http://hdl.handle.net/1893/13 Electronic theses of Aquaculture Students. Fri, 02 Jun 2017 07:48:57 GMT 2017-06-02T07:48:57Z http://hdl.handle.net/1893/25419 Title: Assessment of a novel filter system for recirculating aquaculture Authors: Montorio, Luca Abstract: The aim of this project was to investigate the usage of manganese dioxide ore as a bio-filter media to remove metabolites in aquaculture closed system, and to determine whether manganese toxicity would at the same time represent a risk to fish. Initial work investigated the physical properties of manganese dioxide and its chemical interaction with ammonia and nitrite in the absence of biological activity. Subsequently, two pilot-scale pressurised filters were installed in a commercial scale hatchery in order to compare the metabolite removal performance of manganese dioxide against silicate sand in the presence of biological activity commonly found in aquaculture conditions. The investigation suggests that Mn medium is more reliable in converting ammonia to nitrate without producing a residual output of nitrite. The superior performance ofMn media compared with sand appears to be mainly related to the physical structure of the manganese ore. Furthermore, the Mn medium did not appear to be soluble in the ambient conditions normally found in aquaculture-closed system. From the design point of view, due to the higher ammonia and nitrite removal rates, a shorter retention time and a lower volume of media are required in the case of manganese dioxide technology compared with sand media. As a result, it is much easier to size a biofilter with Mn media. Manganese systems have a comparable total costs to conventional sand media, but using the Mn technology provides a more reliable control of toxic nitrite, thereby reducing risks offish loss and hence with reduced expected produ Thu, 01 Jan 2004 00:00:00 GMT http://hdl.handle.net/1893/25419 2004-01-01T00:00:00Z http://hdl.handle.net/1893/25417 Title: Virulence and required genes in the fish pathogen Vibrio anguillarum Authors: McMillan, Stuart Abstract: Vibrio anguillarum infects many fish species in aquaculture, reducing farm productivity and negatively impacting fish welfare. Deeper understanding of the biology of V. anguillarum, particularly during infections in vivo, will help to improve disease prevention and control. Thus, the aim of this thesis was to provide further insight into the infection biology of V. anguillarum with a view to identifying better ways to reduce the impact of this pathogen in aquaculture. Conventional studies on virulence, particularly those aiming to identify novel virulence factors, often employ transposon mutagenesis where the functions of individual genes in the bacterium are disrupted. These mutant libraries are screened to identify those with attenuated virulence, allowing subsequent identification of the gene responsible. Usually the native fish host would be used but such studies are increasingly difficult to perform due to regulations on vertebrate experiments and ethical concerns. As a result, alternative invertebrate hosts are now an important means to studying microbial infections, but few models have been assessed for bacterial pathogens of fish. In this thesis, larvae of the greater wax moth Galleria mellonella were evaluated as an alternative host to investigate V. anguillarum virulence. Wild-type V. anguillarum isolates killed larvae in a dose-dependent manner, replicated in the haemolymph, and larvae infected with a lethal dose of bacteria could be rescued by antibiotic therapy, thus indicating that V. anguillarum established an infection in G. mellonella. Crucially, virulence of 11 wild-type V. anguillarum isolates correlated significantly between larva and Atlantic salmon infection models, and studies with isogenic mutants knocked out for various virulence determinants revealed conserved roles for some in larva and fish infections, including the pJM1 virulence plasmid and rtxA toxin. Thereafter, 350 strains from a V. anguillarum random transposon insertion library were screened for attenuated virulence in G. mellonella. In total, 12 strains had reduced virulence and in these mutants the transposon had inserted into genes encoding several recognised and putative virulence factors, including a haemolytic toxin (vah1) and proteins involved in iron sequestration (angB/G and angN). Importantly, the transposon in one strain had inserted into an uncharacterised hypothetical protein. Preliminary investigations found this putative novel virulence factor to contain a GlyGly-CTERM sorting domain motif, with sequence similarity to VesB of Vibrio cholerae which is involved in post-translational processing of cholera toxin. Finally, three transposon insertion libraries were mass sequenced on a MiSeq platform to identify V. anguillarum genes lacking transposon insertions. These genes were assumed to be ‘required’ for viability in the conditions under which the mutants were selected, in this case tryptone soya agar. In total, 248 genes lacked a transposon insertion and were the putative ‘required’ genes, and these may be important chemotherapeutic targets for new approaches to combat V. anguillarum infections. This thesis has furthered our understanding of the biology of the important fish pathogen V. anguillarum using an ethically acceptable approach, and the findings may assist with new ways to reduce the burden of this bacterium in aquaculture. Mon, 31 Oct 2016 00:00:00 GMT http://hdl.handle.net/1893/25417 