Bt-maize is engineered to express insecticidal toxins derived from the bacterium Bacillus thuringiensis and has been shown to be very effective against pests like Busseola fusca. However, resistance of this pest against Bt-maize has developed and spread throughout South Africa. This study was inspired by the lack of knowledge over the microorganisms associated with the gut of these insects as they play a vital role in insect growth and development. Microbial-derived enzymes may have a role during an insect‟s adaption in different environmental conditions and to new diets. Previous studies suggest (1) that gut bacteria are required for B. thuringiensis-induced mortality in most Lepidoptera species and (2) that the toxicity of B. thuringiensis depends on microbial community interactions within the gut. The aim of this study was to determine the microbial diversity present in the midgut of B. fusca larvae occurring in maize. Busseola fusca larvae were collected from 30 sites throughout South Africa and dissected to collect their midgut contents. Serial dilutions were made of the contents and spread plated onto nutrient agar after which morphotypes were identified. One-hundred and five morphotypes were identified; DNA were extracted from the selected morphotypes and subjected to PCR analysis followed by secquencing. Sequencing results revealed the dominance of Enterococcus spp., specifically Enterococcus casseliflavus and Enterococcus gallinarum, Klebsiella spp., espesially Klebsiella pneumoniae and Klebsiella oxytoca and Bacillus spp. such as .B. thuringiensis and B. subtilis. Other organisms isolated, included Achromobacter spp., Brevudimonas spp., Caulobacter spp., Enterobacter spp., Halomonas spp., Ochrobactrum spp., Pantoea spp., Pseudomonas spp., Serratia spp., Stenotrophomonas spp., Arthrobacter spp., Brevibacterium spp., Leucobacter spp., Microbacterium spp., Planomicrobium spp. and Staphylococcus spp. The microbial diversity of larvae collected at the respective sampling sites were determined with the Shannon diversity index. The data were compared to several factors regarding the sampling sites. No significant differences were observed between the microbial diversities isolated at the respective sites. This may imply that the microbial community within B. fusca larvae are relative consistent throughout the maize production area. It is important to understand the distribution and structure of gut microbial communities within insects and whether the gut community is influenced by the geographical distribution of the insects. A better understanding of the distribution of the insects and community structure of their gut microbiota may aid in the development of better insect control strategies. / MSc (Environmental Sciences), North-West University, Potchefstroom Campus, 2015

Bt-maize is engineered to express insecticidal toxins derived from the bacterium Bacillus thuringiensis and has been shown to be very effective against pests like Busseola fusca. However, resistance of this pest against Bt-maize has developed and spread throughout South Africa. This study was inspired by the lack of knowledge over the microorganisms associated with the gut of these insects as they play a vital role in insect growth and development. Microbial-derived enzymes may have a role during an insect‟s adaption in different environmental conditions and to new diets. Previous studies suggest (1) that gut bacteria are required for B. thuringiensis-induced mortality in most Lepidoptera species and (2) that the toxicity of B. thuringiensis depends on microbial community interactions within the gut. The aim of this study was to determine the microbial diversity present in the midgut of B. fusca larvae occurring in maize. Busseola fusca larvae were collected from 30 sites throughout South Africa and dissected to collect their midgut contents. Serial dilutions were made of the contents and spread plated onto nutrient agar after which morphotypes were identified. One-hundred and five morphotypes were identified; DNA were extracted from the selected morphotypes and subjected to PCR analysis followed by secquencing. Sequencing results revealed the dominance of Enterococcus spp., specifically Enterococcus casseliflavus and Enterococcus gallinarum, Klebsiella spp., espesially Klebsiella pneumoniae and Klebsiella oxytoca and Bacillus spp. such as .B. thuringiensis and B. subtilis. Other organisms isolated, included Achromobacter spp., Brevudimonas spp., Caulobacter spp., Enterobacter spp., Halomonas spp., Ochrobactrum spp., Pantoea spp., Pseudomonas spp., Serratia spp., Stenotrophomonas spp., Arthrobacter spp., Brevibacterium spp., Leucobacter spp., Microbacterium spp., Planomicrobium spp. and Staphylococcus spp. The microbial diversity of larvae collected at the respective sampling sites were determined with the Shannon diversity index. The data were compared to several factors regarding the sampling sites. No significant differences were observed between the microbial diversities isolated at the respective sites. This may imply that the microbial community within B. fusca larvae are relative consistent throughout the maize production area. It is important to understand the distribution and structure of gut microbial communities within insects and whether the gut community is influenced by the geographical distribution of the insects. A better understanding of the distribution of the insects and community structure of their gut microbiota may aid in the development of better insect control strategies. / MSc (Environmental Sciences), North-West University, Potchefstroom Campus, 2015

