No description available.

The present investigation was carried out to study the inflammatory response of carp (Cyprinus carpio L. ) to various stimuli. The stimuli were; surgical wound, talcum powder, Freund's complete adjuvant (FCA), a bacterium Aeromonas hydrophila, and spores of fungus Aphanomyces invaderis. Following exposure to these stimuli, fish were sacrificed over a period of time, and sequential tissue samples were processed for histopathological examinations. The surgical wounds at 27°C, re-epithelialized at 4 hours. Macrophage infiltration began at 2 hours, and myophagia at 6 hours. Fibroplasia and muscle regeneration were initiated at 2 days. After 16 days epidermis was normal and dermis was completely linked. The wounded area restored its main components by 16 to 24 days after wounding. Injection of talcum powder at 26.5°C, and FCA at 24°C, produced chronic granulomatousin flammatory reaction. The following events started in both experiments at the same time; myophagia at 6 hours, macrophages had changed into epithelioid cells, active fibroplasia and muscle regeneration at 3 days. Regenerated muscles filled the defects by 14 to 28 days post-injection (p. i. ). Langhans and foreign body giant cells, were observed after 3 days in talc and 4 days in FCA experiment. New capillaries formed at 3 days in talc and 2 days in FCA study. At the end of the experiments (42 days) the encapsulation of the irritants was very advanced, and the granulomata were entirely surrounded by normal muscle tissue. Inoculation of the Aeromonas hydrophila at 27.5°C, provoked a lethal acute inflammatory response within 48 hours. The surviving fish showed well developed capacity for dealing with bacteria. An ulcerative wound developed by 48 hours in the surviving fish. Polymorphonuclear cells (PMNs) were observed at 1 hour p.i . and remained up to 7 days. PMNs were one of the dominant inflammatory cells and participated in myophagia and micro-abscess formation. The acute inflammation then developed into chronic inflammation characterised by fibroplasia which was active at 5 days. Process of wound healing began and developed by 5-10 days and was completed by scar formation at 28 days. A chronic inflammatory response occurred after inoculation of spores of the fungus Aphanomyces invaderis at 27°C. Limited growth of the fungus in tissue occurred in the early stages p. i. but was then halted by fish's defence mechanisms. Macrophages had changed into epithelioid cells at 3 days p. i. Presence of Langhans, foreign body and intermediate giant cells, muscle regeneration, fibroplasia, and vascularization also started at 3 days. Developing granulomata formed by 10 days, and fully matured granulomata were observed by 18 days.

No description available.

Cereal aphid (Sitobion avenae) and wheat bulb fly (Delia coarctata) are serious pests of wheat in the UK. At the present, chemical pesticides are used to control these insects, but they are limited in effectiveness, and have undersirable ecological impacts. There is a need to improve wheat genetically to be resistant to such inset pests. The objectives of this work were to investigate digestive biochemistry in the selected insect pests of wheat, and to determine effects of potential endogenous resistance factors in wheat on digestion, nutrition and other insect metabolic processes. The aim was to develop new strategies for crop protection. Digestive biochemistry in S. avenae and D. coarctata was studied to characterise gut proteases and their inhibition by host plant proteinase inhibitors (PIs). Investigation of proteolytic digestion in S. avenae gut showed that in spite of being a phloem-feeding insect, cereal aphid could digest ingested protein, using cysteine proteases. D. coarctata larvae contained mainly serine protease activity. A serine protease (DcSP) and a cysteine protease (DcCathL) from D. coarctata gut tissue were expressed as recombinant proteins. Only DcCathL was recovered in active form. DcCathL was insecticidal to Mamestra brassicae when injected into hemolymph, causing systemic and extensive melanisation. DcCathL selectively degraded recombinant serpins from M. brassicae in in vitro assays, and is suggested to interfere with regulation of the proteolytic cascade leading to phenoloxidase activation and melanin production in vivo. DcCathL has potential as a biopesticide if it could be made effective when orally delivered. A cationic amino acid transporter from D. coarctata gut (DcCAAT) was also cloned as a target for RNA interference. Potential resistance factors in wheat were characterised by expression as recombinant proteins. Two PIs from wheat (subtilisin/chymotrypsin inhibitor; WSCI, and cysteine proteinase inhibitor; WCPI) were expressed in the yeast Pichia pastoris, and purified. WSCI inhibited gut protease activity of both insects in in vitro and in vivo assays, whereas WCPI only inhibited S. avenae gut extract activity. On feeding, WSCI was antimetabolic to both insects, affecting both survival and growth, whereas WCPI was antimetabolic to S. avenae only. Wheat Hessian fly responsive (Hfr) genes are up-regulated in response to herbivory by Hessian fly (Mayetiola destructor). The protein product Hfr-3 was expressed and purified, and showed antimetabolic effects on survival and growth of both S. avenae and D. coarctata. Both accumulated and induced defence proteins, like WSCI, WCPI and Hfr-3, have the potential to act as endogenous resistance factors in wheat towards a range of insect pests. Developing a wheat variety constitutively expressing these defence proteins by using traditional breeding methods and/or modern biotechnological tools is discussed.

