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The study of biological invasions is a major research topic, both because of the ecological and economical damage caused by invasive species and also as a great natural experiment to study evolutionary responses of non-native populations to their new environment, and the factors influencing invasions. Introduced species often evolve rapidly, despite the assumed loss of genetic variation associated with bottlenecks during the invasion process. In order examine the processes and mechanisms affecting the outcome invasions I studied two non-native fish species, the topmouth gudgeon (Pseudorasbora parva) is an Asian cyprinid that is found in most European countries as a result of accidental introductions. Rainbow trout (Oncorhynchus mykiss) has been introduced from the United States for aquaculture and angling, however, despite numerous introductions, it has only been able to establish in few European waters. I used mitochondrial DNA and microsatellite markers to understand the invasion history of these species and the factors that influence their establishment success/failure. Part of the cytochrome b gene was analysed in European and native Asian P. parva populations and microsatellite markers were used to investigate the source populations of the species. The analyses elucidated the colonisation pattern of P. parva in Europe and supported the hypothesis that the species spread through long-distance and stepping-stone methods and originate from admixed source populations. In O. mykiss, part of the d-loop region of the mitochondrial genome was analysed to compare the phylogeographic structure of native US and introduced European populations to examine the spread of the species outside its native range, as well as to find out whether the resistant Hofer strain is the source population of the European rainbow trout populations. I found that European populations are likely to originate from various sources, mainly from California. The Hofer strain is likely to have contributed to some of the wild European populations. Assessing the role of these processes is fundamental in understanding invasive species and finding suitable management practices to control them. From an evolutionary point of view, I was able to detect some of the processes that are important during invasions, in these studies particularly the role of multiple introductions and introduction from genetically admixed source populations.

Understanding that disease is a limiting factor to the aquaculture industry together with the knowledge that drugs and chemotherapeutics can cause newly resistant bacterial strains, has driven attention to finding new prophylactic measures to control diseases that include vaccination and the use of “functional feeds” to modulate the fish immune system. The supplementation of immunostimulants into fish diets is already widely used in aquaculture. However, searching for new and effective substances is one of the targets of many fish feed suppliers, including EWOS Ltd. who have co-funded the work presented in this study. There are a number of immunostimulant molecules used at present. Some bacterial components such as LPS are used to enrich fish diets and have been described to improve the natural immune defences. In contrast, peptidoglycan (PG), another ubiquitous component of the bacterial cell wall, has so far received less attention and is therefore investigated in this present study. Its ability to stimulate innate immunity is assessed using antimicrobial peptides (AMPs) as molecular markers, which are known to be involved in the early response against a broad range of pathogens. To date, AMPs in fish are not well characterised and in most cases the mechanisms of pathogen killing as well as the pathways inducing their expression still remain to be elucidated. Initially the cloning and characterisation of three novel trout β-defensin genes (omDB-2, omDB-3, omBD-4) was performed, and the molecules compared to the previously reported omDB-1. Each β-defensin gene was fully cloned and preliminary expression work in vivo and in vitro revealed the ability of these genes to be induced by bacteria and viruses. Analysis of the gene organization found that all three new genes contained three exons divided by two introns. Constitutive expression of these genes was detected by real time PCR ofmucosal and systemic tissues from healthy fish, with omDB-3 and omDB-4 showing the highest expression levels. Following bacterial challenge in vivo, the defensin genes were induced at the three mucosal sites examined (skin, gill, gut), with levels of omDB-2 and omDB-3 increased some 16-fold in gut and gill respectively. Using polyinosinic polycytosinic RNA (polyI:C) as a viral mimic, all of the four trout -defensin genes were induced in head kidney primary leucocyte cultures at 4h post-stimulation, with omDB-1 and omDB-3 showing particularly high expression. To determine the -defensin spectrum of activity against 10 strains of Gram negative and Gram positive bacteria, transfected RTG-2 cell lines over expressing GFP and the target genes omDB-1, omDB-3 and omDB-4 -defensins were produced and their supernatants used. Results showed highest bioactivity against Gram negative bacteria, in particular the supernatant from omDB-1 transfected cells showed the widest range of activity towards the majority of selected bacteria. In