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The growth of aquaculture industry, and in particular of salmonids, has dramatically increased in the last decades to supply the growing worldwide demand for fish products. The expansion of aquaculture needs a good management to ensure the sustainability of this activity. In particular, improving feeds for aquaculture to enhance health and performance of reared fish is imperative for this industry. Molecular biological approacches can help interpret responses to new diets and immune responses. Ubiquitin ligases (UbE3s) play a pivotal role in the ubiquitin proteasome pathway of protein degradation and are markers that could be linked to health and performance of fish. Six UbE3s were characterized, 4 are related to muscle catabolism and two involved in NF-kB activation. Atrogin-1 and MuRF genes were increased following food deprivation and a proinflammatory stimulation. MULAN and Mul1b, are mitochondrial activators of NF-kB and the expression of both genes was increased following a proinflammatory immune challenge both in vivo and in vitro. These genes have been used as markers in dietary traits to understand responses. A transcriptome study using Atlantic salmon fed a commercially available functional feed designed for optimal health status compared to a standard feed was performed. Both the liver and muscle were chosen for the transcriptomic analysis. In liver, a number of process were found down regulated, including protein turnover and innate immunity. No significant alterations were found in muscle. To determine the effect of a high plant protein diet (PP), compared to a marine profile diet (MP) in Atlantic a transcriptomic analysis was performed. Fish grew equally well on both diets and no significant histological differences were seen in intestine. The PP diet induced tissue specific changes in gene expression, with the mid intestine showing activation of the adaptive immune response and protein turnover. In liver cell proliferation and apoptosis indicate cellular reorganization and also a general suppression of processes such as immune response was observed. In contrast muscle tissue showed reduced protein metabolism and decrease in immune gene expression suggesting less energy expenditure in this tissue. To assess fish response to Piscirickttsia salmonis, the etiological agent of the salmonid rickettsial seeticemia (SRS), a transcriptomic analysis was carried out. P. salmonis infection has profound effects on transcription in the head kidney, liver and muscle of salmon. Head kidney, liver and muscle tissue showed a decrease in expression of mRNAs related to acquired immune function indicating the pathogen is down regulating this response controlled in part by the inhibition of the G-protein signalling pathway, whereas the innate immune response was increased in head kidney. Liver and muscle also responded, including specific responses in each tissue type. These results improve understanding of the mechanisms by which this bacterium survives and replicates within host cells and may assist selection of molecular biomarkers useful for the development of a diagnostic tools, vaccines and therapeutants for the aquaculture industry. Using markers selected from the previous experiments, the effect of four different diets, a marine profile diet (MP/FO), a plant protein diet (PP/FO), a vegetable oil diet (MP/VO) and PP/VO diet, in Atlantic salmon were examined. Markers for protein and lipid metabolism and immune response were studied in mid intestine, liver and muscle of fish fed for 77 days the four diets. Small but significant changes were observed, but despite of this, fish fed all the four diets showed similar growth rates. Moreover, to study the possible effect of the diets on fish health, the expression of specific SRS markers was studied in head kidney and liver of fish fed the four diets and infected with P. salmonis. The SRS challenge demonstrated that all groups of fish were able to respond efficiently to the pathogen. In conclusion, these new diets may represent good alternatives to fish meal (FM) and fish oil (FO) diets indicating that a replacement of marine diets with vegetable sources is possible in Atlantic salmon without affecting neither fish growth and performance nor the health of fish. The findings presented in this thesis suggest that E3 ligases are good molecular markers, allowing to study performance and health of fish following feeding trial and pathogen challenge. In addition, transcriptomic analysis performed to select further key molecular markers involved in a variety of processes and improved our knowledge of fish physiology and immunology, providing useful information for the development of new alternative diets for aquaculture.

