues were utilised to suggest the probable biochemical characteristics of the gland contents and revealed that mucus and protein were components of the gland secretions. TEM analysis revealed a considerable mucus layer covering the body cuticle of L. salmonis. This layer was presumed to be derived from the exocrine glands of this species. To characterise the secretion more precisely specific enzyme assays, selective staining procedures and high performance thin layer chromatography (HPTLC) were employed. These techniques indicated that the peroxidatic enzyme catalase was present in the glands at significantly higher levels than in the general body tissues. This enzyme was shown to be contained within the secretory vesicles of the glands. Having positively identified an enzymatic component of the gland tissues, acrylamide gel electrophoresis was undertaken to specifically determine the molecular weight and quaternary structure of the enzyme. Further studies using this technique focused on identifying secreted enzymes of L. salmonis and correlated such proteins to those identified in samples of gland tissue. A four subunit, catalase of between 260 and 280kDa was demonstrated to be present in the secretions of L. salmonis. The final stage of the work tested the hypothesised functions of the secretions of the identified glands of L. salmon is. The gland secretions of L. salmonis were demonstrated not to possess an antibacterial activity against some common species of marine bacteria whilst the deliberate removal of the external mucus layer significantly affected the hydrodynamic characteristics of L. salmonis.