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With the culture of marine fish species increasing, interest in ways to combat infection in these species has grown. One such avenue is the search for novel antimicrobials. Antimicrobial peptides (AMPs) are host derived molecules with a significant role in innate immune responses and have been studied extensively in mammals. Well known AMP families include β-defensins, cathelicidins, liver expressed antimicrobial peptides and, specific to fish, the piscidins. While these molecules have been found in some fish species mining of expressed sequence tag (EST) databases was carried out to identify them in other fishes. In this study we have found new members of the β-defensin, cathelicidin and piscidin families of AMPs, while also identifying new hepcidins and LEAP-2 molecules in a diverse range of fish species. The rest of this thesis focused on one major AMP family, the cathelicidins, which in fish have been mainly identified in salmonids. They are stored in secondary granules of neutrophils as inactive pro-peptides and when infection occurs, a cleavage event releases the active mature form that effects microbicidal activity. Characterization of the cathelicidin genes present in Atlantic cod (Gadus morhua) and haddock (Melanogrammus aeglefinus) was undertaken. A number of ESTs with significant homology to cathelicidins were found in Atlantic cod. Primers were designed to amplify these sequences by PCR in both gadoid species, and the gene products were cloned and sequenced. One and two cathelicidin genes were identified in cod (gmCath1) and haddock (maCath1; maCath2), respectively, which differ in the length of their active mature peptides. Gadoid cathelicidins were constitutively expressed in many tissues and were upregulated by a range of immunostimulants. Analysis of the gmCath1 promoter revealed numerous putative transcription factor binding sites, some of which were used in an expression vector construct to show upregulation of gene expression when stimulated with the viral RNA mimic Poly I:C. Synthetic and recombinant gadoid cathelicidins were produced but did not appear to display antimicrobial activity, so this requires further investigation. The discovery of new members of the major AMP families in such a diverse range of fish species bodes well for future research into the use of these antimicrobials as potential disease control molecules in the aquaculture industry. Particularly for Atlantic cod this is crucial as the major cod farms in Norway are experiencing difficulties with bacterial infections for which there is no vaccine at present.

Immune systems are thought to be costly to maintain and express in a variety of taxa. Evidence for this comes from observations that mechanisms which deal with pathogenic challenge are often extremely variable and are induced in the presence of an immune challenge, rather than being constitutively active. This thesis presents work aimed at testing predictions arising from these ideas, using the mealworm beetle T. molilor (Coleoptera: Tenebrionidae) as a model system. Assays were developed which reflected three specific aspects of immunity (haemocyte count, phenoloxidase levels and resistance to a generalist fungal pathogen), thereby allowing aspects of immune function to be quantified. It was shown that the level of fungal resistance differed in beetles reared gregariously (higher resistance) and solitarily (lower resistance). Conspecifics are a source of disease (Freeland 1983), so this is an example of immune defences being induced in situations with a higher risk of pathogenesis (density dependent prophylaxis). A strong predictor of fungal resistance was the degree of melanisation of the adults' cuticles. This trait was shown to be highly heritable (59%), as was the total haemocyte count of an individual: an important aspect of general invertebrate immunity. Selection for cuticular melanisation resulted in a rapid response, confirming the existence of large amounts of additive genetic variance for this trait. Fungal resistance showed a correlated 111 response to selection for cuticular melanisation, indicating that this too has additive genetic variance. Lines selected for darker cuticles showed higher levels of fungal resistance than those selected for lighter cuticles. Cuticular melanisation and fungal resistance are therefore genetically correlated, and the former can be used as an indicator of the latter in T. molitor. No specific costs of cuticular melanisation or fungal resistance were identified. A correlated response to selection for cuticular melanisation on larval competitive ability was investigated, but no such response was seen. Thus the mechanisms maintaining variability and inducibility in cuticular melanisation and fungal resistance are unknown. This thesis has therefore identified patterns of immune expression consistent with the hypothesis that immunity has associated costs, although these costs have not been shown. It has also identified a potentially novel role for cuticular melanisation, as an indicator of immunity to fungal pathogens.