Thesis (M.Sc.)--Memorial University of Newfoundland, 2004. / Bibliography: leaves 106-115.

Atlantic cod (Gadus morhua L.) is one of the most promising new fish species introduced to cold water aquaculture due to the large established market in Europe and the USA and the decline in wild stock. So far, the production of farmed cod has been relatively low, with the main hindrance due to diseases. Vibrio anguillarum has been recognised as the biggest disease problem of farmed cod and has slowed the development of a successful cod aquaculture industry. When the first incidences of V. anguillarum occurred in cod aquaculture, vaccines designed for vibriosis in Atlantic salmon (Salmo salar L.) were used in an attempt to combat the disease. However, these vaccines did not provide sufficient protection, possibly because they lacked serotype O2b, which is known to affect cod and to a lesser extent salmonids. Recently, vibriosis vaccines specifically designed to protect Atlantic cod have been formulated, but outbreaks of vibriosis in vaccinated fish are still being reported, suggesting that these formulations are inadequate. The aim of this project was to develop a whole cell inactivated vaccine formulation specifically tailored to protect Atlantic cod against Vibrio anguillarum. The serological classification of V. anguillarum was first investigated by producing a set of monoclonal antibodies (mAbs). Using lipopolysaccharides (LPS) extracted with butan-1-ol, 4 mAbs were selected and shown to react specifically with V. anguillarum serotypes O1, O2a and O2b. A collection of over 150 V. anguillarum isolates were screened using these, which revealed that most of the isolates had been previously correctly classified. A new sub-serotype of V. anguillarum O2 was identified from isolates recovered from outbreaks of vibriosis in Norway as well as Scotland. This new sub-serotype was referred to as O2d since the subserotype O2c has been recently identified in vibriosis cases from Atlantic cod. However, it was shown that the O2c sub-serotype might not belong to the O2 serotype, but in fact belongs to another serotype. To protect Atlantic cod against all the V. anguillarum serotypes (and subserotypes) which they are susceptible to, it is recommended that isolates from serotypes O1, O2a, O2b, O2c and O2d should all be included in a bacterin vaccine for cod. In order to determine which isolates from each of the serotypes to include in the vaccine, a variety of virulence factors of V. anguillarum were investigated in vitro. The interaction of some candidate isolates from O1, O2a and O2b serotypes (O2c and O2d were not identified at the time this part of the study took place) with cod phagocytic cells were studied using flow cytometry. Phagocytosis and respiratory burst of cod macrophages and neutrophils as well as cod serum killing of V. anguillarum were quantified. It was found that isolates within the same serotype displayed varying degrees of resistance to phagocytosis and the subsequent respiratory burst activity as well as that all the V. anguillarum strains tested were resistant to Atlantic cod serum killing. These in vitro assays were found to be very useful in assessing the virulence of V. anguillarum. The isolate within each serotype eliciting the highest percentage of positive phagocytic cells was selected in order to increase the antigen presentation pathway, thus theoretically enhancing the protection elicited by the vaccine. A multivalent formalin-inactivated non-adjuvanted vaccine was prepared which included all the serotypes previously described and was injected intraperitoneally into Atlantic cod. A bath challenge was performed on vaccinated and mock-vaccinated fish, 6 weeks post immunisation, using V. anguillarum isolates from the serotypes O2b, O2c and O2d that were not included in the vaccine. An excellent level of protection was obtained against O2b and O2d (relative percentage survival 100% and 96.4%, respectively), but the challenge with the sub-serotype O2c isolate did not produce any mortality in the control group and needs to be repeated. The vaccine formulation was very efficient at protecting Atlantic cod against vibriosis but further challenges need to be performed with other serotypes included in the vaccine (O1 and O2a), as well as with more isolates from the O2b, O2c and O2d sub-serotype. To conclude, Atlantic cod is a species which will certainly have a major influence in marine aquaculture, but many areas have to be improved. The development of an effective and broad range vaccine to protect cod against Vibrio anguillarum offers another advance which should help Atlantic cod aquaculture to reach its full potential.

