Genetically modified (GM) crops attracted interest globally when use of these crops resulted in significant increases in yield and production. These increases were due to protection of crops from pests, weeds and diseases. However, evolution of resistance by pests threatens the continued efficacy of GM crops. One such example is the resistance to Cry1Ac toxin in Helicoverpa armigera (Lepidoptera: Noctuidae). Resistance in this pest was due to a mutation in the aminopeptidase N1 (APN) Cry receptor gene, encoding the receptor for Cry1Ac. Laboratory studies have indicated that species in families Noctuidae, Pyralidae and Plutellidae can develop resistance to Bttoxins. To date, field-evolved resistance has only been reported in Busseola fusca (Fuller) (Lepidoptera: Noctuidae) in South Africa, Helicoverpa zea (Boddie) (Lepidoptera: Noctuidae) in the south-eastern United States, Spodoptera frugiperda (J.E. Smith) (Lepidoptera: Noctuidae) in Puerto Rico, Pectinophora gossypiella (Saunders) (Lepidoptera: Gelechiidae) in India, Helicoverpa armigera (Hübner) (Lepidoptera: Noctuidae) in northern China and Plutella xylostella (Linnaeus) (Lepidoptera: Plutellidae) in The Philippines and Hawaii. Resistance development in lepidopteran species is thus a common phenomenon. The stem borer B. fusca is a major insect pest to Bt-maize in the Vaalharts irrigation scheme (South Africa). The first official report of B. fusca resistance to Cry1Ab toxin was recorded in 2007, although farmers observed increased damage to Bt-maize from stem borers as early as 2004. A second report of resistance in an area nearby followed in 2009. No study has yet been done to determine the molecular mechanism of B. fusca resistance to Cry1Ab. As mentioned, a mutation in the APN receptor gene is responsible for H. armigera resistance to Cry1Ac. Although B. fusca has developed resistance to the B. thuringiensis Cry1Ab toxin, the binding-patterns and -sites of Cry1Ac and Cry1Ab are similar. Thus a similar mutation may be responsible for B. fusca resistance to Cry1Ab. Aminopeptidase, cadherin and alkaline phosphatase are the major Cry toxin receptors that have been identified in lepidopteran species. The present study was concerned with the investigation of mutations in these receptor genes. However, in order to study mutations, sequence data of receptor genes are essential. Degenerate primers were designed based on conserved regions observed in multiple protein sequence alignments of aminopeptidase N (isogenes 1 to 6), cadherin and alkaline phosphatase of several lepidopteran species. Primers were degenerate to take into consideration the variant regions in receptor gene sequences among lepidopteran species. These primers were used to amplify genomic DNA (gDNA) from susceptible and resistant larvae by using PCR. Sequences of PCR amplicons were determined through Sanger sequencing reactions and subjected to BLAST searches. Results of the BLAST searches showed some similarities to the respective receptor genes. These sequences were also used in phylogenetic analysis. This analysis intended to determine the phylogenetic relationship of the respective receptor genes between B. fusca and other lepidopteran species. Mutations could not be identified in the present study, due to a lack in receptor gene sequence data for B. fusca. Thus a goal of the present study was to generate sequence data for B. fusca. In addition to the proposed objectives, cytochrome b gene sequences of B. fusca were used to determine the phylogenetic relationship between B. fusca and other lepidopteran species. Genome sequencing of B. fusca is recommended, as this will provide a platform for genomic, transcriptomic and proteomic studies on this species. These studies will provide much needed information, which can be used to formulate strategies to prevent resistance development in and spread of resistance to other B. fusca populations in sub-Saharan Africa. / Thesis (MSc (Environmental Sciences))--North-West University, Potchefstroom Campus, 2013.

