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The decreasing supply and high cost of fishmeal, as well as the sustainability issues of this finite raw material, will continue to push the industry to concentrate their efforts on finding alternative sources of protein to substitute fishmeal in aquafeeds. From all the possible alternatives, e.g. animal by-products, fishery by-products, single-cell protein and the recent insect meal options, plant-based meals seem to be one of the major contributors to reduce reliance upon marine sources. However, when considering plant-based meals for aquafeeds it is commonly agreed that one of the negative aspects is the presence of anti-nutritional factors (ANF’s; e.g. cyanogens, saponins, tannins, etc.) that are detrimental to fish and shrimp (Krogdahl et al., 2010). Although there are processes to remove or inactivate many of these ANF’s, the same does not apply for mycotoxins which are reasonably stable to processing conditions (Cheli et al., 2013). The effects of mycotoxins in fish and shrimp are diverse, varying from immunosuppression to death, depending on toxin-related (type of mycotoxin consumed, level and duration of intake), animal-related (animal species, sex, age, general health, immune status, nutritional standing) and environmental-related (farm management, biosecurity, hygiene, temperature) factors. Therefore, it is often difficult to trace observed problems back to mycotoxins. The main goal of this thesis is to increase the awareness of mycotoxin contamination in aquafeeds and its consequences to aquaculture species, especially characterizing the impact of deoxynivalenol in rainbow trout (Onchorhynchus mykiss). In Chapter 1 the known mycotoxin occurrence and co-occurrence in aquaculture finished feeds are described and critically reviewed by correlating the extent of mycotoxin contamination in aquaculture feeds to its impact in aquaculture species. This chapter also contributes to understanding the risk of mycotoxin carry-over to aquaculture seafood products. Additionally, this chapter aims to expose the scientific community, the regulatory authorities and the aquaculture industry, to the main challenges and myths that the industry faces in developing mycotoxin management strategies. Chapter 2 presents the results the impact of deoxynivalenol (DON) in rainbow trout and the difficulties to diagnose DON ingestion. Here was explored two different DON contamination scenarios, i.e., the effect of short-term feeding of high levels of DON and the effects of long-term feeding of low levels of DON (a more common scenario in aquaculture industry). It was concluded that the ingestion of DON by trout over short-term periods at high dosages (50 days; 1,166 μg kg -1 and 2,745 μg kg -1) impacts growth performance, especially feed intake, with minor or variable biochemical changes in trout blood and histopathological changes. In this case, some fish did exhibit clinical symptoms (i.e., anal papilla), which could be attributed to the DON treatment (reported for the very first time). This chapter also reports, for the first time, the effects of the long-term exposure of rainbow trout to low concentrations of DON (168 days; 367 μg kg -1 DON). Ingestion of DON in the long-term study was asymptomatic; however, the fish started to reduce their growth performance 92 days after ingesting DON. Such low contamination levels, which might be unnoticed by farmers, may have economic consequences for aquaculture. Here it was concluded that the short-term effect of DON ingestion cannot be extrapolated to other contamination scenarios, e.g., long-term exposure. Chapter 3 aimed to elucidate the impact of DON on rainbow trout and study the reasons behind the apparent lack and/or high variability of clinical signs during DON ingestion (as reported in chapter 2). With this experiment, we further confirmed that ingestion of DON by rainbow trout is mainly characterised by impaired growth performance and reduced feed intake, with the total absence of any visible clinical signs (up to 11,412 ± 1,141 μg kg -1). Proteolytic enzyme activities (pepsin, trypsin and chymotrypsin) in trout were altered by DON ingestion. The impact of DON on the abundance of specific measured mRNA transcripts was unexpected with higher expression levels for insulin-like growth factors, igf1 and igf2, which are directly related to elevated insulin levels in plasma. This can also in part be influenced by the trypsin activity and by npy, given its higher mRNA expression levels. The apparent digestibility of dry matter, protein and energy was not affected by dietary levels of DON, however, nutrient retention, protein, fat and energy retention were significantly affected in animals fed DON. Adenylate cyclase-activating polypeptide (PACAP) expression seems to play an important role in controlling feed intake in DON fed trout. In the present study, we have shown for the first time that DON is metabolized to DON-3-sulfate in trout. DON-3-sulfate is much less toxic than DON, which helps to explain the lack of clinical signs in fish fed DON. Being a novel metabolite identified in trout makes it a potential biomarker of DON exposure. It was suggested that the suppression of appetite due to DON contamination in feeds might be a defence mechanism in orderto decrease the exposure of the animal to DON, therefore reducing the potential negative impacts of DON. In Chapter 4, this thesis further explored the current knowledge of DON toxicokinetics and rainbow trout DON metabolism, accessing the organ assimilation rates, excretion and possible biotransformation kinetics. Here we found that an hour after tube-feeding, [3H]-DON was detected in the liver samples of fish, indicating a rapid absorption of DON. In the first hour, [3H]-DON was present in the GIT (20.56 ± 8.30 ng). However, 6.19 ± 0.83 ng was also detected in the water at this sampling point. The fast excretion of [3H]-DON (faster than the average trout gastric emptying time) suggests that DON, as a water-soluble compound, is excreted with the liquid phase of the chyme. The presence of [3H]-DON in the GIT was stable during the first six hours. This long residence time of DON in the GIT may compromise the health of the GIT and favour absorption. Our data suggest that an effective DON detoxifying method should have a period of action of ≤ 6 h. Furthermore, as most of the excretion can be expected to happen after six hours, the detoxification should be irreversible at GIT conditions. Results of this PhD study contributes to better understand the importance and the risk of mycotoxin occurrence and co-occurrence in aquaculture finished feeds, highlighting concerns regarding the unvalued risk of mycotoxin carry-over to aquaculture seafood products. Here is highlighted that long-term exposure to DON is normally asymptomatic which might be unnoticed by farmers, however, representing economic consequences for aquaculture (reduced growth performance). Suppression of appetite due to DON contamination in feeds seems to be a defence mechanism in order to decrease the exposure of the animal to DON, therefore reducing the potential negative impacts of DON. The biotransformation of DON to DON-3-sulfate helps to explain the lack of clinical signs in fish fed DON and may be used as a novel biomarker of DON exposure.