In Scotland, environmental regulation restricts commercial cod culture to the equivalent of 66 % of that granted for commercial Atlantic salmon (Salmo salar L.) farms. This calculation is based on estimations of nitrogen discharge from the difference in protein content between salmon and cod diets, with the higher levels of protein in cod diets suggesting a higher nitrogen discharge compared to that observed for salmon diets. In turn, this could potentially result in increased nitrogen enrichment of a marine ecosystem. The aims of this study (quantifying and modeling of nitrogenous wastes associated with the commercial culture of Atlantic cod (Gadus morhua L.) were achieved through a series of tank and cage investigations, each of which studied juvenile and adult Atlantic cod. The study provided data with respect to nitrogen excretion from juvenile and adult fish in both systems. This would allow the development of dispersion models and the calculation of nitrogen budgets for commercial cod culture, thus providing environmental regulators data independent of salmon models to create regulations that would be specifically applied to cod farming. The tank - based studies investigated three diet formulations produced by EWOS® Innovation in Norway, as a 4 mm pellet (juvenile study) and as a 7 mm pellet (adult study). The three iso -energetic diets varied primarily in protein content (40%, 50% and 60%). Two tank studies, one on juvenile and one on adult cod, investigated growth, condition and tissue composition, and the production of dissolved nitrogenous wastes over a 5 and 7 month period respectively. At the beginning of the acclimation period prior to the adult tank study commencing, the fish had a mean weight of approximately 1275 g. The difference in the final weight promoted by each diet was not significant (with an approximate final weight of 2400g), suggesting that a low protein diet (40 % protein) promoted similar growth to a high protein diet (60 % protein). Other growth and condition parameters were also similar for all diets with the condition improving over the course of the study. As the fish completed spawning immediately prior to the commencement of the study, an increase in condition was not observed until approximately day 90 of the 210-day investigation. Over the course of the 5-month juvenile study, growth was approximately 224g for the 40% protein diet and approximately 275g for the 50% protein and 60% protein diets. This suggests that a higher protein diet is required for optimal growth of juvenile cod and that 50 % and 60% protein diets promote similar growth and condition, potentially reducing the protein requirement of juvenile diets. In the juvenile investigation, condition increased over the full range of the study. In both tank studies, nitrogen digestion was directly related to protein (and associated nitrogen) content of the diets in the juvenile study expressed as a percentage of the nitrogen content of the diet as 59.19% (40% protein), 56.90% (50% protein) and 52.23% (60 % protein) suggesting that nitrogen digestion is more efficient at lower protein content in the diet. When expressed as a percentage of the nitrogen content of the diet, nitrogen digestion observed in the adult study was 60.55%, (40% protein) 60.92% (50% protein) and 60.60% (60% protein) respectively, suggesting protein digestion is similar regardless of protein content in adult cod. In the adult tank study, under a manual feeding regime, a post-prandial-peak is observed at 105 min. following the cessation of feeding. Thereafter, ammonia levels drop over the course of the sampling period. Following the afternoon meal commencing at 420 min., ammonia levels rise at least until the final samples are collected at 450 min. Under an automated regime, a lesser post-prandial-peak is observed but the ammonia concentration is lower over the sampling period compared to the respective profile under a manual regime. The ammonia profile produced throughout the juvenile tank study follows a very similar trend to that observed in the adult study under the manual feeding regime. Two cage - based investigations took place at the No Catch® Ltd. commercial organic cod farm in Vidlin Voe on the east coast of Shetland. Both studies investigated growth, condition and tissue composition, as well as the production of dissolved nitrogenous and particulate wastes associated with the culture of juvenile and adult Atlantic cod in cage systems. Sampling for the adult study occurred over three days during three sampling trips (September 2005, November 2005 and February 2006). Sampling for the juvenile study took place over three days on a single trip to Vidlin in late April 2006. The diets used at No Catch® Ltd. were produced by Biomar® in Grangemouth. A relationship between feeding and ammonia concentration is less evident in the cage studies than in the tank studies, and similarly, the relationship between feeding and ammonia concentration is less evident in juvenile fish than in adult fish. As ammonia values were converted to (µg/L/tonne biomass), the ammonia concentration recorded is largely dependent upon the biomass of the sampled cages at both the nursery site and production site. Deposition rates of organic carbon and nitrogen around the production and nursery cage sites in Vidlin Voe are related to the position of the sediment trap relative to its location and proximity of the trap to the specific cage site. Weather condition also had an impact on deposition rates with calmer weather producing lesser deposition rates. Around the production site, deposition rates of organic carbon and nitrogen are greatest in the direction of the prevailing current. Deposition rates decrease with an increasing distance from the cages. Although sediment trap results were inconsistent, a similar trend is observed for each of the three sampling trips, although actual deposition values were different. Differences between the deposition rates at the highly dynamic production site and the low energy nursery site indicated that sedimentation of waste from cod culture is highly dependent on water currents. Models of particulate waste deposition associated with the production cage site in Vidlin Voe were produced using the spreadsheet - based Cage Aquaculture Particulate Output and Transport (CAPOT) model, developed at the Institute of Aquaculture. The models were parameterised using the data collected and tested against an established regulatory model, DEPOMOD. The similarity in results illustrated the robustness of the highly flexible spreadsheet waste model for cod culture.

