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

The evolution of pest resistance to Cry proteins threatens the long-term use of Bt crops. Busseola fusca developed resistance to Bt maize in South Africa but the mechanism of resistance is not well understood. According to the gut microbiota theory, extensive cell lysis caused by Cry proteins provide gut microbes access to the more favourable environment of the hemocoel where they germinate and reproduce, causing septicemia and death of the host. This theory brought on questions about the role of gut microbes in the efficacy of Bt maize against target lepidopteran pests. The aim of this study was to determine whether microbes present in the mid-gut of B. fusca influence the efficacy of Cry 1Ab proteins. Larvae were collected from 30 different geographical locations, dissected to excise the midgut and mid-gut content which was separated according to morphological types. The morphological types were used to test the antibiotic susceptibility of the bacteria and proved that ciprofloxacin, ampicillin and doxycycline were the most effective bacteriostatic and bactericidal antibiotics. These three antibiotics were exposed to the morphological types at different concentrations to visualise the possible deleterious effects of the antibiotics on the bacteria. This visualisation was performed by observing the growth curve of the bacteria in the presence of the combination of antibiotics. The antibiotics concentration of 500 μg/ml showed the highest efficacy compared to the other concentrations tested. An antibiotic concentration of 500 μg/ml of ciprofloxacin, ampicillin and doxycycline was incorporated into an artificial diet for the larvae to feed on for 7 days. This method was used to rid the larvae of gut microbes before allowing them to feed on Bt maize (MON810) plant material expressing Cry proteins. The results suggests that by placing antibiotic reared larvae on a Bt plant, the absence of the mid-gut microbes contributed to larvae survival on Bt maize. This observation will contribute to understanding the role of gut microbes on the efficacy of Cry proteins. / MSc (Environmental Sciences), North-West University, Potchefstroom Campus, 2015

The evolution of pest resistance to Cry proteins threatens the long-term use of Bt crops. Busseola fusca developed resistance to Bt maize in South Africa but the mechanism of resistance is not well understood. According to the gut microbiota theory, extensive cell lysis caused by Cry proteins provide gut microbes access to the more favourable environment of the hemocoel where they germinate and reproduce, causing septicemia and death of the host. This theory brought on questions about the role of gut microbes in the efficacy of Bt maize against target lepidopteran pests. The aim of this study was to determine whether microbes present in the mid-gut of B. fusca influence the efficacy of Cry 1Ab proteins. Larvae were collected from 30 different geographical locations, dissected to excise the midgut and mid-gut content which was separated according to morphological types. The morphological types were used to test the antibiotic susceptibility of the bacteria and proved that ciprofloxacin, ampicillin and doxycycline were the most effective bacteriostatic and bactericidal antibiotics. These three antibiotics were exposed to the morphological types at different concentrations to visualise the possible deleterious effects of the antibiotics on the bacteria. This visualisation was performed by observing the growth curve of the bacteria in the presence of the combination of antibiotics. The antibiotics concentration of 500 μg/ml showed the highest efficacy compared to the other concentrations tested. An antibiotic concentration of 500 μg/ml of ciprofloxacin, ampicillin and doxycycline was incorporated into an artificial diet for the larvae to feed on for 7 days. This method was used to rid the larvae of gut microbes before allowing them to feed on Bt maize (MON810) plant material expressing Cry proteins. The results suggests that by placing antibiotic reared larvae on a Bt plant, the absence of the mid-gut microbes contributed to larvae survival on Bt maize. This observation will contribute to understanding the role of gut microbes on the efficacy of Cry proteins. / MSc (Environmental Sciences), North-West University, Potchefstroom Campus, 2015

The maize stem borers, Busseola fusca (Fuller) (Lepidoptera: Noctuidae) and Chilo partellus (Swinhoe) (Lepidoptera: Pyralidae) are economically important pests of maize in South Africa. Genetically modified Bt maize (MON810) expressing Cry1Ab protein is used to control these pests on approximately 425 000 hectares in South Africa. Before this study no information was available on the diversity of Lepidoptera on maize in South Africa or the potential impact of Bt maize on non-target Lepidoptera species under field conditions. There was also no information on the susceptibility to Bt maize of another stem borer species, Sesamia calamistis (Hampson) (Lepidoptera: Noctuidae), which is not a target species of Bt maize. The aims of this study were to determine which Lepidoptera species occur and feed on maize and could be directly exposed to Bt toxin as well as to assess the levels of infestation of target stem borer species and non-target Lepidoptera species on Bt- and non-Bt maize fields. Field collections of Lepidoptera that were directly exposed to Bt toxin through feeding on Bt maize plants were done between January 2004 and May 2006. Surveys were conducted in the North-West, Free State, Gauteng and Limpopo provinces. In order to quantify infestation levels and incidence of larvae on plants, sampling was done by inspecting between 300 – 900 plants per field. Studies were also done to compare the incidence of damaged plants and larvae on plants between Bt- and adjacent non-Bt maize fields. The susceptibility of S. calamistis to several Bt maize hybrids was evaluated under laboratory and greenhouse conditions. Fifteen species of Lepidoptera were recorded on maize plants. The following six species were recorded to feed on Bt maize and were reared on Bt maize until the adult stage: Acantholeucania loreyi (Noctuidae), Agrotis segetum (Noctuidae), B. fusca (Noctuidae), Helicoverpa armigera (Noctuidae), Eublemma gayneri (Noctuidae) and Nola phaeocraspis (Nolidae). Although Bt maize was damaged by several species of leaf, stem and ear feeding Lepidoptera in this study, the incidence of damage was always significantly lower on Bt maize fields than susceptible fields. This study provided base line data on Lepidoptera that feed on Bt maize in South Africa. Non-target Lepidoptera species that are directly exposed to Bt toxin was identified. An ecological model wasused to develop a preliminary risk assessment for Bt maize through which priority species for research and monitoring was identified as well as species that are at risk of resistance development. / Thesis (M. Environmental Science (Plant Protection))--North-West University, Potchefstroom Campus, 2007.