This thesis describes a number of experiments designed to further our knowledge of the complex relationship that exists between nutrition and the immune system of fish, with a view to finding a practical means of improving disease resistance in farmed fish. In particular, vitamins are known to play an important role in vertebrate immune responses, and in the present study the practical value of vitamin C as an immunostimulant was investigated. A study was also made of the capacity of vitamin C to ameliorate stress-induced immunosuppression, and of the effect of stress on the demand for and distribution of vitamin C amongst various tissues of Atlantic salmon (Salmo salar L). Taken together, the results of these studies revealed no significant evidence for a fundamental role for vitamin C in regulating the stress response of salmon, and no significant evidence that high vitamin C diets have any value as immunostimulants in aquaculture. The practical value of diets containing high levels of vitamin A and carotenoids were also investigated. Whilst there was some evidence that vitamin A deficiency resulted in increased disease susceptibility, there was no evidence to suggest that feeding elevated levels of these compounds was immunostimulatory. Hence, the practical value of these compounds in promoting disease resistance in aquaculture also appears limited. The immunomodulatory effect of differential food acquisition by individual fish kept in different ration groups (i.e. as determined by management feeding strategy), and also within those ration groups (as determined by social interaction) was also investigated. The results obtained indicate that feeding very high ration levels may have deleterious consequences for disease resistance in salmonids, a finding which may have major implications for the aquaculture industry. Overall, the results presented in this thesis have served to eliminate several avenues of research which were popular at the time of its conception, and to suggest new approaches which may lead to real improvements in disease resistance of farmed fish in the future.

