Algal/invertebratee ndosymbioseas re common in the marinee nvironmenta nd appeart o be nutritional in nature. The giant Tridacnid clams form a mutualistic extracellular endosymbiosis with dinoflagellate algae (zooxanthellae) of the species Symbiodiniunt microadriaticum. The large size of the Tridacnids is generally attributed to the nutritional role of their endosymbionts. This thesis examines the nature and mechanisms by which important biochemicals, such as lipids, are translocated from the symbionts to the host. Methodologies have been developed to enable the determination of the carbon isotope composition of individual saturated and polyunsaturated fatty acids to enable natural abundance isotopic variations in these compounds to be investigated in the Tridacnid-algal symbiosis. Compound specific isotope analysis of fatty acids in different species of Tridacnids, their zooxanthellae and non-symbiotic species reveal that several fatty acids are apparently directly translocated from algae to host. Evidence is presented for the synthesis of fatty acids from acetate by the clam. Carbon isotopic data also reveal that carbon limitation may play a role in lipid metabolsim in giant clams. Compound specific isotope analysis has also been used to investigate the contribution of translocated compounds during a diel cycle in clam haemolymph and reveals that concentrations of key metabolites vary over the diel cycle as a function of irradiance. The cyclic sugar alcohol scyllo-inositol was detected in clam haemolymph, which may be related to osmoregulation in the clam or to a signalling role in cell proliferation. Oxygen and carbon stable isotope compositions and strontium abundances of shell carbonate are examined in several clams subjected to varying degrees of phosphate and ammonium nutrient supplementation to investigate the relationship between growth rate and stable isotope compositions and strontium contents.

Heavy metal contamination in food is a worldwide concern. Man-made ponds are domestic sites in the production of Procambarus clarkii and Procambarus zonangulus, two edible species of crayfish. Ponds may be constructed in former sugar cane or rice fields. Crayfish farming is an ancillary seasonal business within the rice-growing season. The use of products to control insects, pests, and weeds in rice and sugar cane production, may cause an accumulation of heavy metals in the crayfish tail within pond structures. Arsenic, cadmium, copper, and zinc are heavy metals that are absorbed through the roots of and distributed through rice products. Metabolites associated with rice products are absorbed in the human body. Research suggests that metabolites associated with heavy metals cause disease in animals and humans.

Most microbial organic molecules are secondary metabolites which consist of diverse chemical structures and a range of biological activities. Actinobacteria form a large group of Eubacteria that are prolific producers of these metabolites. The recurrence of pathogens resistant to antibiotics and a wider use of these metabolites apart from their use as anti-infectives, has been the impetus for pharmaceutical companies to search for compounds produced by rare and existing actinobacterial cultures. Accessing microbial biosynthetic pathway diversity has been possible through the use of sensitive and innovative molecular detection methodologies. The present study evaluated the use of molecular based screening as a rational approach to detect secondary metabolite biosynthetic genes (SMBG) in uncharacterised natural Actinobacterial populations. A polymerase chain reaction (PCR) approach was selected for ease of application and high sample processivity. Rational designed screening approaches using PCR in the discovery of SMBG, involved identifying common functions in secondary metabolite biosynthetic pathways, such as condensation reactions in polyketide synthesis, genes encoding these functions, and using conserved regions of these genes as templates for the design of primers to detect similar sequences in uncharacterised actinobacteria. Design of primers involved rigorous in silico analysis followed by experimentation and validation. PCR screening was applied to 22 uncharacterised environmental isolates, eight of these displayed the presence of the ketosynthase (KS) gene belonging to the type I polyketide synthases and eight contained the ketosynthase (KSĄ) gene belonging to the type II polyketide synthases, six of the isolates contained the presence of a presumptive dTDP-glucose synthase (strD) gene which is involved in the formation of deoxysugar components of aminoglycoside antibiotics and one isolate contained the presence of a presumptive isopenicillin N synthase (pcbC) gene involved in beta-lactam synthesis. Alignments of partially sequenced PCR products from isolates A1488 and A3023 obtained using type II PKS primers showed close similarities with KSĄ genes from antibiotic producing actinobacteria. Similarly, alignments of sequences from isolates A1113 and A0350 showed regions of similarities to KS genes from antibiotic producing actinobacteria. Fermentation techniques were used for inducing expression of secondary metabolites from the uncharacterised actinobacteria isolates. By using antimicrobial guided screening it was determined that most of the isolates possessed the capacity to produce antimicrobial metabolites. Dominant antagonistic activity was detected against Gram positive bacteria and to a minor extent against fungi. Optimal fermentation liquid media were identified for certain isolates for the production of antimicrobial metabolites. Two alternative fermentation methods; solid-state and liquid-oil fermentations were evaluated to improve secondary metabolite production in the uncharacterised isolates. Solid-substrate fermentation showed that it could induce a complex metabolite pattern by TLC analysis, however this pattern varied according to the substrate being used. Liquid media supplemented with refined oils, showed a positive response indicated by higher antibacterial activities detected. Evaluation of semi-purified organic extracts identified two isolates A1113 and A0350 producing similar antimicrobial metabolites as detected by HPLC/UV/MS, a literature database search of similar compounds containing the same molecular weight identified the compound as belonging to the actinomycin group of compounds. A complex metabolic pattern was identified for isolate A2381, database searching identified some of the compounds as having similar molecular weights to actinopyrones, trichostatins, antibiotics PI 220, WP 3688-5 and YL 01869P. Drug discovery screening can serve to benefit from PCR detection of biochemical genotypes in initial screens, providing a rapid approach in identifying secondary metabolite producing capabilities of microorganisms prior to the commencement of costly and time consuming fermentation studies. Additionally the identification of biochemical genotypes allows a directed approach in using fermentation media designed to induce biosynthetic pathways of specific classes of compounds.

Thesis (M.S.)--University of Delaware, 2007. / Principal faculty advisor: William W. Saylor, Dept. of Animal and Food Sciences. Includes bibliographical references.

Graduation date: 1997

<p>The aim of this project is to characterise novel psychrotrophic actinomycetes isolated from Antarctic Dry Valley soils and to isolate and characterize secondary metabolites produced by these actinomycetes.</p>

Graduation date: 1995

Graduation date: 1993

Thesis (Ph. D. (Chemistry))--Rhodes University, 2003.

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