The development of a cost-effective off-the-shelf micro-particulate diet with a nutritionally optimal formulation for larval North African catfish, Clarias gariepinus (Burchell, 1822) is one of the most important requirements for the aquaculture sector in Uganda. North African catfish contribute ~50% of Uganda's production of farmed fish, both in terms of tonnage and economic value, however, studies conducted thus far to develop a micro-diet have taken no account of the digestive physiology of the fish itself, but rather have dwelt more on the composition of the diet and its apparent performance in terms of fish growth. Under these conditions, however, large mortalities are experienced during early development. Only a few studies have been conducted concerning the digestive physiology of the larvae or have characterised aspects of the key digestive enzymes produced by this species. Hence a major mismatch has existed between the general approach to creating micro-diets and the level of knowledge of the functional development of the sensory and digestive system in larval fish, including their theoretical digestive capabilities. This study sought to characterise the ontogeny of key digestive enzymes in North African catfish larvae from hatching to 21 days post-hatch (dph) with special emphasis on the period 2-4 dph where live feed is the more accepted diet for the fish larvae. In real-world terms, however, extensive use of live-feed makes farming extremely labour-intensive and it is costly to operate a catfish hatchery providing a consistent product and at the same time generate profit for farmers. The work described in this thesis was conducted at the University of Stirling's Tropical Aquarium, a facility of the Institute of Aquaculture, which houses a population of North African catfish introduced into the facility over 30 years ago and assumed to be, but not fully verified as, Clarias gariepinus Burchell 1822. In order to ensure that the results of the research presented in the thesis could be effectively applied in Uganda, the identity of this species needed to be confirmed. From a morphological and meristic characterisation, in addition to use of a molecular taxonomic classification, the species held at the Institute of Aquaculture and employed for the present study was confirmed to be Clarias gariepinus, (Burchell, 1822) rather than a hybrid with, Clarias anguillaris which is a situation that is common in African populations. Studies were undertaken to determine the point at which when exogenous feeding should be commenced and to establish when fish could be weaned on to an inert formulated diet in order to improve growth and survival during larval rearing. From the growth studies conducted, findings revealed that at 2Dph a mixed diet of (live) Artemia and (inert) Coppens diet gave better growth than both Coppens and Artemia fed alone, which gave similar growth rates. The histomorphology of both adult and larval C. gariepinus was examined in order to determine the course of development of digestive structures in the larva. In addition, new in situ labelling procedures were developed to highlight the localisation of expression of transcripts for key digestive enzymes comprising two alkaline proteolytic enzymes, namely chymotrypsin and trypsin, one acidic proteolytic enzyme, pepsin, and one chitinolytic enzyme, gastric chitinase. Larval development followed a time-course similar to that described by other researchers with the pancreas being discrete in the larval stages but being more dispersed throughout the intestinal region in the adults. The stomach was present by 4 to 5 Dph, supporting a suggestion that alkaline protease digestion dominates early in development, switching to a more acidic digestive mode following development of the stomach. In situ labelling protocols showed trypsin and chymotrypsin to be localised in the pancreas by as early as 2 Dph, while pepsin and gastric chitinase were localised in the stomach. More specifically, pepsin was localised to oxynticopeptic cells and gastric chitinase to the gastric pits with the former being present by 4Dph and the latter being present by 6Dph. Quantitative real-time PCR (qRT-PCR) was employed to investigate the timing and level of transcript expression for trypsin, chymotrypsin, pepsin and gastric chitinase. First expression was observed, perhaps unsurprisingly, to occur largely at a time when the tissue representing the site of production was first observable by histological examination and expression increased from this time. Trypsin and chymotrypsin expression were detectable from 1Dph, with clear expression by 2Dph, coinciding with the appearance of the first anlage of the pancreas in histology. Pepsin and gastric chitinase mRNA was detected at 3Dph and was highly expressed from 4dph onwards. This coincided with the appearance of the first anlage of the stomach in histology. Important new resources for North African catfish, including the first assembled and annotated transcriptome are now available for this species. These were created from a mixed tissue / stage transcriptome library. Using this transcriptome resource as a basis, an oligo-microarray was constructed, which allowed broad-scale transcriptome analysis to be conducted on samples from early larvae fed using two diet regimes. During development on the standard diet, a number of pathways were observed to be over-represented in terms of transcript expression between 1 Dph and 4 Dph. These included protein digestion