| Summary: | Formulated feeds with a relatively low (< 5 % of dry mass) kelp (Ecklonia maxima) inclusion level are widely used on commercial abalone (Haliotis midae) farms in South Africa. Although the use of kelp, a major constituent of the natural diet of H. midae, as a dietary supplement is considered to enhance abalone growth and feed utilisation, there are no published studies which quantify the effects of kelp inclusion in formulated feeds. Furthermore, the physiological mechanisms by which kelp supplementation may positively influence abalone digestive physiology and growth are largely unknown. As the kelp supplement is comprised mostly of soluble fibres and abalone gut bacteria associated with macroalgae (and its fibrous polysaccharides) are known to play a key role in digestion, it was hypothesised that the kelp supplement influences the gut-bacterial community profiles of cultured abalone through prebiotic and other metabolic effects. The present thesis thus examined the effect of kelp supplementation on the performance of abalone (Haliotis midae) fed formulated feeds and explored the influence of a kelp supplement on the abalone gut microbiota and its function in the gastrointestinal tract. The key hypotheses of the study were that kelp supplementation in formulated feed: 1) enhances abalone growth; 2) causes a shift in abalone gut-bacterial community composition through a prebiotic-like effect; 3) may induce changes in crop morphology as a result of potential bacterial-associated increases in volatile short-chain fatty acids, and 4) alters digestive enzyme activities in the abalone gut through changes in bacterial-derived (exogenous) digestive enzymes. The growth-promoting efficacy of low-level kelp supplementation was tested by feeding isonitrogenous and isoenergetic experimental feeds containing 0.00 – 3.54 % kelp (dry mass) to sub-adult abalone (~43 mm shell length) for eight months under commercial farm conditions. The growth trial established that kelp supplementation (0.44 – 3.54 % of dry mass) promoted faster growth and improved feed conversion and protein efficiency ratios in cultured abalone compared to abalone fed the non-supplemented control diet, while there were no significant differences in growth for abalone fed the different kelp-supplemented diets (0.44, 0.88, 1.76 and 3.54 % of dry mass). Feed conversion and protein efficiency ratios displayed significant correlations with kelp level in the range of 0.00 – 3.54 % dry mass, and it is therefore recommended that kelp be included in the formulated feeds of cultured South African abalone at a rate of up to 3.54 % of dry mass. A kelp-supplemented (0.88 % dry weight inclusion) feed was fed to abalone under farm conditions to compare gut physiological parameters (crop morphology, digestive enzyme activities and the gut microbiota) in abalone against that of abalone fed an isonitrogenous and isoenergetic non-supplemented control feed. To establish if the observed higher abalone growth rates were related to improved gastrointestinal tract epithelium activity and integrity, as reflected by epithelial cell growth in response to potential changes in bacterial-derived short-chain fatty acid production, crop epithelial morphology was compared between abalone fed the kelp-supplemented and control feeds. Kelp supplementation did not induce any observable changes in crop epithelial cell height for farm-reared sub-adult abalone fed the experimental diets on-farm for seven weeks. This was attributed to the similar macronutrient compositions of kelp-supplemented and control diets and/ or the common diet history of experimental abalone from weaning to the initiation of the experiment. Digestive enzyme activity was compared between abalone fed a kelp-supplemented and a control feed during an on-farm feeding trial with sub-adult abalone. Gut samples were collected after seven weeks and colorimetric enzyme assays were performed for the polysaccharide-degrading enzymes amylase, alginate lyase, laminarinase and fucoidanase, and for acid protease, trypsin and chymotrypsin activity. Amylase and alginate lyase activities were relatively high, compared to the other enzymes. Polysaccharidase and acid protease activity levels did not differ significantly between abalone fed kelp-supplemented and control feeds, but a greater variability in enzyme activity levels was observed in abalone fed the control diet. It was hypothesised that this might be due to the kelp supplement promoting a more stable and less opportunistic gut-bacterial community than the control diet. Pooled gut samples of abalone fed the kelp-supplemented diet were used for proteomic analyses to identify the composition of enzyme proteins of both endogenous and exogenous origin in the abalone digestive system. The key polysaccharidases and proteases in the gut samples of kelp-supplemented formulated feed-fed abalone were all of abalone origin, whereas the bacterial enzymes were of the types that form part of intermediate reactions in metabolic pathways. The results suggested that bacterial enzymes play a different role to abalone endogenous enzymes in the digestion of formulated feed. While abalone enzymes appear to be the main degraders of carbohydrate and protein macromolecules, the profile of exogenous enzymes suggests that they perform bioconversions of smaller organic compounds. The profiles of gut-bacterial communities of farm-reared sub-adult abalone fed kelpsupplemented and control feeds on-farm for seven weeks were analysed with metagenomic pyrosequencing and DGGE analyses, using 16S rDNA-targeted amplified DNA. The results indicated a shift in gut-bacterial composition with a higher abundance of Mollicutes in abalone fed kelp-supplemented feed compared to those fed the control feed. DGGE band patterns displayed a greater within-group similarity in gut bacteria for abalone fed the kelpsupplemented diet and the presence of unique and variable bands for bacteria in the guts of abalone fed the control diet. It was concluded that when cultured abalone are fed kelpsupplemented formulated feeds, more stable gut bacterial communities are present compared to a more opportunistic gut-bacterial community in abalone fed non-supplemented feeds, and that the observed increase in Mollicutes could reflect the restoration of the abalone gut microbiota to a more natural state. The novel application of proteomics to abalone nutrition in the present study demonstrated that gut-bacterial enzymes may form part of many different metabolic pathways and suggests that the metabolism of the gut microbiota serves as an extension of the abalone’s digestive metabolism. Future studies should quantify the contribution of commensal gut-bacteria to cultured abalone nutrition by employing metabolomic studies to characterize the utilisation of bacterial-derived metabolites by the abalone host.
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