Genetically modified (GM) crops attracted interest globally when use of these crops resulted in significant increases in yield and production. These increases were due to protection of crops from pests, weeds and diseases. However, evolution of resistance by pests threatens the continued efficacy of GM crops. One such example is the resistance to Cry1Ac toxin in Helicoverpa armigera (Lepidoptera: Noctuidae). Resistance in this pest was due to a mutation in the aminopeptidase N1 (APN) Cry receptor gene, encoding the receptor for Cry1Ac. Laboratory studies have indicated that species in families Noctuidae, Pyralidae and Plutellidae can develop resistance to Bttoxins. To date, field-evolved resistance has only been reported in Busseola fusca (Fuller) (Lepidoptera: Noctuidae) in South Africa, Helicoverpa zea (Boddie) (Lepidoptera: Noctuidae) in the south-eastern United States, Spodoptera frugiperda (J.E. Smith) (Lepidoptera: Noctuidae) in Puerto Rico, Pectinophora gossypiella (Saunders) (Lepidoptera: Gelechiidae) in India, Helicoverpa armigera (Hübner) (Lepidoptera: Noctuidae) in northern China and Plutella xylostella (Linnaeus) (Lepidoptera: Plutellidae) in The Philippines and Hawaii. Resistance development in lepidopteran species is thus a common phenomenon. The stem borer B. fusca is a major insect pest to Bt-maize in the Vaalharts irrigation scheme (South Africa). The first official report of B. fusca resistance to Cry1Ab toxin was recorded in 2007, although farmers observed increased damage to Bt-maize from stem borers as early as 2004. A second report of resistance in an area nearby followed in 2009. No study has yet been done to determine the molecular mechanism of B. fusca resistance to Cry1Ab. As mentioned, a mutation in the APN receptor gene is responsible for H. armigera resistance to Cry1Ac. Although B. fusca has developed resistance to the B. thuringiensis Cry1Ab toxin, the binding-patterns and -sites of Cry1Ac and Cry1Ab are similar. Thus a similar mutation may be responsible for B. fusca resistance to Cry1Ab. Aminopeptidase, cadherin and alkaline phosphatase are the major Cry toxin receptors that have been identified in lepidopteran species. The present study was concerned with the investigation of mutations in these receptor genes. However, in order to study mutations, sequence data of receptor genes are essential. Degenerate primers were designed based on conserved regions observed in multiple protein sequence alignments of aminopeptidase N (isogenes 1 to 6), cadherin and alkaline phosphatase of several lepidopteran species. Primers were degenerate to take into consideration the variant regions in receptor gene sequences among lepidopteran species. These primers were used to amplify genomic DNA (gDNA) from susceptible and resistant larvae by using PCR. Sequences of PCR amplicons were determined through Sanger sequencing reactions and subjected to BLAST searches. Results of the BLAST searches showed some similarities to the respective receptor genes. These sequences were also used in phylogenetic analysis. This analysis intended to determine the phylogenetic relationship of the respective receptor genes between B. fusca and other lepidopteran species. Mutations could not be identified in the present study, due to a lack in receptor gene sequence data for B. fusca. Thus a goal of the present study was to generate sequence data for B. fusca. In addition to the proposed objectives, cytochrome b gene sequences of B. fusca were used to determine the phylogenetic relationship between B. fusca and other lepidopteran species. Genome sequencing of B. fusca is recommended, as this will provide a platform for genomic, transcriptomic and proteomic studies on this species. These studies will provide much needed information, which can be used to formulate strategies to prevent resistance development in and spread of resistance to other B. fusca populations in sub-Saharan Africa. / Thesis (MSc (Environmental Sciences))--North-West University, Potchefstroom Campus, 2013.

Busseola fusca is an indigenous lepidopteran pest species in tropical Africa, attacking several grain crops. Crop loss caused by this pest can be as high as 100 % depending on conditions. Despite it being a major pest in Africa, occurring in contrasting climatic zones, only a few studies have been published on its developmental biology. The effect of temperature on the development of B. fusca was studied at five different temperature regimes namely 15, 18, 20, 26 and 30 ± 1 °C and 70 ± 30 % relative humidity (RH) with 14L: 10D photoperiod. The number of instars for B. fusca was also determined. The most favourable temperature as well as the upper threshold temperature for larval development was found to be between 26 and 30 °C. Total development period was 152.6 to 52.6 days, respectively, at 15 °C, and 26 - 30 °C. The thermal constants for B. fusca was 99.50, 536.48, 246.25 and 893.66 °D and lower temperature threshold was 10.36, 8.14, 8.99 and 8.84 °C, for completion of the egg, larval, pupal, and egg-to-adult stages, respectively. The number of larval instars was determined by using head capsule widths that ranged from 0.31 - 2.68 mm. Clear distinctions of head capsule widths could be made from instar 1 to 3, yet overlapping occurred from instar 4 to 6. No distinction could be made between instars 7 and 8 in terms of head capsule width. All successive instars, except for instar eight, increased in size according to Dyar’s ratio. The effect of temperature on reproduction of B. fusca was studied at 15, 20, 26 and 30 ± 1 °C, 70 ± 30 % RH with 14L: 10D photoperiod. Oviposition occurred at all the temperatures evaluated, but no fertility was recorded at 30 °C. The total number of eggs laid by B. fusca females was 300 - 400 eggs and the optimum temperature for oviposition and fertility was determined to be between 20 and 26 °C. Results from this study on the thermal constants and lower and upper threshold temperatures of B. fusca can be used to predict the impact of climate change on the distribution and population growth of this pest. / 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