Doctor of Philosophy / Department of Entomology / Larry L. Buschman / Kun Yan Zhu / Genomic information for lepidopteran insects, particularly agricultural pest species, is very limited but urgently needed due to their economic importance and biodiversity. The huge economic losses ($ 1-2 billons / year) caused by the European corn borer (Ostrinia nubilalis, Hübner, ECB) makes this insect species one of the major pests of corn in the United States and western world. Management of ECB by conventional methods is limited but has had a great success by transgenic Bt (Bacillus thuringiensis) corn, which targets insect gut. However, the widespread use of Bt corn may lead to the development of Bt resistance in ECB. Knowledge of genes expressed in the insect gut is considered crucial for understanding basic physiology of food digestion, their interactions with Bt toxins and pathogens, and for discovering new targets for pest management. A large database of 15,000 expressed sequence tags (ESTs) was established from the ECB larval gut. To our knowledge, this database represents the largest gut-specific EST database from a lepidopteran pest. Analysis of 10 aminopeptidase-like genes between Cry1Ab–resistant and –susceptible ECB larvae revealed that aminopeptidase P-like (OnAPP) gene is a strong candidate for its role in Bt toxicity and resistance. The RNA interference mediated reduction in the transcript level of OnAPP gene in ECB larvae resulted in their reduced susceptibily to Cry1Ab. Analysis of the chitinase-like gene (OnCht) revealed its essential role in regulating chitin content of peritrophic membrane (PM). Our results suggest that OnCht may influence food digestion, nutrient absorption or movement of digestive enzymes through the PM and can be an important target for insect management. We also identified and characterized six genes involved in the innate immune defense response in ECB and showed that the expression of these genes were induced when challenged with bacteria. In addition to these results, this research generated significant genomic information for the development of microarray from the larval gut of ECB. The establishment of the feeding-based RNA interference technique could potentially help in delivering dsRNA orally to ECB for high throughput screening of effective genes to be targeted for insect pest management.

In all living organisms, biological activities such as proper functioning and co-ordination of different organs will depend on different cells and molecular interactions. In some organisms the loss of functional organs or damage of organs can be lethal, whereas in others a special process called regeneration can retrieve lost organs. The molecular details of regeneration are still not completely understood in many organisms. Echinoderms are close to vertebrates in the evolutionary tree and are well known for their amazing regeneration capacity. So we chose to investigate the molecular processes of regeneration mechanism with an interest towards our favorite groups of molecules, glycosaminoglycans (GAGs). GAGs are linear polysaccharides, expressed on all cell surfaces and extracellular space and are also known to be involved in many cellular activities. We aimed to characterize the GAGs present in Echinodermata species Amphiura filiformis and investigated their role during arm regeneration. In Paper I we characterized the structure and function of GAGs from A. filiformis and identified that A. filiformis contains CS/DS type of GAGs, but no HS. The sulfation degree of these CS/DS is close to the one of heparin, i.e. they are highly sulfated. These chains are able to bind FGF-2 growth factor and induce FGF-2 mediated cell signaling. In Paper II we further characterized these GAGs for their localization and for their role in arm regeneration in A. filiformis. Immuno- and histochemical stainings on arm sections revealed that CS/DS GAGs are localized around the podia, surrounding the water vascular system, and around the muscle tissues. Inhibition of sulfated GAG biosynthesis by chlorate treatment affected the regeneration efficiency of the arms, which may be an indication of the importance of CS/DS structures in A. filiformis arm regeneration. We also characterized some bacterial sulfatases in Paper III and a lyase in Paper IV from human and canine gut symbiotic bacteria. Here we sought to find the substrate specificity and optimal conditions for these enzymes’ activities. Our findings suggest that these polysaccharide lyase and sulfatases can be used as potential tools to characterize different GAG structures and their application could further add knowledge on diseases mechanisms related to host pathogen interactions.

