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In 2005, Atlantic salmon (Salmo salar L.) migrating to the United Kingdom exhibited swollen, haemorrhagic vents, symptoms not previously recorded. The condition was latterly termed Red Vent Syndrome (RVS), and subsequently observed across the North Atlantic. RVS has been pathognomonically associated with one of the most abundant parasites within the marine environment, the ascaridoid nematode Anisakis simplex, which also causes Anisakiasis in humans. Although A. simplex is commonly found in Atlantic salmon, heavy infestation of the vent region is novel, and the expression of RVS has not been prevalent in other fish species. Red Vent Syndrome has been well studied, however, the causes of the condition, and the reasons driving the novel site of infestation exhibited by A. simplex, have not been clarified. The aim of this PhD therefore, is to provide new information regarding the underlying factors of the infestation of the vent region by A. simplex, and the emergence of RVS. This study therefore: i) assessed the relationship between nematode burdens within the viscera and musculature, in comparison to the vent in 117 adult Atlantic salmon; ii) compared the genetic structure of A. simplex present in the vent region and the viscera using the entire nuclear internal transcribed spacer (ITS) region; iii) investigated migratory route and feeding ground of Scottish salmon populations using stable isotope analysis of dorsal muscle tissue and parasite component communities and, iv) assessed the expression of the cytokine TNF-α1 within vent muscle tissue using (q)RT-PCR, in relation to RVS severity. Phylogenetic analyses have shown that it is A. simplex sensu stricto infesting the vent region. The results show that there is a significant positive relationship between the nematodue burden in the body (viscera and musculature) and in the vent region. Isotopic signatures of salmon populations showed no significant differences, however, A. simplex intensities between populations on the East and North coasts of Scotland suggest geographical differences in A. simplex transmission pathways. Finally, the expression of TNF-α1 is not significantly different between RVS severity, and nematode burden. Out of the four studied factors, increasing nematode intensities in Atlantic salmon populations, and the significant positive relationship of nematode intensities between the body (viscera and musculature) and the vent, are likely to explain the infestation of the vent by A. simplex. The underlying causes of RVS however remain uncertain and require further research. With incidences of RVS observed across a number of populations over a large spatial area, regional and global effectors such as warming sea surface temperatures, and the North Atlantic Oscillation are expected to play key roles in its aetiology.

Das Flussneunauge (Lampetra fluviatilis) ist eine nach europäischem Recht besonders geschützte Tierart. Es wurde untersucht, ob Flussneunaugen in Sachsen ablaichen bzw. regelmäßig vorkommen. In 16 Nebenflüssen der Elbe, die als Laichgewässer für das Flussneunauge in Sachsen in Frage kommen, konnten jedoch keine adulten Flussneunaugen nachgewiesen werden. Eine regelmäßige Fortpflanzung des Flussneunauges in sächsischen Elbnebenflüssen muss deshalb gegenwärtig in Frage gestellt werden. Der Aufstieg einzelner Flussneunaugen in sächsische Elbnebenflüsse ist dennoch belegt und auch für die Zukunft nicht auszuschließen. Die Ergebnisse stützen die These, dass Bach- und Flussneunauge einer Art angehören und sich nur durch ihr Wanderverhalten unterscheiden.

Cryopreservation of fish ovarian tissue fragments can be a viable alternative to cryopreservation of oocytes and embryos. The ability to cryopreserve both maternal and paternal gametes would provide a reliable source of fish genetic material for scientific and aquaculture purposes. The main aim of the present study was to develop an in-vitro culture protocol and cryopreservation protocol for zebrafish ovarian tissue fragments. In-vitro culture protocol for the tissue fragments containing stage I and stage II follicles were developed and the growth assessment of follicles were evaluated using biomarkers. To develop the cryopreservation protocol using control slow cooling method, the effect on freezing medium, cryoprotectants and cooling rate on the tissue fragments were investigated. The in-vitro culture experiments showed that L-15 medium (pH 9) containing 100mIU/ml FSH along with 20% FBS was effective for tissue fragments containing stage I and II follicles to grow in-vitro. The growth of the ovarian follicle stages was confirmed by the level of expression of p450aromA and vtg1 gene. The optimal cryopreservation protocol for the ovarian tissue fragments was found as 2M methanol+ 20%FBS in 90% L-15 medium with the cooling rate of 4°C/min. Although the highest survival rate obtained for stage II follicles within the fragments was 68.2±1.9% and stage I follicles within the fragments was 55.4±2.3% using TB staining, it showed a significant decrease in their ATP levels. This is the first study carried out on the zebrafish ovarian tissue fragments. Study on cryopreservation of the ovarian tissue fragments and development of the in-vitro culture protocol and use of biomarkers for the ovarian tissue fragments were reported here for the first time. The outcomes of this study have provided useful information for future cryopreservation protocol development.

