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The aim of this project was to study the mechanisms of disease resistance in plaice (<i>Pleuronectes platessa</i> L.). Within this broad area, the work concentrated on investigating the migration, phagocytic activity and luminol-dependent chemiluminescence (CL) of plaice phagocytic leucocytes <i>in vitro</i>. In these studies two sources of leucocytes were used: (i) glycogen-elicited, peritoneal exudate cells and (ii) kidney leucocytes that had been purified on a discontinuous Percoll gradient. Both cell isolates comprised of at least 70% neutrophils. Several factors were found to chemoattractants for both peritoneal and kidney neutrophils. Endogenous attractants included leukotriene B<SUB>4</SUB> and two neutrophil-derived factors, which differed in their solubilities in organic solvents. Exogenous attractants included the formyl-peptide, N-formylmethionyl-leucyl-phenylalanine, and an organic solvent soluble factor released by viable <i>Vibrio alginolyticus</i>. The interactions of these attractants and their <i>in vivo</i> activities were also investigated. The migration of neutrophils <i>in vitro</i> was significantly reduced in the presence of cortisol at levels equivalent to those found in stressed plaice (> 400 ng/ml). The opsonic activity of plaice serum was investigated by analysing the phagocytosis and CL of plaice neutrophils. Sheep erthrocytes were only opsonized by the presence of specific antibody and an activatable factor(s) found in normal serum. The bacterium, <i>V. alginolyticus</i>, was opsonized by a factor(s) in normal serum, the activity of which was not further enhanced by the presence of specific antibody. In fact, two different opsonins were identified in normal plaice serum: (i) a very heat-stable activity (stable at 70<SUP>o</SUP>C for 30 min) recognised by kidney neutrophils and (ii) a heat-labile activity (50<SUP>o</SUP>C for 30 min) recognised by peritoneal neutrophils. Plaice neutrophils were shown to produce several reactive oxygen species: superoxide anion (O<SUP>-<SUB>2</SUB></SUP>), hydrogen peroxide (H<SUB>2</SUB>O<SUB>2</SUB>) and their derivatives, possibly including hydroxyl radical (OH<SUP>bullet</SUP>).

The work described in this Thesis was conducted to investigate aspects of copper metabolism in the plaice Pleuronectes platessa (L). Three major aspects were studied. The first was to investigate and partially characterize the distribution of copper and copper-dependent enzymes in the various tissues of the plaice. The second was to study the effect of cadmium on copper-dependent enzymes as a base line for understanding the competition between an essential metal ion and a non-essential toxic metal ion. This has established a foundation for the interpretation of the toxicity of pollutants to a marine organism. The third and the major part of the investigation was to purify and characterize the copper-dependent enzyme, ceruloplasmin, and to compare it with the isolated enzyme from the mammalian system. The results from the first part of the investigation showed that (i) Copper is relatively evenly distributed in the plaice. Apart from brain which has the highest concentration of copper, the concentrations of copper in the other tissues generally reflect the copper-dependent enzyme activities. (ii) The presence of the copper-dependent enzymes in the plaice are confirmed. The assay system used for the mammalian enzyme systems apply reasonably well to the plaice enzymes. (iii) The limited study on the relatively crude preparations of copper-dependent enzymes show that they are similarly distributed in mammals and that they have similar properties. Cytochrome oxidase, however, has a higher pH optimum than that of the mammalian enzymes. Exposure of the plaice to sublethal concentrations of cadmium as cadmium chloride results in initial increase in ceruloplasmin, cytochrome oxidase, monomaine oxidase and tryptophan oxygenase activities followed by a decrease after a prolonged exposure. Changes in the copper-dependent enzyme activities are accompanied by parallel changes in the concentrations of copper in the serum and liver. Thus cadmium alters copper metabolism in the plaice.