Aquaculture is an essential developing sector for world food production, however the attainment of sexual maturity during commercial on-growing is a major bottleneck to industry expansion. Sexual maturation brings a commercial loss due to reduced growth performance as well as reduced immune function. Furthermore, serious concerns exist over potential genetic interaction with native stocks through broadcast spawning or spawning interaction by escapees. In the north Atlantic region, the Atlantic cod (Gadus morhua) and Atlantic halibut (Hippoglossus hippoglossus) are key aquaculture species in which industry expansion is limited by pre-harvest sexual maturation. However, through a species specific combination of modern technologies and refinement in management practices it is possible that this sexual maturation can be controlled and on-growing potential enhanced. Thus the overall aim of this thesis was to conduct novel research that will improve our understanding of the underlying mechanisms that regulate sexual maturation, whilst also advancing the optimisation of technologies for the management of maturation in cod and halibut. In Atlantic cod, owing to the inconsistent inhibition of maturation in commercial conditions, ever increasing intensities of light and in some cases narrow spectrum technologies are being used to try to combat this problem. Firstly, this PhD project investigated the potential welfare impacts of high intensity artificial lighting which have not been studied to date (Chapter 2). The work specifically investigated the effect of traditional metal halide and novel green cathode lighting on the stress response, innate immunity, retina structure, feeding activity and light perception of Atlantic cod. Results indicated that although acute responses to light were observed, there were no clear significant long term effects of any of the lighting treatments on these parameters. Regarding light perception, interestingly even when subjected to high intensity constant lighting (metal halide mean tank intensity: 16.6 watts m-2), cod still demonstrated a day/night rhythm in melatonin release which suggests perception of the overlying ambient photoperiod. The second trial of this PhD project investigated the efficacy of shading of ambient photoperiod in addition to constant lighting to inhibit maturation of cod outdoors (Chapter 3). This aimed at improving the performance of artificial lighting regimes in the open cage system during commercial on-growing by reducing the relative difference between day/night light intensities. The trial was conducted over a one year period where a low and high shade treatment were tested in outdoor tanks. Shading increased the relative night time illumination to 6.6% and 31.3% of daytime levels respectively, compared to <2% in an unshaded set-up. Both shading treatments were effective at suppressing sexual development in cod as confirmed through measurements of gonadosomatic index, histological analysis of gonadal development, oocyte diameter measurements and sex steroid profiles as well as measurements of growth. In addition to research at the applied level in Atlantic cod, this thesis has also extended to the fundamental level and explored one of the potential mechanisms relaying photoperiod signal to the endogenous regulation of sexual maturation in cod, namely the kisspeptin system (Chapter 4). Partial sequences for the signal peptide Kiss2 and its receptor Kissr4 were isolated and described showing similarity to other teleost species such as the medaka, Oryzias latipes and stickleback, Danio rerio. Novel molecular qPCR assays were designed and developed to measure the expression of both genes in male and female cod over a maturation cycle and compared to cod under constant lighting which remained immature. Interestingly, expression patterns of kiss2 and kissr4 did not reveal any clear association with season or photoperiod treatment. However, pituitary expression of gonadotropins (FSH, follicle stimulating hormone; LH, luteinising hormone) did show a differential expression in relation to treatment from early winter approximately 4-6 months after the photoperiod change. These new results are in contradiction with the hypothesis that the kisspeptin system would be involved in the initiation of gametogenesis, as shown in mammals. However, the FSH/LH data defines a window during which time kisspeptin or another GnRH stimulating