Freshwater streams in the developed world are becoming increasingly dominated by treated wastewater. Continually discharged into most surface waters, these effluents contain a suite of bioactive man-made chemicals, including steroid and non-steroid oestrogens, which have been found to feminise male fish, skew sex ratios, and cause reproductive failure. However, the consequences of reproductive disruption remain poorly explored at the population level. This thesis was initiated to evaluate how oestrogenic contaminants might influence the population ecology of a common cyprinid, the roach (Rutilus rutilus). An investigation encompassing population structure, multigenerational exposure and the role of additional drivers of fish population dynamics was undertaken to contextualise the effects of oestrogenic effluents on wild fish populations. Population genetic analysis of UK roach found they exhibit moderately high levels of genetic diversity and significant intra-river genetic structure. Genetically differentiated local subpopulations indicate little interbreeding and limited gene flow, consistent with a typical metapopulation that has not been homogenised by restocking. Similarly, my thesis demonstrates no significant relationship between effluent exposure and Ne (effective population size) or genetic diversity of roach populations, albeit a 65% reduction in Ne is possible at highly polluted sites. River stretches contaminated with high levels of effluent can support breeding populations, which recruit successfully with minimal immigration from less contaminated sites. Multigenerational effects of effluent exposure on roach were also evaluated experimentally using reproductive success from breeding adults over three generations. Lifelong exposure to 100% treated effluent resulted in feminised phenotypes (ovarian cavities and intersex condition) in males but no observable effect on females. Additionally, despite gonadal disruption in males and effluent exposure of their mothers, I found no detrimental effect on their ability to compete with control fish. Instead, reproductive success was primarily determined by body size. A novel approach considering additional fish population drivers suggests that genetic diversity and species diversity decline in parallel with an increasing presence of disturbed land, when combined with geographical isolation. In conclusion, group assemblage and genetic structure of fish populations appears multi-causal and cannot be disaggregated, such that a single environmental characteristic can be shown to drive patterns of population success.

Non-native parasites pose a significant threat to aquatic bio-diversity and fishery development. Many factors have facilitated the introduction of non-native parasites into England and Wales. Identifying the effects of these parasites and the importance of any changes to wild fish populations represents a considerable challenge. However, in order for the Environment Agency to identify future disease threats, effectively manage resources and implement practical and sustainable control measures, the risks posed by these parasites to fisheries must be better understood. A structured, risk-assessment process for non-native freshwater parasites is proposed. This approach includes procedures for hazard identification, impact assessment, risk management and communication. A quantitative approach to hazard identification provides an initial prediction of impact at the time of introduction to inform decisionmaking and guide preliminary control measures. This is followed by a more comprehensive framework for impact assessment that promotes understanding of changes at host, population and fishery levels. These are placed into context with the economic and ecological value of native resources. An independent 'non-native parasite review group' has been convened to ensure consistency of policy decisions and clear communication of disease risks to interested bodies. It is hoped that this wil assist the management of future invasions and provide a scientifically robust foundation on which to base proportionate control measures. The Category 2 parasites are defined as "species having a significant disease potential when introduced into waters where they do not already exist, or are non-indigenous species with unknown pathogenicity and distribution". Understanding of the dangers posed by these parasites varies considerably. Ergasilus sieboldi, Anguilicola crassus, Bothriocephalus acheilognathi and Lernaea cyprinacea, are well recognised non-native fish pathogens that pose a considerable threat to fisheries. Conversely, the effects of other introduced parasites remain poorly understood. In many cases, a paucity of published literature at the time of introduction has limited a reliable assessment of impact. Four non-native parasites were considered a priority for further study. These were Paraergasilus longidigitus (Copepoda: Poecilostomatoida), Ergasilus briani (Copepoda: Caryophylldae) Poecilostomatoida ), Atractolytocestus huronensis (Cestoda: and Philometroides sanguinea (Nematoda: Philometridae). Pathological, epidemiological and experimental investigations were undertaken to assess the effects of these parasites at host and population levels. P. longidigitus can cause pronounced pathological changes to the olfactory epithelium of infected fish. Although this damage provides the potential for disruption to spawning through reduced sensitivity to reproductive chemical cues, experimental observations combined with seasonality studies suggest that the parasite is unlikely to disrupt reproduction within infected cyprinid fisheries. Similarly, studies conducted on the caryophylidean cestode A. huronensis provide little evidence to suggest that the parasites poses a threat to car fisheries. Damage within the intesinal tract was characterised by relatively mild mechanical and inflammatory changes. Infections of up 213 parasites had no adverse affect upon the conditon of common carp. Based on these findings and the application of the aformentioned risk model, P. longidigitus and A. huronensis are considered to be of low disease risk to fisheries. The pathology of E. briani and P. sanguinea are described for the first time. Literature suggests that both parasites are pathogenic to juvenile fIsh. E. briani causes a number of pathological changes within the gils of juvenile cyprinids, although these remain localised due to the very strict site specificity of the parasite. Migrations of the nematode P. sanguinea causes damage to the fins and caudal musculature of juvenile crucian car. For both parasites, the extent and severity of pathological changes are inversely proprtional to host size. Further studies at the population level are needed before an assessment of impact to fisheries can be made. The difficulties associated with studying the effects of introduced parasites to juvenile fish populations in the wild are recognised. Recommendations for further study are given.