Gut hormones are implicated in the regulation of energy balance. The studies in this thesis have examined the effects exercise on gut hormones (acylated ghrelin and peptide YY3-36), appetite and food intake, over extended durations. Sixty-nine young, healthy, predominantly Caucasian males were recruited to six studies. The age, height and body mass of the participants were: 22.4 ± 0.3 y, 1.80 ± 0.1 m, 76.2 ± 1.0 kg (mean ± SEM). In study one, 90 min of resistance exercise did not influence appetite or energy intake over 24 h of assessment, yet stimulated a latent preference for carbohydrate rich foods. Study two demonstrated that appetite was suppressed during 60 min of swimming but was elevated after consuming a post-exercise meal. Plasma acylated ghrelin was suppressed during swimming but was unaltered after. Energy/macronutrient intake remained unchanged. In study three, 60 min of brisk walking (45 ± 2% of max) did not influence appetite, energy/macronutrient intake or plasma concentrations of acylated ghrelin during an eight hour observation period. Study four showed that 90 min of treadmill running (69 ± 1% of max) transiently suppressed appetite and acylated ghrelin but did not influence these variables, or energy/macronutrient intake within 22.5 h after exercise. The findings of study five suggest that the suppression and subsequent rebound in plasma acylated ghrelin after exercise may be related to a delayed voluntary decision to eat after. Finally, study six showed that appetite, food intake and circulating concentrations of acylated ghrelin and peptide YY3-36 are responsive to acute deficits in energy induced by food restriction but are not sensitive to equivalent energy deficits induced by exercise. This thesis has shown that exercise transiently alters circulating levels of acylated ghrelin and peptide YY3-36 in directions expected to inhibit appetite however no changes are seen after exercise. Conversely, food restriction elicits marked compensatory changes in circulating acylated ghrelin and peptide YY3-36. This thesis also demonstrates that resistance exercise, brisk walking and running do not stimulate appetite or energy intake over defined periods, even when the energy expenditure elicited is high. Swimming appears to increase appetite in the latter hours after exercise.

Most animals contain chronic microbial infections that inflict no harm on their hosts. Recently, the gut microflora of humans and other animals have been characterized. However, little is known about the forces that shape the diversity of these bacterial communities. In this work, comparative genomics was used to investigate the evolutionary dynamics of host-adapted bacterial communities, using Wolbachia infecting arthropods and Lactobacteria infecting bees as the main model systems. Wolbachia are maternally inherited bacteria that cause reproductive disorders in arthropods, such as feminization, male killing and parthenogenesis. These bacteria are difficult to study because they cannot be cultivated outside their hosts. We have developed a novel protocol employing multiple displacement amplification to isolate and sequence their genomes. Taxonomically, Wolbachia is classified into different supergroups. We have sequenced the genomes of Wolbachia strain wHa and wNo that belong to supergroup A and B, respectively, and are present as a double-infection in the fruit-fly Drosophila simulans. Together with previously published genomes, a supergroup comparison of strains belonging to supergroups A and B indicated rampant homologous recombination between strains that belong to the same supergroup but were isolated from different hosts. In contrast, we observed little recombination between strains of different supergroups that infect the same host. Likewise, phylogenetically distinct members of Lactic acid bacteria co-exist in the gut of the honeybee, Apis mellifera, without transfer of genes between phylotypes. Nor did we find any evidence of co-diversification between symbionts and hosts, as inferred from a study of 13 genomes of Lactobacillus kunkeei isolated from diverse bee species and different geographic origins. Although Lactobacillus kunkeii is the most frequently isolated strain from the honey stomach, we hypothesize that the primary niche is the beebread where the bacteria are likely to contribute to the fermentation process. In the human gut, the microbial community has been shown to interact with the immune system, and likewise the microbial communities associated with insects are thought to affect the health of their host. Therefore, a better understanding of the role and evolution of endosymbiotic communities is important for developing strategies to control the health of their hosts.

Doctor of Philosophy / Department of Human Nutrition / Mark D. Haub / Little is known about the impact of dietary fiber (DF) on children. Current recommendations are based on extrapolations from adult studies. Research is needed to provide science based evidence to determine how DF impacts the gut of children. Two studies were conducted to investigate the interactions of DF in the child large intestine. In the first study, the dose response of DF on breath hydrogen, methane, and total hydrogen content was investigated relative to Institute of Medicine (IOM) recommended intakes in free-living preschool children. Only four of the 18 participants were able to comply with the treatment protocol. Although, no significant differences were noted in breath measures of fermentation across fiber consumption levels, there was a numerical pattern for increasing levels of DF to evolved increased gas production in the four compliers. In free-living individuals measures of acute fiber fermentation through breath was not sensitive enough over 6 hours to distinguish a difference in fermentative rate. Children, parents, and child care centers found this approach apparently acceptable. In study two, the impact of DF (10 g) fed over three weeks in children and their parents on metabolic markers of fermentation were evaluated. The body was able to adapt to 10 g/day DF consumption as bloating (p < 0.05) and flatulence (p = 0.06) decreased each week of the study. Fecal propionic acid was significantly increased over three weeks of DF supplementation. There was also an interaction (p=0.05) between time and age for butyric acid. Dietary fiber supplementation (10 g/day) over three weeks via a commercially available extruded cereal was well-tolerated by the participants, with no disturbances in bowel habit in children or adults. Alternatively, there were no improved bowel habit measures with increased DF consumption. This study provides evidence that this tolerable dose of DF supplementation over three weeks had similar impacts in free-living children and adults. However, the presence of increased butyric acid only in children may be reflective of different production or absorptive capacities between children and adults. Although not presented here, the bacterial ecological analysis may shed further insight into the interactions occurring in the large intestine. These are the first studies to my knowledge to have investigated these outcomes in young children. In addition to the gut health outcomes, this research provided a framework into the apparent feasibility of studying children in a gentle, non-invasive, and cost-effective manner.