mechanism must be active, this compels the need further investigation. In Atlantic halibut farming, all-female production removes the concerns of production losses through sexual maturation. Accordingly, this thesis investigated the potential/feasibility of generating monosex populations by FACS (fluorescence activated cell sorting) semen sexing based on cellular DNA content, as proven in terrestrial agriculture. Results however did not show any clear differences between the DNA of sperm in a range of species tested (Atlantic halibut, cod, sea bass, perch) suggesting that this technique may not be applicable in such species. The project also focussed on the production of a population of sex reversed halibut broodstock (neomales) that will generate, in the long term, a basis for traditional monosex population generation in the UK. Two in feed MDHT (17α-methyldihydrotestosterone) treatments were tested with the aim to reduce the use of hormone. Results were very successful with a hormone treatment of 5ppm MDHT generating a 97% phenotypic male population thus suggesting the presence of sex-reversed halibut which can be used for future monosex production. Overall, this work aimed to develop and/or refine potential remediation techniques for sexual maturation in two key commercially important farmed marine fish species, cod and halibut, as well as further our understanding on the regulation of puberty. The knowledge gained from this work provides a means to optimise the techniques employed in the industry and has the potential to increase production and profitability without compromising farmed animal welfare, thus ultimately promoting the sustainable expansion of the Atlantic cod and halibut aquaculture.

The persistence of a species in the face of environmental change is a function of the extent to which populations respond differently to changes in their environment and the spatial correspondence between the scale of disturbance and the scale of adaptation. The pattern by which a population, or genotype, expresses a range of phenotypes across an environmental gradient is called a norm of reaction. The level of phenotypic plasticity displayed within a population (i.e. the slope of the reaction norm) reflects the short-term response of a population to environmental change while variation in reaction norm slopes among populations reflects the spatial scale of variation in these responses. Using a reaction norm framework, I examined the spatial scale of genetic variation in plasticity for life-history traits in Atlantic cod (Gadus morhua), a marine fish of global biological and socioeconomic importance. Through common-garden experiments, I found evidence of both adaptive and non-adaptive plasticity for larval growth rate and survival in two cod populations that experience contrasting thermal environments in nature. A comparison of these reaction norms with those of four cod populations studied previously revealed significant genetic divergence in adaptive traits at a smaller spatial scale than has previously been shown for a marine fish with no apparent physical barriers to gene flow (<250 km). This fine-scale genetic structure is likely the result of populations being locally adapted to seasonal changes in temperature during the larval stage caused by differences in spawning times and may be maintained by behavioural barriers to gene flow. Implications of variation in life-history trait plasticity to fisheries management in the face of predicted changes in climate are discussed.

The populations of Atlantic cod (Gadus morhua) in the Baltic Sea has been heavily exploited for decades, with fishing mortalities close to, and for several occasions, even above one. The larger part of the spawning stock biomass is consequently being removed each year. The issue of fisheries induced evolution (FIE) has been gaining attention from researchers lately. The selection pressure driving this evolution is powered by a connection between high mortality rates and heritable traits. The fishing in the Baltic Sea is mainly performed with size selective gear that can impose selection on traits like size-at-age or size-at-maturity. In this review I show how FIE may affect the Baltic cod towards decreased size-at-age/maturity and how size selective fishing on stocks at low levels can increase the inherent instability of the population as well as deprave the Baltic Sea of ecosystemic services from cod.