The toxicities of several oil dispersants (solvent emulsifiers or 'detergents,) to the young stages of six species of commercially important marine fish, haddock, herring, lemon sole, pilchard, plaice and sole, have been examined. Details of the laboratory rearing and maintenance of the embryos and larvae of, those species are given. The chemical nature of dispersants and their behaviour in sea water are discussed in relation to toxicity testing. The main factors influencing the acute toxicity of a dispersant were the type and aromatic content of the solvent. Newer dispersants using straight chain alcohols or Water as solvents had very low toxicities. Ageing of dispersant solutions led to a marked decrease in toxicity and this decline could be related to the loss of aromatics from solution by evaporation or degradation. Temperature and salinity had only slight influence on toxicity. For all dispersants differences, of susceptibility between species wore less than differences at different ages within a species. The larvae of all species showed a similar susceptibility when newly hatched but the embryos (within the chorion) wore more resistant. Susceptibility increased throughout the yolk-sac stage and a similar increase was observed when feeding larvae were starved. The transition period from yolk reserves, to an external food supply Was most critical for once larvae had established feeding resistance increased up to metamorphosis. The dispersants appeared to act largely as physical toxins causing, initially at least, e reversible narcosis, Criteria other than survival were used to determine the presence and persistence of sub-lethal toxication. Treatment of developing embryos with dispersants gave rise to abnormalities in cell division, heart rate, eye pigmentation, growth rate and hatching success but did not affect the length of the incubation period. The treatment produced larvae with abnormal flexures of the spine which prevented the larvae from feeding successfully. Feeding experiments demonstrated that in the presence of sub-lethal concentrations the ability of larvae to capture food wan impaired. After returning to clean water they recovered but remained consistently less successful at food capture than untreated larvae. However plaice larvae reared to metamorphosis after a sub-lethal dosing showed no differences in survival, size condition or pigmentation when compared with controls. By using a thermistor technique to monitor activity it was possible to determine the effects of dispersants on the normal phototactic behaviour exhibited by larvae at high and low light intensities. Initially the dispersants caused an enhanced response but with continued exposure the activity of the larvae declined and a breakdown in the negative, then the positive phototaxis followed. Behavioural aberrations were detected at only one hundredth of the lethal concentration. The larvae quickly recovered the normal behaviour pattern when transferred to clean water. Larvae did not exhibit a chemotactic response to horizontal gradients of dispersants generated in a fluvarium but because the dispersants increased their swimming activity the larvae exhibited an avoidance of potentially lethal concentrations. Similarly the larvae swam into dispersants in horizontal gradients. They remained in high concentrations until they became narcotized when they sank into clean water and recovered. The ecological significance of the lethal and sub-lethal effects of dispersants on the eggs and larvae of marine-fish and the problems of extrapolation of laboratory results to field situations are discussed in the light of possible effects on future stock and recruitment.

Lectins have long been established as potential agents for use in control of insect pest species. In this study the use of garlic bulb lectins against phloem-feeding hemipteran insect pests is investigated. There is no Bacillus thuringiensis (Et) toxin available which is effective against hemipteran insects. Plant lectins have been shown to be toxic to some insects, including aphids, in particular lectins from garlic. Subsequently heterodimeric (ASAI) and homodimeric (ASAlI) garlic bulb lectins were cloned and e!Cpressed in 31 recombinant yeast system, Pichia pastoris. Recombinant lectins were successfully purified and demonstrated to be functionally active in vitro by haemagglutination assays and toxic to a range of hemipteran insects: pea aphid (Acyrthosiphon pisum), peachpotato aphid (Myzus persicae) and rice brown planthopper (N. lugens) in ~ificial diet bioassays. To analyse the interactions between recombinant ASAII and the gut of A. pisum a pulldown assay was developed to identify specific interactions between ASAII and solubilized A. pisum gut proteins. Proteins which interacted with ASAll were subjected to matrix-assisted laser desoprtion ionization time of flight (MALDI-TOF) mass spectrometric analysis, this revealed that ASAll bound to alanyl aminopeptidase N (alanyl APN), a major constituent glycoprotein of the aphid gut which is rich in mannose oligosaccharides, and membrane-bound sucrase, an enzyme important in the maintenance of osmotic balance in the aphid gut. It was not possible to establish whether transport of ASAll into the haemolymph of A. pisum occurred using a Western blotting approach due to the lack of immunoreactivity of anti-ASA antibodies to the low levels of ASAII expected to be transported. A fusion protein of ASAll-Avidin was created to enable the conjugation of biotinylated peptides of potential insecticidal interest to lectin carrier proteins. One such peptide, leucomyosuppressin (LMS) from cockroach Diploptera punctata, was conjugated to ASAII-Avidin and toxicity to A. pisum was demonstrated in artificial diet bioassays. It was confirmed that avidin is transported into the A. pisum haemolymph, suggesting that if ASAII was not responsible for transport of LMS in to the haemolymph then it may be mediated by avidin. Using a bioinformatic approach a putative A. pisum LMS gene was assembled.

No description available.

No description available.

No description available.