Busseola fusca is an indigenous lepidopteran pest species in tropical Africa, attacking several grain crops. Crop loss caused by this pest can be as high as 100 % depending on conditions. Despite it being a major pest in Africa, occurring in contrasting climatic zones, only a few studies have been published on its developmental biology. The effect of temperature on the development of B. fusca was studied at five different temperature regimes namely 15, 18, 20, 26 and 30 ± 1 °C and 70 ± 30 % relative humidity (RH) with 14L: 10D photoperiod. The number of instars for B. fusca was also determined. The most favourable temperature as well as the upper threshold temperature for larval development was found to be between 26 and 30 °C. Total development period was 152.6 to 52.6 days, respectively, at 15 °C, and 26 - 30 °C. The thermal constants for B. fusca was 99.50, 536.48, 246.25 and 893.66 °D and lower temperature threshold was 10.36, 8.14, 8.99 and 8.84 °C, for completion of the egg, larval, pupal, and egg-to-adult stages, respectively. The number of larval instars was determined by using head capsule widths that ranged from 0.31 - 2.68 mm. Clear distinctions of head capsule widths could be made from instar 1 to 3, yet overlapping occurred from instar 4 to 6. No distinction could be made between instars 7 and 8 in terms of head capsule width. All successive instars, except for instar eight, increased in size according to Dyar’s ratio. The effect of temperature on reproduction of B. fusca was studied at 15, 20, 26 and 30 ± 1 °C, 70 ± 30 % RH with 14L: 10D photoperiod. Oviposition occurred at all the temperatures evaluated, but no fertility was recorded at 30 °C. The total number of eggs laid by B. fusca females was 300 - 400 eggs and the optimum temperature for oviposition and fertility was determined to be between 20 and 26 °C. Results from this study on the thermal constants and lower and upper threshold temperatures of B. fusca can be used to predict the impact of climate change on the distribution and population growth of this pest. / 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

Thesis (M.Sc. (Environmental Science and Management)--North-West University, Potchefstroom Campus, 2008

Thesis (M.Sc. (Environmental Science and Management)--North-West University, Potchefstroom Campus, 2008

Thesis (M.Sc. (Environmental Science and Management)--North-West University, Potchefstroom Campus, 2008

Genetically modified (GM) maize was developed mainly to control lepidopteran pests such as the maize stem borer (Busseola fusca) (Lepidoptera: Noctuidae). Since the first commercialization of GM crops with insecticidal properties, possible non-target effects such as the effect at the third trophic level on important predators for example lacewing species (Chrysoperla spp.) have been of concern. Contradicting results were reported in previous studies with regard to the effect of Cry 1Ab protein produced by Bt maize on the performance of lacewings. Some studies found that Bt proteins had no effect while others reported that C. carnea performed poorly if they consumed prey that consumed Cry 1Ab protein. In South Africa one of the most common chrysopid species in maize ecosystems is Chrysoperla pudica (Navás) (Neuroptera: Chrysopidae). Evolution of Bt resistant pests, such as B. fusca in South Africa facilitates a new pathway for exposure of predators to healthy prey that consumes Cry 1Ab proteins. The aims of this study was to determine the effect of the Cry 1Ab protein expressed in Bt maize on a non-target organism‟s (C. pudica) biology via indirect exposure, and to determine the concentration of Cry 1Ab protein in the plant, prey and predator. Chrysoperla pudica larvae were indirectly exposed to the Bt-toxin through healthy Bt-maize feeding prey (B. fusca larvae) in two feeding experiments and lacewing survival and life history parameters recorded. Bt had a limited effect on some parameters that were evaluated. The larval and pupal periods of C. pudica larvae that were exposed to the Bt-toxin had a significant difference from that of the control treatment. The Bt-toxin had a significant effect on fecundity, fertility and malformation after emergence of C. pudica adults of which larvae fed only on Bt resistant B. fusca larvae, but not on the mortality rate. Cry 1Ab concentration was the highest in the plant, followed by the prey and lacewing larvae. This study showed that the Cry 1Ab protein had a slight adverse effect only on certain life parameters of C. pudica, and that Cry 1Ab protein was hardly detectable in C. pudica larvae. However, since this study represented a worst-case scenario where diverse prey was not available, insignificant effects is expected under field conditions where prey is diverse. / MSc (Environmental Sciences), North-West University, Potchefstroom Campus, 2014