Marked shifts in the life history traits of fish have been reported in many exploited fish stocks, with a particular trend towards decreasing size and age at maturity. Though other environmental and behavioural factors have been implicated, the key driver of these changes links to fishing pressure, through both the direct selective effects of fishing itself, and indirectly through the manipulation of important biological and environmental factors. Although reproduction itself has been well described in teleosts, the mechanisms of environmental and endogenous entrainment of maturation remain unclear and it was the principal aim of this thesis to improve current understanding of these systems in gadoids. Photoperiod has been identified as the strongest environmental cue for entraining seasonal behaviours, including seasonal reproduction. Over the last decade, several of the key drivers involved in the photoneuroendocrine cascade have been elucidated in mammals and birds, with the Eya3 pathway merging as an important mechanism for entraining maturation. However, little is yet known of their influence on maturation in fish. In the first study, the photoperiodic regulation of the Eya3-Tshβ-Dio2 cascade was analysed in Atlantic cod exposed to either continuous light (reproductive inhibition) or simulated natural photoperiod (reproductive stimulation) from July to December. Monthly expression was measured through QPCR, demonstrating a strong activation of pituitary Eya3 under declining photoperiod. As this coincided with the onset of secondary gametogenesis, these results suggest that Eya3 may play a stimulatory role in the photoneuroendocrine cascade of Atlantic cod. Although photoperiod represents the most reliable and noise free proximate signal to entrain the reproductive process, it is clear that a minimum growth and energetic state must be reached for maturation to progress. This directed the second line of study – a series of diet restriction trials on haddock and cod designed to investigate the influence of naturally occurring lipid levels in the diet on growth and reproduction, define the “critical window” in which fish assess their energetic state and how this is analysed before commencing secondary gametogenesis, and to assess whether Eya3 is regulated by the growth axis in cod. The results of these experiments indicate that overall size around the autumn equinox is the most accurate indicator of maturation commitment in cod. Additionally, Eya3 expression was elevated in maturing fish indicating a role for this marker in linking the energetic signal with entrainment of the reproductive axis. Finally, an analysis of the physiological and genetic stock structure of cod from the North Sea IVa stock region and westward into VIa was performed. The results of this analysis support previous genetic studies, indicating further fine-scale structuring of these stocks, reflective of the structure indicate by the differences in maturation strategies of the component populations. The results identified both environmental and harvest related pressures which may be driving the current stock structure. The results of these studies greatly improves our understanding of the key drivers and mechanisms regulating maturation in cod, highlighting new avenues for future research.

During the last decades marine aquaculture has steadily expanded and diversified to include a wider range of commercial species. Despite the intense effort towards understanding the biological requirements of farmed species, several issues remain to be addressed. Mariculture success is restricted by a number of production bottlenecks including limited seed supply, caused mainly through a combination of compromised productivity in broodstock paired with high mortalities during the early life stages. Productivity and survival success is often dependent on the successful recreation of natural environmental conditions. While in a commercial setting a concerted effort is generally made to simulate key environmental stimuli there remains a lack of understanding of the significance of many potential signals. The overarching aim of this thesis was to investigate the effects of some of the overlooked environmental stimuli on fish performance in enclosed facilities and where possible relate this to the natural setting from which the species have been removed. The studies contained in this text are focused on the effects of anthropogenic noise, light spectral composition and substrate on the performance of broodstock and juvenile development of two valuable commercial marine species Atlantic cod (Gadus morhua) and turbot (Scophthalmus maximus). The aim of Chapter 3 was to test if artificial sound can act as a stressor in Atlantic cod and thereafter to examine if chronic sound disturbances can compromise broodstock spawning performance in land-based facilities. Results showed that anthropogenic noises in a land-based marine farm are within the auditory thresholds of cod and other fish species. Juvenile cod exposed to 10 min of artificial noise (100-1,000 Hz) from 10 to 20 dB 1 re µPa above background sound levels presented a typical acute stress response with a 4 fold elevation of plasma cortisol levels within 20 min, with a return to basal levels after 40 min, while the intensity of the stress response (in terms of amplitude and return to normal levels) appeared to