Cryopreservation of gametes provides a promising method to preserve fish genetic materials, which offers many benefits to the fields of aquaculture, conservation and biomedicine. Although successful cryopreservation of spermatozoa of about 200 fish species has been achieved, systematic studies on cryopreservation of fish oocytes have only recently been undertaken. The objective of the present studies was to use zebrafish as a model system to develop a cryopreservation protocol for fish oocytes and to develop reliable viability assessment methods for monitoring zebrafish oocyte viability both before and after cryopreservation. A simple and rapid enzymatic method for zebrafish oocytes isolation was developed and the investigations on cryopreservation of zebrafish oocytes using improved controlled slow cooling and vitrification were carried out. Oocyte viability following cryopreservation was investigated by ATP assay, oocyte viability molecular signature (OVMS) and cryomicroscopic observation in addition to staining methods. The optimum conditions for oocyte enzymatic separation were identified as 0.4mg/ml collagenase or 1.6mg/ml hyaluronidase treatment for 10min at 22ºC and this method can be used for oocytes at all stages. The use of sodium free medium (KCl buffer), fast warming and 4-step removal of cryoprotectants in an improved controlled slow cooling protocol significantly enhanced oocyte viability (67.5 ± 1.7%) when compared with a previous study (16.3 ± 2.3%) in this laboratory. Mixtures of cryoprotectants (methanol, Me2SO and propylene glycol), stepwise addition and removal of cryoprotectants in combination of a new vitrification system (CVA65 vitrification system) were used in vitrification studies. Oocyte survivals after vitrification assessed by trypan blue staining were relatively high (76.5 ± 6.3%) shortly after warming in KCl buffer. Furthermore, the result of ATP assay showed that ATP levels in oocytes decreased significantly after cryopreservation indicating the bioenergetic systems of oocytes were damaged. Cryomicroscopic observations demonstrated that Intracellular ice formation (IIF) is the main factor causing injuries during cryopreservation of zebrafish oocytes. The results provided by the present study will assist successful protocol design for cryopreservation of fish oocytes in the future.

Cryopreservation of fish germ cells has important applications in aquaculture, conservation of endangered species and human genomic studies. Although investigations on cryopreservation of fish sperm and embryos have been carried out extensively, cryopreservation of fish oocytes has not been studied systematically. The objective of the present study was to develop successful cryopreservation protocol for zebrafish oocytes at temperature of liquid nitrogen (-196°C), or if unachieved, to investigate the limiting factors associated with fish oocytes cryopreservation. In this study, the effects of cryoprotectants exposure and enzymatic treatments on oocytes survival were studied, and new viability tests for zebrafish oocytes were developed. The effects of controlled slow cooling with different cryoprotective agents, in different freezing media and at different cooling rates on cryosurvival of zebrafish (D. rerio) oocytes were investigated. Cryomicroscopic observations on zebrafish oocytes were also carried out. Three reliable vital tests -trypan blue (TB) staining, ATP assay, and in vitro maturation followed by germinal vesicle breakdown observation (GVBD) were found suitable for assessment of oocytes viability. Vitellogenesis (stage III) was found to be the optimal developmental stage for cryopreservation. Methanol was found to be the best CPA for zebrafish oocytes. Combination of 4M methanol and 0.2M glucose in potassium chloride (KCI) buffer was found to be the optimal cryoprotective solution. Controlled slow cooling at 0.3°C/min rate, combined with seeding at -12.5°C and plunge to liquid nitrogen (LN) at-40°C were found to be the optimal conditions for cryopreservation of stage III oocytes. However, even with the optimal protocol, TB-assessed viability, Le. the ratio of oocytes with intact plasma membrane after cooling to -196°C was 19.6±8%. Furthermore, GVBD experiments showed that none of the cryopreserved oocytes can be matured in vitro, and their ATP levels were decreased dramatically, indicating that successful cryopreservation of fish oocytes at liquid nitrogen temperature still remains elusive. Cryomicroscopic observations demonstrated, that the damages of oocytes are associated with intracellular ice formation (lIF). IIF occurred simultaneously with extracellular ice formation (ElF) in nearly 100% of the cases, and formation of lethal hexagonal type of ice was observed. This study was the first systematic attempt to cryopreserve fish oocytes at liquid nitrogen temperature. The results provided will undoubtedly assist successful protocol design for cryopreservation of fish oocytes in the future.