Student Number : 9613963J - PhD thesis - School of Animal, Plant and Environmental Sciences - Faculty of Science / Animals living in cold environments show physiological, morphological and behavioural adaptations to low temperatures. The African ice rat, Otomys sloggetti robertsi, which is endemic to the southern African Drakensberg and Maluti mountains above 2000m, is an interesting exception since, unlike most alpine small mammals, it does not hibernate or display torpor and is physiologically poorly adapted to low temperatures. It is a strict herbivore, feeding on a low quality diet. Ice rats do show some morphological (e.g. short tails) and behavioural (e.g. communal huddling; constructing underground burrows) adaptations, but little else is known about their biology, particularly how they maximise energy gain to meet thermoregulatory requirements, especially during cold periods. Since feeding represents the primary method of energy gain in endotherms, I studied aspects of the foraging biology of ice rats, including gut structure, foraging patterns and habitat choice. The gut structure of O. s. robertsi is well adapted for a high fibre, herbivorous diet and shows broad similarities with those of its mesic- and arid-occurring relatives. However, O. s. robertsi showed increased dimensions of several foregut organs which may be adaptations for increased energy uptake and/or poor diet quality in alpine environments. Furthermore, females had a larger stomach as well as a longer caecum, small and large intestine in summer than in winter but the gut of males was unaffected; such sexual asymmetry may be related to increased energy requirements of females during pregnancy and lactation. Environmental influences on the aboveground behaviour of O. s. robertsi were investigated by recording the duration of behaviours as well as sequential transitions among behaviours. Ice rats spent most of their day foraging and basking, and much time was spent in their underground burrows. Seasonal comparisons revealed that ice rats spent significantly more time acquiring energy through foraging in winter, whereas they remained below ground for longer periods of time during the middle of the day in summer to escape extreme heat and solar radiation. To understand how low temperatures and predation influenced foraging patterns, the behaviour of ice rats was studied in summer and winter in a population where predators were minimal and in another population which experienced higher levels of predation. Ice rats are central place foragers that travel short distances to forage and display significant seasonal variation in their foraging patterns. In the absence of predation risk, ice rats generally returned to a central place with forage, even though returning to a burrow after foraging in winter was energetically costly. However, these costs must be weighed against the benefits of avoiding exposure to low temperatures by feeding under cover as well as the loss of collected food and possible injury associated with aggressive interactions with conspecifics. Under moderate predation pressure in both seasons, ice rats followed a central place foraging strategy to minimise predation risk, always returning to a burrow entrance with forage collected elsewhere. However, when no perceivable threat was observed, ice rats displayed ‘optimal’ foraging patterns in summer similar to those recorded in the absence of predation pressure and only returned to a burrow with forage as distance from that burrow increased, suggesting that ice rats display facultative foraging decision making in response to multiple environmental cues. The distribution of occupied ice rat burrows was correlated against several environmental factors to determine microhabitat requirements. Ice rat burrows were situated in close proximity to herbaceous and wetland plants, but away from woody vegetation, suggesting that habitat choice is related to the presence of food plants and reduction of shade, facilitating short travel distances during foraging as well as promoting basking. Despite the physiological shortcomings of ice rats, the gut structure, foraging behaviour, and habitat choice of the taxon are adapted for life in cold alpine habitats, most likely by maximising energy intake. Similarities in foraging behaviour and habitat use between O. s. robertsi with its closely-related arid-occurring relative Parotomys spp. suggest phylogenetic influences, but it is possibly more a reflection of similar phenotypic responses to the extreme habitats inhabited by these otomyines.