be correlated to the noise level applied. When a similar artificial noise of 35 dB 1 re µPa above background sound level was applied to a broodstock population daily on a random schedule during the spawning season, it significantly impacted on reproductive performances in comparison to a control undisturbed population with notably a reduction in fertilisation rate that correlated with increased egg cortisol contents. Overall, these studies confirmed, for the first time, that artificial noise mimicking anthropogenic sounds generated in marine land-based facilities trigger a typical acute stress response if a similar sound exposure is then applied in a chronic manner it resulted in reduced broodstock spawning performances. Overall this work provides novel evidence on the potential of anthropogenic noise to act as stressor in fish. The possible implications for both captive and wild stock are discussed. In chapter 4 the effects of light spectrum and tank background colour on Atlantic cod and turbot larval performance from hatch until the end of metamorphosis were investigated. In both species larvae exposed to shorter wavelengths (blue and green spectrums) showed significantly enhanced growth in terms of standard length, myotome height, eye diameter and condition factor in comparison to larvae exposed to longer wavelengths (red). Larvae performances in the colour background experiment differed between species. Atlantic cod larvae reared in a red tank background displayed the best growth and survival, while larvae in blue tank background had a significant positive effect on final survival rate. In contrast, turbot larvae survival rates were the highest in the red tank background colour with the lowest growth parameters, while larvae in the blue tank background displayed the best growth. In both species, white tank background colour resulted in the lowest final survival rate. These results highlight the biological relevance of light spectrum and background colour in marine larvae performance and survival, demonstrating the importance of considering the light composition of the light units used in the hatcheries for larval rearing. Subsequently in chapter 5 the effects of light spectrum in juvenile turbot growth, appetite, stress response and skin pigmentation were investigated. Two sets of experiments were performed with post-metamorphosed (1 g) and on-growing (100 g) turbot. Results demonstrated that short wavelength treatments had a significant positive effect on growth parameters (total length and wet weight), food intake and feeding response. Light treatments caused a positive correlation between plasma glucose and cortisol levels with significant differences between the short and long wavelength treatments. Skin pigmentation was affected by the light treatments, showing a relationship between wavelength and brightness (negative) and darkness (positive). Blue light treatment resulted in brighter and lighter skin colouration, while red light had the opposite effect: darkening of the skin. Overall these results confirm that turbot juveniles performance is enhanced by exposing them to a similar photic environment than the one from the natural ecological niche. Light spectrum intervenes in skin pigmentation and the possible mechanisms behind the variations are discussed. In general chapter 5 provides background knowledge of the possible implications of light spectrum in fish juveniles performance and possible commercial applications. The final two experimental chapters turned focus back on the optimisation of broodstock environmental management and subsequent effects on their productivity. In Chapter 6 the importance of crepuscular light simulation was investigated in Atlantic cod broodstock spawning performance. No significant impact could be observed in terms of egg production and quality in association with dawn/dusk simulation compared to abrupt lights on/off. This suggests, at least for Atlantic cod, that crepuscular light simulation is not a key factor affecting spawning performance during the spawning window. The possible implications of twilight on gamete quality prior ovulation are discussed. In Chapter 7 the effect of a “breeding nest” containing a substrate (i.e. sand) in turbot broodstock spawning performance was investigated. Behavioural observation recorded active occupancy of the nests with the suggestion of social structuring as specific individuals (females) occupied the nest preferentially. However no fertilised, naturally released eggs were collected from the overflow during the spawning seasons. This would suggest that the presence of a nest is not enough to induce natural spawning behaviour in turbot in itself however the elective occupancy suggests that nests and/or their substrate was a physical enrichment that was valued by the fish which should be explored further. Overall the studies contained in this thesis highlight further the importance of considering noise and light as crucial environmental factors in marine aquaculture. Results from the different chapters offer a possible application within the enclosed facilities that might contribute to the success of the industry. Present findings contribute towards the understanding of the effects of environmental signals in fish and provide further insight to guide further lines of research on the involvement of light spectrum on fish physiology.