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Gastric emptying of Sprattus sprattus and Arenicola spp. (gastric lavage) and formulated pellets (X-Ray studies) was investigated in a range of sizes of whiting, Merlangius merlangus fed different meal sizes at different temperatures. Emptying of indigestible solids, barium sulphate spheroids and ballotini were also studied. Additionally stomach contents of whiting sampled from Red Wharf Bay, Eastern Anglesey, UK. were analysed to determine diet composition, diel feeding periodicity and feeding strategy of whiting. Daily and total annual intake of main prey taxa, sprat, sandeel and crab, were estimated. Finally feeding behaviour of whiting in captivity was monitored. With respect to the studies conducted under laboratory conditions, it was found that gastric emptying is best described by a linear function. The results indicated significant changes in gastric emptying rates with both prey type and size. The seasonal change in temperature did not yield a significant change in gastric emptying rate. Increase in diet surface area also did not alter the gastric emptying rate significantly. The results have also shown that in continuously- feeding whiting gastric emptying is affected by the second meal such that the arrival of the second meal is accompanied by rapid emptying of a small fraction of the first meal. Despite difficulties encountered with individual variation among whiting of similar size held under similar conditions, gastric emptying times (GET) for both natural food and formulated pellets can be adequately predicted by the equations: GET = 126.47 W -0.111 g 0.26 e -0.068T for the natural food, GET = 6.6 e -0.09T + 2.48 W0.566 e -0.044T for the formulated pellets; where W is the fish weight (g), S is the meal size (g) and T is the temperature (°C) suggesting that a similar meal size of formulated pellet is emptied faster than natural food, Sprattus sprattus. This was interpreted as evidence that natural diets may reflect a combination of 2 factors (a) the relative size of stimulation to the stomach (% distension) and (b) the existence of protective, fibrous skin which resists digestion. Barium sulphate spheroids of diameter 1 mm (ca 20 per g food) and ballotini of diameter 0.029- 0.049 mm (60-100 per 1.58g) were selectively retained by the whiting stomach, suggesting that these indigestible materials can not be used in gastric emptying studies in whiting. Under laboratory conditions with continuous food availability, medium size whiting (155.25±27.98 g) consumed 5.29 g sprat corresponding to 3.41 %bw (approximately 3 sprat, 1.88g each) at 14 °C. The return of appetite after a single meal was found to follow the gastric emptying curve closely. However if food is not offered directly, but made available through demand feeding, the whiting feed rhythmically (every 21 hours), under these conditions it was estimated that feeding activity returns when the stomach is 40 % full. It wa§ found that, in the wild, whiting prefer fish (Sprattus sprattus and Ammodytes spp. ) and crustaceans and only turn to the polychaetes as a third option, even when they are readily available, when preferred food is scarce. The availability of prey was found to be the main criterion in whiting feeding strategy. In March fish were dominant for most whiting, in August and September/October however, Liocarcinus spp. and Corystes cassivelaunus were the main prey items found in whiting stomach reflecting the availability of these prey species. A highly correlated relationship was found between predator (whiting) and prey (sandeel) length, however this was not noticed for sprat; whiting prey on sprat regardless of its length. An increase in feeding intensity was observed in the morning (August), in the evening (September/October), or at both times, crepuscular feeding pattern, (March). However, when feeding behaviour was investigated under laboratory conditions it was found that whiting is mainly a day time feeder with a slight increase in the morning and in the evening, but some feeding also occurs at night. Additionally a strong tendency was found for crustaceans to be found in fish captured during the daylight hours and fish at night in August and September/October. A linear model to estimate food consumption from the field samples was developed. F= (S2-S1) + 1.5 K T, where S2 and SI are average stomach contents at time tI and t2, K is the gastric emptying rate (gh-1) and T is temperature (°C). The average recruited whiting stock in the area of approximately 150 km2 in Red Wharf Bay was estimated to be 129,000 with a total biomass of 22 tonnes. It was estimated that young whiting (groups 0+ to 3+ years) in Red Wharf Bay eat between 1.29 and 6.57 g day-1 in February. By August, when temperature has maximised, these values increase by approximately 70-80 %. Daily intake of sprat, sandeel and crab was estimated to be 0.41,0.14 and 0.14 g for the 0+ group. This was increased to 0.70,0.80 and 0.94 g by the age of 4. On a daily basis the long-term average of the amount of sprat, sandeel and crab consumed were estimated to be 820, 370 and 520 g/km2/day respectively. Total food consumption per year by the recruited whiting stock in the area studied however was 44.8 tonnes of sprat (approximately 11.5 million sprats), 20 tonnes of sandeel and 28.5 tonnes of crab. These figures were compared with the available data from the North Sea as well as from other parts of the Irish Sea and it was concluded that whiting in the Irish Sea eat more than they do in the North Sea.