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The fact that fish possess a sense of smell has been known for some time. Fabricus (1780) described how lampreys and sharks would follow rotting meat and Mono, (1744) demonstrated that fish would react to a worm in the water and show adaptation to its odour. Bateson (1890) showed feeding responses in both elasmobranchs and teleosts to hidden food and juices squeezed through cloth. These reactions are present in both seeing and blind fish. Copeland (1912)and Parker (1914) shoved that dogfish were able to localize hidden food by olfaction, thus proving that it could be a directional sense. Initially the study of chemosense had been performed by observing the occurrence of a definite feeding reaction either as a result of detecting food odour or to extend the type of stimulus studied when the reaction had been conditioned to another stimulus (Göz, 1941; Teichmann, 1959). Conditioning is a long process and requires a suitably hardy species. In many cases where a stimulus not connected with feeding is used to condition a feeding reaction we may learn of sensory acuity in absolute terms but nothing of the natural use of the reaction. To examine more subtle reactions to stimuli. especially those not related to feeding, specialized apparatus had to be developed. This allowed workers to look at how a response would be used by the fish in nature. As well as showing reactions to food and prey organisms fish have been shown to use olfaction in social behaviour both with other species and conspecifics of both sexes. Göz (1941) managed to condition a single, blinded, minnow Phoxinus phoxinus to show a feeding reaction to the odour of another fish species Ictalurus nebulosus not the prey of the minnow. This took many weeks using even the easily-trained minnow. Because of the difficulties and limitations many workers have adopted more direct approaches, Wrede (1932) found blinded minnows (a shoaling species) preferred to visit a compartment in an aquarium where the odour of a conspecific lingered rather than a control compartment. Hemmings (1966a) used a more complex preference trough derived from that of Shelford and Allee (1914) and found that the shoaling, freshwater roach (Rutilus rutilus) showed a preference for the end which had the odour of other roach. By analysis of movement in the trough he showed that this preference was due to increased turning rate. Investigating these pheromones in char, D6ving, Nordeng and Oakley (1974) used electrophysiological methods, recording electrical impulses from the olfactory tract, and found the char was able to identify racial differences in this social identifier. Doving,Enger and Nordeng (1973) proposed a component of mucus to be the pheromone. Interest in amino acids as a possible stimulus to feeding in marine organisms began when Steven (1959) found that glutamic acid produced a feeding reaction in two species of tropical marine fish. Case and (iwilliam (1961) found that a range of amino acids would stimulate a blinded crab to feed when applied to the cheiae This reaction was confirmed electrophysiologically on isolated dactyl preparations, responses being obtained from the dactyl receptors to a range of amino acids. Many workers including Hara et al (1973), Hashimoto et al (1968), Haynes et al (1967) and Suzuki and Tucker (1971) have found similar reactions from the olfactory nerves of teleosts. The long, easily accessible olfactory tract in some teleost species makes them ideal subjects for electrophysiological investigation. The technique of monitoring nerve impulses enables a large number of amino acids to be rapidly tested; the thresholds obtained however may not be those which will stimulate a feeding reaction and hardy species are needed. Most work on chemosense in teleosts has been performed on adult and juvenile fish; this is not surprising since rearing beyond the non-feeding yolk sac stage of many important marine species has only been successful in the last decade. The histology and morphology of the development of the olfactory system has also received little study since Holm (1894) described this process in Salmo salar. He showed that in Salmo salar, which took 90 days from fertilization to hatch, there was no nervous connection between epithelium and brain at 60 days post fertilization but one was found at 83 days post fertilization. The olfactory nerve appeared as the groove closed. Attention has been drawn to this lack of knowledge by Hasler (1957), Johnson and Brown (1962) and Branson (1963). In many of the teleosts so far studied the olfactory system is undergoing development not only for the whole of larval life but beyond into juvenile development. Larval development is a valuable time to study this, and indeed any organ system, since it is changing in physical structure, increasing in complexity and possibly changing in acuity and function. The role of a larva can be in many cases to give the early stages of an organism a different ecological niche from the adult, preventing intraspecific predation and competition for food. Therefore its senses may be used for different purposes to the adult, (for example the use of chemosense and touch in the settlement of Balanus nauplii (Crisp, 1974) a system obviously of no use to the adult). In the case of a nektonic shoaling fish such as the herring with a planktonic non-shoaling larva, there would be a possibility of larvae dispersing over a large area prior to onset of shoaling behaviour. There would obviously be some value in keeping larvae together in loose