and absorption, bile secretion, complement and coagulation cascades, glycolysis, gluconeogenesis, PPAR signalling pathways, and ribosomal pathways. Together these pathways denoted changes in elements of growth, protein production, digestion, the immune system and synaptic transmission inter alia. From the research presented in this thesis, it is clear that the key digestive enzymes studied here are rapidly switched on during the first few days of development and first feeding. Different dietary regimes had important effects on expression of digestive enzymes and knock-on effects for digestion, growth, and immune system development, which may have important consequences for larval health and survival under aquaculture conditions. The results of this work have the potential to assist the Ugandan feed industry in that they can be used as a baseline to make improved diets for early weaning in larval fish, using increased knowledge of which enzymes are involved in larval digestion and when they are produced by the fish. Clues about the course of development of larval immunity, particularly innate immunity, may help to provide strategies to mitigate the high losses experienced by farmers in early development and may assist in the management of high intensity catfish hatcheries. The findings of the work presented in this thesis can help to better formulate inert feeds and optimise timing of introduction to larval stages to maximise growth and survival. Public transcriptomic resources produced in the course of this work can be used by other researchers as a basis for larger broad-scale transcriptomic investigations using microarray, RNAseq or allied technologies to characterise different states including disease, nutrition, physiology and development which could be used to e.g. improve diets, develop vaccines, or increase production values such as growth characteristics.

With increasing world population, the demand for fish is growing thus there is a need to identify products with potential to increase the efficiency of fish production. Phytogenics are among the products being investigated as potential naturally derived growth promoters. The aim of this study was to identify phytogenic compounds and doses with growth-promoting effects in Nile tilapia and investigate relevant pathways underlying their growth promotion effects. The phytogenic compounds limonene, carvacrol and thymol, major constituents of essential oils from the plants citrus, oregano and thyme, respectively, were evaluated. Six Trials (Trials I, II, III, IV, V and VI) were carried out using diets supplemented with varying concentrations of the phytogenic compounds. In Trials I, II and III (Chapter 3), the effects of either limonene (Trial I), carvacrol (Trial II) or thymol (Trial III) on growth performance of Nile tilapia were investigated (objective 1) and performance parameters including final fish weight, daily growth coefficient, growth rate per metabolic body weight, percentage (%) weight gain, % survival, feed intake, feed conversion ratio and protein efficiency ratio were evaluated. Results from Trials I, II and III indicated that dietary supplementation of 400 and 500 ppm limonene and 750 ppm thymol had growth-promoting effects in Nile tilapia but the somatic growth was not associated with enhanced feed intake and feed utilisation efficiency. Trials IV and V (Chapter 4) investigated growth and nutritional physiology pathways in Nile tilapia regulated by individual phytogenic compounds (objective 2). This was accomplished by analysing the effects of limonene (Trial IV) and thymol (Trial V) supplemented diets on the expression of key genes participating in selected pathways of somatotropic axis-mediated growth, appetite regulation, nutrient digestion, absorption and transport, lipid metabolism, and antioxidant enzyme defence system. Limonene was supplemented in the diet at 0, 200, 400 and 600 ppm while thymol was supplemented at 0, 250 and 500 ppm. Trials IV and V found that growth-promoting effects of limonene (400 and 600 ppm) in Nile tilapia involved up-regulation of key genes within pathways including somatotropic axis-mediated growth, nutrient digestion, absorption and transport, lipid metabolism and antioxidant enzyme defence system. Dietary thymol at 250 and 500 ppm did not significantly enhance growth of Nile tilapia nor regulate the nutritional physiology pathways listed above. In Trial VI (Chapter 5), the effects of combined phytogenic compounds (limonene and thymol) on growth and nutritional physiology of Nile tilapia was tested (objective 3) to establish if the compounds had synergistic or additive effects on the growth of the fish as well as complementary effects on the selected nutritional physiology pathways. A candidate gene approach was also used for the selected pathways. Results from Trial VI showed that a diet supplemented with a combination of limonene (400 ppm) and thymol (500 ppm) has neither synergistic nor additive effects on the growth performance of Nile tilapia, with limonene mainly influencing the attained somatic growth. The analysed candidate genes involved in the pathways of nutrient digestion, absorption and transport, lipid metabolism, antioxidant enzymes and somatotropic axis growth also showed no synergistic or additive effects of a dietary combination of limonene and thymol in Nile tilapia. Overall, results from the study suggest approaches for developing functional diets for Nile tilapia using limonene and thymol growth promoters.