<I>Gyrodactylus salaris</I> Malmberg, 1957, a monogenean parasite of salmonid fish, has caused the death of up to 95% of salmon parr in 37 Norwegian rivers. In order to prevent further spread of this parasite, a reliable method of identifying <I>G. salaris</I> and distinguishing it from other closely related species is required. This study, the first investigation of <I>Gyrodactylus</I> genetics, has demonstrated that DNA technology can provide methods of gyrodactylid species identification suitable for routine use. DNA was extracted from <I>G. salaris</I> and two other species common on salmonid fish; <I>G. derjavini</I> and <I>G. truttae</I>. The small subunit ribosomal RNA (srRNA) gene was amplified from this DNA by polymerase chain reaction (PCR). The complete nucleotide sequence of the srRNA gene from <I>G. salaris</I> was determined. This was used to predict a secondary structure for gyrodactylid srRNA and to construct molecular phylogenies of platyhelminths including <I>Gyrodactylus. </I>Fragments of the srRNA gene from each species were compared by Denaturing Gradient Gel Electrophoresis. Mobility differences in <I>G. truttae</I> fragments were found and one fragment showed variation between and within species. The V4 region of srRNA was amplified from single specimens of gyrodactylids using a combined lysis and PCR reaction and sequenced. Examination of these sequences enabled prediction of Restriction Fragment Length Polymorphisms (RFLPs) between species and the design of oligonucleotide probes specific for each species. Digestion of the srRNA gene V4 region with two restriction enzymes produced restriction fragment polymorphisms that can be used to discriminate between <I>G. salaris</I>, <I>G. derjavini</I> and <I>G. truttae</I>. The digoxigenin labelled oligonucleotides GsV4, GdV4 and GdV4 are specific for <I>G. salaris, G. derjavini</I> and <I>G. truttae</I> respectively and can detect PCR amplified DNA from single specimens in dot blots. Both RFLP and probe methods of identifying <I>Gyrodactylus </I>species are suitable for use in diagnostic laboratories.

Singular or dual dietary depletions of vitamins C and E in Atlantic salmon parr increased susceptibility to Aeromonas salmonicida challenge. An array of immune parameters were evaluated to identify the involvement of these vitamin depletions on the immune response. Dietary vitamin E levels in salmon had no impact on haematological parameters, total serum protein or lysozyme levels. Similarly, dietary vitamin E levels did not affect leucocyte antibody production, macrophage activating factor (MAF) release and respiratory burst (RB) phenomena. However, haemolytic and opsonic properties of complement were compromised in vitamin E depleted salmon. Parenteral administration of vitamin E to vitamin E depleted carp did not elevate phytohaemagglutinin (PHA) lymphocyte proliferation responses or complement activity. In vitro additions of vitamin E to lymphocytes from carp fed a commercial diet did not elevate PHA proliferation responses either. The increased disease susceptibility provoked by dietary vitamin C restriction in Atlantic salmon was not correlated with serum protein levels, differential leucocyte numbers or phagocyte functions as tested by RB activity or phagocytosis by macrophages. Lymphocyte functions were operational in these fish as examined by MAF secretion and antibody production. Analogous to vitamin E depletion, dietary vitamin C restriction in salmon compromised complement haemolytic activity. Elevating the vitamin C content of diets above normal levels enhanced complement activity in salmon. Vitamin C was a potent modulator of rainbow trout leucocyte functions. In vitro supplementation of vitamin C in the sodium (NaAsc) or polyphosphate (PPAsc) form was required for PHA-proliferation responses. MAF secretion was also augmented by in vitro additions of NaAsc to leucocytes obtained from vitamin C depleted trout. Injecting NaAsc into depleted fish elevated PHA proliferation responses compared with saline-injected controls. Leucocytes from the latter group could recover proliferative responses to levels associated with NaAsc-injected fish with in vitro additions of 1x10-3 and 1x10-4M PPAsc. Increased disease susceptibility, reduced complement activity and haematocrit values were symptomatic of combined dietary vitamin C and E depletion in salmon parr. Total and differential leucocyte numbers, or serum parameters including antiprotease activity and total protein level were