L’étude du microbiote digestif est un enjeu important de recherche en microbiologie. La première partie de cette thèse porte sur la recherche au sein du microbiote digestif de nouveaux antibactériens, qui apparait comme une des pistes clés dans la lutte contre la résistance aux antibiotiques. Les trois quarts des antibiotiques sont des produits naturels, ou dérivés, sécrétés par des microorganismes de l’environnement. Comme lui, le microbiote digestif représente un écosystème complexe où règne une grande compétition. Nous avons recherché des antagonismes de culture dans le microbiote digestif contre les bactéries les plus pathogènes pour l’homme. Nous avons trouvé une inhibition de S. aureus par P. avidum, de E. cloacae par B. fragilis, E. dispar, L. delbruckii, P. acidipropionici, S. equinus, S. gallolyticus, et enfin de E. aerogenes par B. vulgatus et E. dispar. Nous avons également trouvé des clusters de gène de métabolites secondaires dans le génome de ces bactéries. Ce travail préliminaire confirme que le microbiote digestif est une source potentielle de nouveau antibactériens. En dépit de l’explosion du nombre d’espèces isolées dans le microbiote digestif grâce à la culturomics, certaines restent fastidieuses à cultiver. Nous avons analysé par métagénomique et culturomics une selle avant et après incubation anaérobie en présence de 5% de rumen et 5% de sang de mouton. Ce travail montre une dynamique de croissance des bactéries très hétérogène. Le milieu d’enrichissement utilisé était efficace et permettait la culture d’un plus grand nombre d’espèces bactériennes. Ce travail apporte des éléments nouveaux permettant l’optimisation de cette étape de culturomics. / Gut microbiota is a major health concern for microbiologists. Its alterations were previously related to diseases. In the first step of this thesis, we have searched for new antimicrobials within the gut microbiota. Indeed, antibiotic resistance is a global health concern and research for new antibiotics is a cornerstone for fight against it, according to the WHO. Three quarter of all current antibiotics are natural products, or derived from them, synthesised by bacteria and fungi from soil. Gut microbiota is another complex ecosystem with strong competition. We have searched for antagonism in the gut microbiota species against most human pathogenic species. We found an inhibition of growth of S. aureus by P. avidum, of E.cloacae by B. fragilis, E. dispar, L. delbruckii, P. acidipropionici, S. equinus, S. gallolyticus,and an inhibition of E. aerogenes by B. vulgatus and E. dispar. We also found BGCs for all these species. This preliminary work confirm that gut microbiota is a potential source for new antibiotics. Despite the explosion of bacterial species isolated from gut, some fastidious species remains difficult to grow. We performed a metagenomic and culturomics analysis of a fresh stool sample before and after incubation into an anaerobic blood bottle supplemented with sheep blood and rumen fluid. This medium used in culturomics for enrichment was effective, allowing the isolation of higher number of species. This work show that the dynamic growth of bacteria is very variable. This work brings some precisions in the dynamic of bacterial growth that could improve the culturomics process.

Les infections digestives à Clostridium difficile nécessitent une première étape de colonisation de l’écosystème intestinal. Avant l’âge de deux ans, la colonisation par C. difficile est fréquente mais paradoxalement le plus souvent asymptomatique. Nous avons montré que plus d’un tiers des enfants de 0-3 ans, et plus de 70% de ceux de 7 à 9 mois (15% pour les souches toxinogènes), étaient porteurs sains de C. difficile à l’hôpital et dans la communauté. Deux périodes d’acquisition de C. difficile ont été identifiées : néonatale ou 3-6 mois. Les souches infantiles de C. difficile étaient identiques aux souches isolées chez l’adulte, faisant du jeune enfant un réservoir potentiel de souches infectieuses. Nous avons également montré par méthode moléculaire que des changements en espèces dominantes du microbiote étaient associés à la colonisation par C. difficile. Bifidobacterium longum caractérisait le microbiote des enfants non colonisés, et pourrait participer à la résistance à la colonisation par C. difficile. / Gastrointestinal infections with Clostridium difficile require a first step of colonization of the intestinal ecosystem. Under the age of two years, C. difficile colonization is frequent but paradoxically most often asymptomatic. We have shown that more than a third of children 0-3 years and more than 70% of those from 7 to 9 months (15% for toxigenic strains) were healthy carriers of C. difficile in the hospital and in the community. Two C. difficile-acquisition periods were identified: neonatal or 3-6 months. The C. difficile strains from infants were identical to strains isolated from adults, making infants a potential reservoir of infectious strains. We also showed by molecular method that changes in dominant species of the microbiota were associated with colonization by C. difficile. Bifidobacterium longum characterized the microbiota of children not colonized by C. difficile, and could be involved in the colonization resistance process.