addition immune relevant genes (Toll-like receptors, genes involved in the anti-inflammatory response and in the apoptosis process) were screened in normal cell lines stimulated with the supernatant of omDB-1, as well as in the RTG-2 cells transfected with the three different defensins. Results showed for all the cell lines, a clear link with the viral recognition receptors TLR 3 and TLR 9, which supported the poly I:C data reported in Chapter 2 and by the induction in omDB-1 and omDB-3 transfected cell lines of the IFN- gene known to be involved in the antiviral response. Trout β-defensins also up-regulated MHC II and the CCR6 receptor. To determine the effects of fish diets enriched with different concentrations of PG, three in vivo feeding trial experiments in rainbow trout were carried out. Effectiveness of the diets was assessed using gene expression of selected AMPs, including β-defensins, cathelicidins and liver expressed antimicrobial peptide molecules. Fish fed with diets containing either 10 mg/Kg or 50 mg/Kg of PG respectively, showed the highest up-regulation of AMPs at 14 days of feeding. Data showed omDB-2 in the gut as the most inducible gene in agreement with the results obtained in the first experiment and omDB-3 was the fastest to respond in skin and gill. In addition, after ceasation of feeding the enriched diet, modulation of AMP expression was still detectable 28 days later, although a lower degree of induction was found in such fish relative to those maintained on the enriched diet. A final PG feeding trial was combined with a Yersinia ruckeri bacterial challenge which used two PG supplemented diets containing 10 mg/Kg and 50 mg/Kg of immunostimulant, and a commercial β-glucan supplemented diet (as a positive control), and fed to trout for 7 and 14 days before intraperitoneal injection challenge of the fish. Only a delay in the mortality rate was found in fish fed for 14 days with the 10 mg/Kg diet, with no clear protection from any of the functional feeds assessed. Finally, at least 500 bp of the regulatory 5’ end flanking region of two defensin (omDB-1 and omDB-2) and two liver expressed (hepcidin and LEAP-2A) genes were cloned and sequenced. In addition, the promoter sequence already known for the cathelicidin-1 gene was used in this study. Bioinformatic tools were used to search for putative transcription factor binding sites, and revealed the presence in all promoters of regulatory elements which could enhance or inhibit the expression of these genes, in response to different stimuli.

This study investigated the biological activity of a macrophage activating factor (MAF) produced by activated lymphocytes from the rainbow trout (Oncorhynchus mykiss) and attempts to discover its molecular source. Peripheral blood lymphocytes were shown to release factors with MAF activity following incubation with a variety of stimulants and were subsequently shown to activate macrophages using at least two different methods, the nitroblue tetrazolium (NBT) colourimetric assay and the luminol-dependent chemiluminescent assay. The latter technique detected an immediate response which decayed over a 40 minute period on the addition of cell-free supernatants from activated lymphocytes to macrophages. A number of molecular approaches, including degenerate PCR primer amplification, DNA cross-hybridisation and cDNA library screening were used in this study to try to isolate any cytokine genes from Oncorhynchus mykiss. As a control β-actin cDNA was successfully amplified from Oncorhynchus mykiss using primers based on the salmon sequence. The Oncorhynchus mykiss orthologue of IFN-y was initially targeted. However, although a PCR product of the appropriate size was amplified using degenerate primers based on mammalian and avian IFN-y sequences, the sequence was not related to IFN-y or any other known Oncorhynchus mykiss sequence. A similar strategy was used to try and amplify the Oncorhynchus mykiss orthologue of mammalian IL-15. Again despite amplification of a DNA fragment of approximately the correct size there appeared to be no relationship between it and the known IL-15 sequences. As an alternative strategy a cDNA library from stimulated peripheral blood lymphocytes (PBLs) was constructed and screened using cDNA probes derived from stimulated and non-stimulated PBLs in order to detect mRNAs which might have been upregulated as a result of in vitro stimulation. A number of positive clones were obtained from the differential screening of the library including cDNAs showing similarity to other unidentified fish sequences as well as to a number of proteins predicted to be involved in regulation of cell proliferation, neocorticogenesis and embryo development. Additionally. the library was also screened using ovine cytokine cDNA probes. although no positively hybridising clones were obtained. The ovine IFN-y gene was also used to probe genomic DNA from Oncorhynchus mykiss. but unlike previous studies with human IFN-y gene no hybridisation between the ovine IFN-y gene and Oncorhynchus mykiss DNA was observed. This investigation highlights the potential difficulties of using various molecular strategies such as DNA cross-hybridisation or PCR techniques for the cloning of fish cytokine sequences. Consequently, future strategies for cloning fish cytokine genes may require targeting the biological activity through expression libraries.