unaffected by this dietary regime. Although poorly adherent, macrophages obtained from dually depleted salmon had similar RB values and expressed greater responsiveness to a MAF-containing supernatant than vitamin sufficient counterparts. Lymphocyte functions were impervious to dual vitamin depletion as antibody and MAF production responses were intact. In vitro additions of NaAsc and PPAsc were shown to elevate RB responses in macrophages from vitamin-restricted and -adequate salmon. MAF secretion was demonstrated to be a temperature-dependent phenomenon in rainbow trout leucocytes. After 48h acclimation at low in vitro temperatures (6 C), leucocytes obtained from trout at 14C expressed impoverished MAF production. Acclimation of trout to 7 C did riot rescue MAF production. Also, normal RB activity was reduced in macrophages obtained from fish at 14 C after 48h at 6 C in vitro. However, if allowed to acclimate, these macrophages become more responsive to MAF and recovered RB activity to levels associated with macrophages held at 10 and 18C. RB responses of macrophages from fish at 7 C functioned equally well across a wide range of in vitro temperature regimes. Phagocytic activity of macrophages obtained from fish at 14 C and placed at 6 C in vitro for 48h expressed temperature sensitivity. This was especially apparent in fish fed a dietary ?-3/?-6 fatty acid ratio of 2.0. However, this dietary regime also elevated phagocytic activity of these macrophages to such an extent, that their response surpassed that of macrophages from fish fed commercial diets or ?-3/?-6 ratios of 0.5 and 1 even at low in vitro temperatures.

No description available.

The initial aims of the present study were to investigate the problems associated with salt water transfer in Sarotherodon spp. with the objective of maintaining high rates of survival and satisfactory growth rates. Members of the euryhaline commercially cultured Sarotherodon species viz. Sarotherodon aureus, S. mossambicus, S. spilurus, S. niloticus and the hybrids of S. aureus/S. niloticus were used. The specific salinity tolerance and the capability of tolerating direct transfers to specified salinities and the comparative abilities of surviving gradual increases of salinity up to full strength sea water were investigated. The involvement of the plasma osmotic concentration in the osmoregulatory process, and the physiological changes following the direct transfer to salt water were examined with special reference to the possibility of using changes of plasma osmotic concentration as an indicator of fish transferability. S. aureus proved to be best able to withstand salinity changes, though in all cases gradual transfer was required to limit mortalities. Feeding of high dietary sodium chloride diet was evaluated for S. mossambicus and S. aureus/S. niloticus hybrids as a method of stimulating osmoregulatory process prior to salt water transfer. This was found to alleviate only slightly the osmotic stress following direct transfer to a known lethal salinity. Water deterioration and temperature fluctuation during fish transportation are inevitable, especially after long journeys. The effects of water quality deterioration, temperature fluctuation and food deprivation, typical of transport conditions, were studied in combination with salinity transfer effects. Effects were significant in all cases though, of the species studied, niloticus appeared to show the highest resistance to the combined effects. Following full acclimation to full strength sea water, the effects of prolonged exposure to sea water on the subsequent survival, growth rates and food conversion were investigated. S. niloticus and the hybrids of S. aureus/S. niloticus were found to be less tolerant to long-term exposure than the other candidates, which were selected for further detailed study. Light and electron microscopic studies of the gills and chloride cells were carried out in S. mossambicus and S. spilurus. This study showed the modified role of the chloride cells in fresh and sea water environments. Increases in number and developments of the ultrastructure of these cells were observed in sea water adapted samples from both of the species. Consequently the significance of the chloride cells in the osmoregulatory process was discussed. The significance of these results was considered in terms of aquaculture practice and suggestions are made for improved transfer methods in this context.

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