2016-10-31T00:00:00Z http://hdl.handle.net/1893/25393 Title: Introgression patterns in Scottish blue mussel (Mytilus edulis) populations Authors: Wilson, Joanna Abstract: Background: The blue mussel, Mytilus edulis L., is an important contributor to the shellfish sector of Scottish aquaculture, with 7,270 tonnes worth £8.8 million being produced for the year 2015. Since 2010, production values have fluctuated as a result of inconsistent spat settlement, several business closures, and heightened levels of marine toxins in some areas. On Scotland’s west coast, some farms (most notably Loch Etive) have suffered production losses from the appearance of non-marketable mussels with particularly fragile shells and poor quality meat. Recent research has demonstrated that these undesirable traits have a genetic factor, linked to the presence of a non-native but related species Mytilus trossulus (Gould, 1850) and often its hybrids with the native M. edulis. M. trossulus has been classed as a commercially damaging species under Scottish law, but there is insufficient data on hybridisation and introgression patterns in Scottish mussel populations to evaluate any possible impacts this could have on production. Existing research has focused on single locus genotyping to identify Mytilus spp. and their hybrids in Scotland. By instead utilising multilocus genotyping, introgression could be identified and a better understanding of population structure could be gained, with implications for management to maintain productivity and profitability. The aim of the research presented here was to develop and validate a suite of new species diagnostic markers for multilocus genotyping of field populations of Scottish mussels, thereby establishing a more complete picture of the taxonomic relationships between species than previous studies have permitted. Results: Analysis of SNPs identified with RADseq confirmed the presence of three genetically distinct Mytilus species in Scotland: M. edulis, M. galloprovincialis and M. trossulus. RADseq and KASP genotyping technology successfully identified and validated a suite of 12 highly robust diagnostic SNP markers for multilocus genotyping of Mytilus mussel populations. These markers permitted more comprehensive genotyping than previous studies had, allowing presumed pure species individuals to be distinguished from first generation (F1) hybrids and introgressed (FX) genotypes in reference populations, and subsequently presented the possibility of exploring introgression in a wider scale study. Multilocus genotyping of mussel populations from around Scotland revealed widespread introgression of M. edulis with both M. galloprovincialis and M. trossulus. No pure M. galloprovincialis was identified and pure M. trossulus was restricted to a single site in Loch Etive, possibly part of a relict population. F1 hybrids between M. edulis and M. trossulus were identified in Loch Etive and in Loch Fyne on the west coast. This was evidence of ongoing hybridisation and suggested an active hybrid zone existed in Scotland, something that previous single locus genotyping studies had not acknowledged. A link between shell fragility and M. trossulus introgression was recognised at a single site outside of Loch Etive, but this was not apparent anywhere else and the actual causes of shell fragility remain unevaluated. There was a clear difference between the genetics of most farmed stock and wild populations, which indicated an anthropogenic effect on introgression and subsequent species composition, and had implications for future farm site selection and broodstock sourcing. Temporal species composition in Loch Etive differed over a short time period, but high proportions of M. trossulus alleles were observable some 25 months after a major fallowing event had taken place. Pure M. trossulus was also identifiable, which was consistent with the presence of an established population of M. trossulus existing in this area. Conclusion: Multilocus genotyping has produced a more in depth picture of species diversity in Scottish mussel populations. SNP assays revealed widespread introgression between three genetically distinct species – M. edulis, M. galloprovincialis and M. trossulus – and furthermore recognised that, to date, single locus genotyping has overestimated the abundance of pure Mytilus mussels in Scottish waters. However, this hitherto unidentified genetic complexity does not appear disadvantageous to mussel production, despite the prevalence of M. trossulus introgression among farmed populations, and it is somewhat unlikely that genetics are the sole cause of undesirable shell characteristics among Mytilus spp. mussels. Fri, 30 Sep 2016 00:00:00 GMT http://hdl.handle.net/1893/25393 2016-09-30T00:00:00Z http://hdl.handle.net/1893/25380 Title: The isolation, identification and exploration of the biophysiological significance of plasma biliverdin in the ballan wrasse (Labrus bergylta) Authors: Clark, William D Abstract: Labrus bergylta (ballan wrasse) have recently emerged as a key resource to aquaculture through proven efficacy in controlling infestations of sea lice (Leclercq et al., 2014a). However, due to complex ecology, and a complete lack of sexual dimorphism gender identification endures as a key restriction to optimising broodstock management therefore male selection and establishing optimal sex ratios is difficult (Talbot et al., 2012). L. bergylta, are noted to demonstrate unusually coloured plasma ranging in hue from green to blue with the haem