Lernaeocera branchialis (Linnaeus, 1767) is a parasitic copepod possessing a complex dual-host lifecycle. The “definitive” gadoid hosts, including Gadus morhua (Atlantic cod), Melanogrammus aeglefinus (haddock) and Merlangius merlangus (whiting), are infected by the fertilised female, which penetrates the host’s ventral aorta or bulbus arteriosus whilst undertaking extensive metamorphosis and a haematophagous lifestyle. The pathogenic effects of this activity upon the host have been well documented and mortality may occur, especially when multiple parasites are present. These negative impacts on cod, particularly juveniles, by L. branchialis have the potential to adversely affect cod aquaculture in the future, and already vulnerable wild cod stocks. This PhD project therefore, investigated the immune response of wild haddock and cultured-cod post-infection by L. branchialis, and the possible mechanisms by which the parasite modulates/evades the host’s immune response. The systemic immune response of both wild haddock and cultured-cod post-infection by L. branchialis depended on the maturation stage of the parasite, and in the former host species, upon the infection intensity. Wild haddock harbouring fully metamorphosed females showed an increase in circulating thrombocytes and a decrease in serum protein levels however; if multiple mature L. branchialis were present the haddock possessed reduced circulating monocytes, and increased circulating thrombocytes and serum anti-trypsin activity. Infection by L. branchialis was also associated with a suppressive effect on haddock serum spontaneous haemolytic activity. These responses were thought to be due to the host trying to counteract the increased damage caused by the massive increase in size and the feeding of the mature parasite, which is more pronounced when multiple parasites are present, resulting in the increase in some parameters and the ‘consumption’ of others. However, the effect of parasite-derived secretions and other pathogens due to observations on wild fish could not be discounted. The laboratory-infection of cultured-cod from two different sources was also performed in order to study the immune response over time. The two groups of cod showed differences in their immune response to L. branchialis. The first group showed suppressed respiratory burst activity of phagocytes, as the parasite reached the early penella sub-stage, whilst no suppression in phagocyte respiratory burst activity was found in the second group. The parasite was found to migrate along the afferent branchial artery of the cod where a thrombus formed and was present throughout its migration into the ventral aorta. At 14 d post-infection, leukocytes expressing Interleukin 8 mRNA were observed within the free-flowing blood at the periphery of the organising thrombus within the lumen of the ventral aorta. This was speculated to aid the recruitment and activation of leukocytes to the site, and the maturation and neovascularisation of granulation tissue. The infection of the second group subsided with the death of the parasite, and none of the parasites metamorphosed past the early penella sub-stage. The live parasites infecting the first group of cod did not possess IgM or complement component C3 binding on their cuticle, however, both IgM and C3 binding occurred on the dead parasites in the second infection trial. This may highlight the importance of these opsonins and the cytotoxic effect of phagocytes in the elimination of L. branchialis by some cod. However, the first infection was terminated as the parasite reached the early penella sub-stage due to a loss of stock cod prior to the study, so the long-term success of the infection can not be concluded. Therefore, the immune response to infection needs to be determined over the entire metamorphosis of L. branchialis to determine whether the infection was successful or not, and preferably in populations with varying susceptibility to L. branchialis. This will not be possible without further studies into the resistance of different stocks of cultured-cod. Many arthropod parasites, such as ticks and salmon lice, have been previously documented to produce pharmacologically active secretions, aiding host invasion and parasite feeding, preventing the host immune response from working effectively against the parasite, all aimed at improving survival of the parasite. Therefore, the effects of the secretory/excretory products (SEPs) produced during the initial infective stage and by the mature, fully metamorphosed female on the immune response of cultured-cod in vitro, and the location of exocrine glands associated with the oral region of the parasite were investigated. The SEPs from the infective stage of the parasite were found not to affect the intracellular hydrogen peroxide (H2O2) production of phagocytes. The practical difficulties in collecting large quantities of the SEPs from the infective stage meant that their effects could not be tested on the other host immune parameters studied. The SEPs from fully metamorphosed female L. branchialis, however, had a number of suppressive effects on the host immune response in vitro including: 1) suppression of the intracellular production of cytotoxic H2O2 during the respiratory burst of phagocytic leukocytes post-PMA stimulation, 2) suppression of the production of macrophage activating factor by leukocytes with a priming effect on naïve phagocyte function, and 3) suppression of the chemo-attraction ‘power’ of zymosan activated cod serum, i.e. anaphylatoxin activity, on head kidney-derived leukocytes. These effects were dose-dependent, and highlight the capacity of L. branchialis to suppress its host’s innate immune response at the local feeding area. Further work is required to establish the mechanisms by which the parasite-derived SEPs suppress these host immune parameters, and to identify which molecules produced by the parasite are responsible. The correlation between these in vitro results, and systemic immune parameters measured from laboratory-infected Atlantic cod and wild infected haddock are discussed. Host immuno-modulation by other arthropod parasites is mediated by pharmacologically active secretions produced by exocrine glands. Therefore, the exocrine glands of the infective and fully metamorphosed female L. branchialis were also investigated in order to identify those that might be responsible for the secretion of host-modifying products. Adult female exocrine glands were mapped using diaminobenzidine (DAB), most commonly known to stain peroxidases and catalases. These compounds are known to be involved in the neutralisation of harmful free radicals which are released during the respiratory burst and tissue damage. Such products may therefore be important protective secretory components at the site of feeding / infection. Exocrine glands were located in the infective stage associated with the oral region, one pair termed the anterior gland complex (AGC), and the other pair extending either side of the oral cone termed the circum-oral glands (CG). These were further investigated using light microscopy and transmission electron microcopy. The AGC and CGs possessed multi-component secretions and they possessed secretory vesicles, abundant and highly active rough endoplasmic reticulum and Golgi apparatus suggesting that protein is an important component of the secretory products. These glands were also observed in the fully metamorphosed females where they had increased in size within the cephalothorax post-metamorphosis. It is hoped that the identification of these glandular structures, which are thought to secrete within the local vicinity of the oral cone, will aid future studies regarding the identification and secretion kinetics of parasite-derived molecules during the infection and feeding process.