Anthropogenic structures fragment river connectivity, impeding the migration of diadromous fish between essential habitats. Tide gates are used worldwide primarily for flood protection and land reclamation by closing under hydraulic pressure during the flood tide and opening when head differential is sufficient during the ebb. Although tide gates are known to decrease fish species richness, abundance, and movement, their impacts on the migration of ecologically and socioeconomically important diadromous fish in terms of passage efficiency and delay have not been reported elsewhere. Acoustic and passive integrated transponder telemetry revealed that passage efficiencies of upstream migrating adult brown trout, Salmo trutta (92%), and downstream migrating juvenile sea trout smolts (96 - 100%) and adult European eel, Anguilla anguilla (98%), were high at top-hung tide gates in two small English streams. However, these fish experienced delay at the gates (adult brown trout, median = 6.0 h; sea trout smolts, mean = 6.5 and 23.7 h; eels, mean = 66.2 h) when compared to migration through unimpeded reaches. The percentage of time the gates were closed and mean angle of opening were positively related to delay in both species and life stages. Diel periodicity also influenced delay for smolts and eels, which were more active at night. For adult trout, water temperature was positively associated with delay. Upstream and downstream water temperature and salinity were influenced by the temporal operation of the gates. Orifices installed in the gates did not mitigate delay for adult or juvenile trout. For adult eels, delay was decreased when an orifice was operational, although this coincided with more eels first approaching the gates when open, higher tides and greater saline intrusion upstream of the gates. When gates were open, fish would not pass immediately through, indicating the potential influence of a behavioural avoidance component. To examine the effect of hydrodynamics created by top-hung tide gates with different aperture sizes, wild sea trout smolt behaviour was observed by video cameras in an experimental flume at night. Avoidance responses occurred within an average of 1.4 fish body lengths upstream of the gate. Fish were more likely to exhibit avoidance (switch in orientation from negative to positive rheotaxis, increased tail beat frequency and/or retreat upstream) in the vicinity of a model gate with a smaller angle of opening and passage aperture. Overall, top-hung tide gates delayed the migration of diadromous fish, potentially increasing energy expenditure and predation risk. Delay was not decreased by orifices. Modifying or replacing top-hung tide gates with designs that allow them to open wider and for longer could reduce migratory delay and improve the environmental conditions that cause behavioural avoidance.

Changes of habitats are amongst the main drivers of evolutionary processes. Corresponding shifts in the behaviour and life history traits of species might in turn also alter ecosystem attributes. The reproduction of Western European fire salamanders (Salamandra salamandra), in small pond habitats instead of first order streams, is one example of a recent local adaptation. Since fire salamander larvae are important top-predators in these fish free habitats, their presence likely changes various aspects of ecosystem functioning. Here, it was analysed how the ecological performance of salamander larvae in ponds in the Kottenforst in Western Germany changed in comparison to sympatric stream populations. Further, it was analysed how their presence in ponds influenced key ecosystem attributes such as prey density and diversity and aquatic-terrestrial linkage. To assess the impact of the life cycle shifts in salamanders on the pond functioning, detailed investigations of salamander larvae population dynamics, phenology, and macroinvertebrate community development in ponds were combined with experimental manipulations of the salamander presence. In the first part of this study, the impact of pond presence of fire salamanders in terms of ecosystem functioning focussing on aquatic terrestrial subsidy transfer was calculated. The study could show, that the adaptation of fire salamanders to breed in pools led to strong increases of animal-mediated import of terrestrial matter into the aquatic habitats. The hypothesis about the impact on macroinvertebrate communities derived from these calculations was then tested experimentally. It was shown, that presence of salamander larvae could influence some taxa of macroinvertebrates but they had only limited effects on the food web structure in their aquatic habitats. Yet, a high relevance of the subsidy exchange from aquatic to terrestrial and its high relevance for the predator persistence in the system could again be confirmed. Moreover, it was demonstrated, that the larval behaviour and performance could have a high inter-annual variability as a reaction to contrasting ecosystem constraints in comparison to the stream habitats. A fact that integrally separates the pond ecotype from stream ecotype conspecifics.