catabolite biliverdin established as the causal pigment in the majority of cases (Abolins, 1961). As most vertebrates excrete biliverdin, or rapidly metabolise it to prevent toxicity, accumulation to such excess is a phenomenon which merits attention. Notably, correlation between plasma biliverdin and gender has been reported in some Labridae. Although patterns vary between species, the abundance or characteristics were such that sexual identity could be established (Gagnon, 2006). Pigment analysis was therefore proposed as a potential sex-marker in L. bergylta. In the initial experimental phase (Chapter 3), the ultimate aim was to isolate and identify the blue pigment from L. bergylta plasma, and to develop a method of quantification. The initial phase confirmed the target pigment was biliverdin IXα by visible spectroscopy, TLC, HPLC, MSMS, and a series of reactions. Following this, a protocol was developed (Chapter 2) to quantify the pigment. This method was applied accross plasma sampled from four geographically distinct wild populations with established biometrics including age, mass, length, gender and external phenotype. Subsequent analysis revealed that although pigment abundance did not vary relative to ontogeny, and there was no difference in concentration between the binary genders, plasma biliverdin was depleted in individuals undergoing sex change. Although this conclusion was complicated by significant biliverdin variation relative to origin and phenotype, which were interrelated based on relative distributions across populations, further analysis of plasma pigment in related species identified that biliverdin accumulation was associated with protogynous species. Considering the anti-oxidant capacity of biliverdin and other potentially relevant functions, this was indicative of association with the tissue remodelling processes which accompany inversion. During Chapter 3 it was noted that the biliverdin appeared tightly bound to a protein moiety. Based on the hypothesis that the pigment was actively managed and accumulated in L. bergylta plasma by this association, the next phase of experiments (Chapter 4) was an exploration of biliverdin and its binding protein in L. bergylta. The experiments revealed plasma biliverdin comigrated with the protein such that it was depleted from solution at the same rate indicating that all of the pigment was associated. Subsequent electrophoretic experiments using the fractionation products supported this, and UV fluourescence identified fragments of interest in the 25-28 kDa region. To confirm observations from the previous cross species comparison, the study was similarly expanded to include other Labrini. This revealed that although the 25 kDa band was common to all species, and genders, the 28 kDa band was collocated with the protogynous, and as such hyperbiliverdinaemic species. The 28 kDa band was sequenced using MSMS, and was identified as similar to the lipocalin Apolipoprotein A1. In combination with the properties of biliverdin, and considering that ApoA1 is analogous to serum albumin in many telesots, this supported the chromoprotein association as the main mechanism of biliverdin accumulation in such species. Further to the proposed function of biliverdin with inversion processes, and considering relevant literature, the active properties of ApoA1 suggested additional associations with prolonged altered states of metabolism which considering the ecology of L. bergylta would include gender transition, overwintering torpor and prolonged micronutrient limitation, all of which occur simultaneously. Other potential roles include modulating inflammatory responses, inhibiting pathogenic incursions and acting as an external point of contact innate immune response. From this, it was concluded that the data fully supported the previous assertions of biliverdins relevance in protogynous species, and identified a number of properties which could be of great interest to the industry in terms of welfare. The final experimental phase (Chapter 5) had two main aims. The first was to establish whether protogynous inversion could be artificially induced in L. bergylta as a means of generating male fish, and whether size had any effect on the process. The second was then to utilise controlled induction for tracking biliverdin mobilisation across the process to test the previous hypothesis. The preliminary trial demonstrated that both androgen inhibition and non-aromatisable testosterone could stimulate inversion in female L. bergylta. From this, the second trial then determined that although there was a dose dependant effect in that high androgen dosages appeared to compress the inversion process, relative size was not a factor. Gonad histology was used to create a unified scale of protogynous transition which could be expressed as a gradient to structure the biliverdin analysis. Although the biliverdin data demonstrated cryptic trends at the higher resolution gender scales, when the endpoint was condensed back to the binary gender scale employed previously (Chapter 3), the prior assertion of depletion during transition, and therefore the association with sex change associated tissue remodelling was supported. Ultimately this thesis revealed links between the biliverdin macromolecule and the highly unusual metabolic and physiological demands of gender transition in sequentially protogynous hermaphroditic temperate wrasse species. Thu, 01 Sep 2016 00:00:00 GMT http://hdl.handle.net/1893/25380 2016-09-01T00:00:00Z