Intensive aquaculture of Atlantic cod is fast developing in both Northern Europe and Canada. The last six years have seen major improvements in the larval rearing protocols and husbandry techniques for this species. Although breeding programmes are currently being developed by both governmental and private institutions in the main cod producing countries (i.e. Norway, Iceland and Canada), most hatcheries still rely on the mass spawning of their own broodstock. Mass spawning tanks are complex systems where fish are left to spawn naturally and fertilised eggs are collected with the overflowing water, with little or no control over the matings of the animals. Few published studies in other commercial marine species (i.e. turbot and sole) have attempted to analyse the output from such systems using microsatellite markers and several parentage analysis software programs. A review of these publications exposed a lack of consistency in the methods used to analyse such complex datasets. This problem was addressed by carrying out a detailed comparison of two analytical principals (i.e. assignment by strict exclusion and assignment by probabilities) and four parentage software programmes (i.e. FAP, VITASSIGN, CERVUS and PAPA), using the DNA profiles, at 5 loci, from 300 cod fry issued from the mass spawning of a large hatchery cod broodstock tank (consisting of 99 fish). This study revealed large discrepancies in the allocation outcomes between exclusion-based and probability-based assignments caused by the important rate of typing errors present in the dataset. Out of the four softwares tested, FAP (Taggart, 2007) was the most appropriate to use for handling such a dataset. It combined the most conservative method of assignment with the most informative output for the results displayed. In an attempt to study the breeding dynamics in a cod commercial hatchery, parental contributions to five groups of 300 fry (from five single days of spawning and from two commercial mass spawning cod tanks) were analysed, based on the genotyping data from eight loci. The parentage results from the exclusion-based analyses revealed that, on a single day, at least 25 to 30% of the total breeding population contributed to fertilised eggs that resulted in viable offspring at 50 and 83 days post-hatch. Family representations were highly skewed - with the marked dominance of a few males - and effective breeding populations were consistently low (approx. 5% of the total breeding population). Parental contribution to a group of 960 codlings - produced following intensive commercial practices (i.e. including successive size gradings and mixing of batches) and belonging to a single graded group - was also analysed, based on the genotyping data from eleven loci. The effective breeding population size of the juvenile batch (c. 14% of the total broodstock population) was two to three times greater than the effective size observed on a single day of mass spawning. The per-generation rate of inbreeding was however relatively high, for this batch alone, at 2.5%. Based on these results, suggestions were made to manage hatchery cod broodstock populations and implement genetic selection. Early maturation of farmed cod in sea cages (at two or three years old) is a major concern for ongrowers. Understanding the mechanism(s) behind sex determination in cod would probably help the development of a method to control sexual maturation. In an attempt to elucidate sex determination in cod, a protocol to induce gynogenesis was developed. Gynogenetic fish were successfully produced by irradiating cod milt with UV and applying a cold shock (at -6oC) to newly fertilised eggs. However, due to poor survival during larval rearing, only one gynogenetic fish survived long enough to be sexed; not enough to conclude anything on the sex determination mechanism(s) in cod.