groups and it is likely chemosense may play some part in this. Aggregations of planktonic marine organisms are a well documented phenomena (Barnes and Marshall, 1951; Weibe & Holland, 1968) from the longevity of these aggregations it seems unlikely that this is caused by physical boundaries of water parcels. It seems possible that chemosense acts to keep aggregations intact and in some organisms is retained in adult shoaling life, perhaps to maintain aggregations when the shoals disperse at night (Harden-Jones, 1962). Feeding patterns in larvae and adults can also differ; for example herring and plaice larvae will snap at food organisms in the water column, taking selected individual prey; in the case of herring almost stalking. As adults, herring mainly feed by filtering, although evidence from other filter feeding fish suggest a proportion of particulate feeding will also occur depending on prey size and density (O'Connell, 1972), and plaice move along the sea bed eating epibenthic organisms, mollusc siphons and sedentary worms, Both modes of feeding require good vision. Newly hatched herring (Blaxter and Jones, 1967) and plaice (Blaxter, 1968a) have very different eyes from the adult, the eye developing throughout larval life. In Sardinops caerulea, Schwassmann (1965), the eye is a very rudimentary structure when feeding begins. Although the feeding act is visual, the volume which can be searched using sight alone is small (in herring 0.3- 2.0 litres per hour and in plaice 0.1-1.8 litres per hour (Blaxter and Staines, 1971). It seems possible that olfaction could assist in the search for food either by directionally guiding the fish larva to a concentration of food organisms or restricting energy-requiring searching behaviour to periods when food organisms can be detected by presence of their odour. With these possibilities in mind the outline of study below was adopted.

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Plankton sampling established the presence of a plaice spawning ground off the west coast of the Isle of Man in the Irish Sea. The total production of stage I plaice eggs from this spawning ground in 1993 was estimated at 7.6 x 101 • Theoretically this spawning ground could supply the local nurseries with plaice larvae. Spawning was most intense in March, before the peak in the plankton bloom in the Irish Sea. Predation on plaice eggs by clupeids was studied in March in an area of high plaice egg density to the east of the Isle of Man. Fish eggs formed the bulk of the stomach contents of sprat and herring due to the lack of alternative zooplankton food at this time of year. The later developmental stages of plaice eggs were more vulnerable to predation. This was thought to be due to the increase in pigmentation with embryonic development increasing the contrast between the egg and the water to predators that detect prey visually. Sprat and herring >80 mm showed a strong selection for plaice eggs over smaller pelagic fish eggs. However, the large eggs of plaice had a refuge in size from predation by sprat <80 mm. Smaller clupeids were feeding more actively than larger clupeids, as indicated by stomach fullness and the total number of fish eggs in stomachs. Stomach content data was combined with published biomass estimates and the daily instantaneous mortality rate of plaice eggs due to predation was estimated at 0.023 for sprat and 0.001 for herring. Sprat were therefore an important predator of plaice eggs in the Irish sea, whereas herring seemed of limited significance. Predation by fishes on O-group flatfishes was studied on a Scottish nursery ground by stomach content analysis. Fish predation was shown to be a significant source of mortality and I-group grey gurnards and gadoids were the major predators. Flounder suffered higher predation rates than plaice or dab due to the smaller size at settlement. Predation on plaice and dab was size-selective, concentrated on the smaller individuals. Smaller flatfishes were vulnerable to a greater size range and greater taxonomic range of predators. Predation by O-group cod on O-group dab was limited by the sizes of predator and prey. It was hypothesised that the times of recruitment of flatfishes and their predators to nurseries, and relative growth rates, would affect predation and hence mortality. The handling time of O-group flatfishes by O-group cod in laboratory experiments was positively related to flatfish size and negatively related to cod size. The handling time of plaice was longer than for dab of a similar size due to the difference in body shape. Profitability of flatfishes (wet weight gained per unit handling time) decreased monotonically with flatfish size over the range of prey and predator sizes used. It was therefore predicted that when O-group cod forage in the field the smallest O-group flatfishes would be the most profitable and would be behaviourally selected for. However, the selection of prey by cod was suggested to be determined by both behavioural and physical processes. There was evidence that behavioural selection of prey occurred at the ingestion stage of feeding. The current evidence for density-dependent processes in the juvenile stage of plaice was reviewed. Both the data in support of, and the processes thought to cause, density-dependent mortality in the juvenile phase were equivocal. It was hypothesised that density-dependent mortality occurs in the early egg stages of plaice and evidence, and the rationale for a potential predatory process resulting in such mortality, were presented.

No description available.