The maize stem borers, Busseola fusca (Fuller) (Lepidoptera: Noctuidae) and Chilo partellus (Swinhoe) (Lepidoptera: Pyralidae) are economically important pests of maize in South Africa. Genetically modified Bt maize (MON810) expressing Cry1Ab protein is used to control these pests on approximately 425 000 hectares in South Africa. Before this study no information was available on the diversity of Lepidoptera on maize in South Africa or the potential impact of Bt maize on non-target Lepidoptera species under field conditions. There was also no information on the susceptibility to Bt maize of another stem borer species, Sesamia calamistis (Hampson) (Lepidoptera: Noctuidae), which is not a target species of Bt maize. The aims of this study were to determine which Lepidoptera species occur and feed on maize and could be directly exposed to Bt toxin as well as to assess the levels of infestation of target stem borer species and non-target Lepidoptera species on Bt- and non-Bt maize fields. Field collections of Lepidoptera that were directly exposed to Bt toxin through feeding on Bt maize plants were done between January 2004 and May 2006. Surveys were conducted in the North-West, Free State, Gauteng and Limpopo provinces. In order to quantify infestation levels and incidence of larvae on plants, sampling was done by inspecting between 300 – 900 plants per field. Studies were also done to compare the incidence of damaged plants and larvae on plants between Bt- and adjacent non-Bt maize fields. The susceptibility of S. calamistis to several Bt maize hybrids was evaluated under laboratory and greenhouse conditions. Fifteen species of Lepidoptera were recorded on maize plants. The following six species were recorded to feed on Bt maize and were reared on Bt maize until the adult stage: Acantholeucania loreyi (Noctuidae), Agrotis segetum (Noctuidae), B. fusca (Noctuidae), Helicoverpa armigera (Noctuidae), Eublemma gayneri (Noctuidae) and Nola phaeocraspis (Nolidae). Although Bt maize was damaged by several species of leaf, stem and ear feeding Lepidoptera in this study, the incidence of damage was always significantly lower on Bt maize fields than susceptible fields. This study provided base line data on Lepidoptera that feed on Bt maize in South Africa. Non-target Lepidoptera species that are directly exposed to Bt toxin was identified. An ecological model wasused to develop a preliminary risk assessment for Bt maize through which priority species for research and monitoring was identified as well as species that are at risk of resistance development. / Thesis (M. Environmental Science (Plant Protection))--North-West University, Potchefstroom Campus, 2007.

Based on surface area, South Africa is currently ranked 8th in planting genetically modified (GM) crops in the world. The stem borer, Busseola fusca (Fuller) (Lepidoptera: Noctuidae) is of economic importance throughout sub–Saharan Africa. Bt maize (MON810) has been grown to control lepidopterous stem borers in South Africa since its first release in 1998. The first report of resistance to Bt maize was made in the Christiana area of South Africa in 2007. The objectives of this study were to evaluate the status of resistance of populations of B. fusca to Bt maize; to evaluate farmers' perceptions of the regulatory aspects guiding the planting of Bt maize and refugia and how the field situation developed between 1998 and 2010; to compare the fitness of the fertility, fecundity and longevity of Bt–resistant and susceptible B. fusca populations and to determine if there are fitness costs associated with resistance of B. fusca to Bt maize. Questionnaire surveys were conducted amongst 185 farmers in seven districts throughout the maize production region. The questionnaire addressed signing of contracts upon purchasing GM seed, refuge compliance, pest management practices, perceived benefits and risks relating to Bt maize. In order to study fitness and fitness costs that may be associated with resistance development, the life history parameters of known Bt–resistant and susceptible populations were compared in the laboratory using a diapauses–as well as second–generation populations collected in maize fields. The following parameters were compared between different stem borers populations and treatments: pupal mass, moth longevity, fecundity, fertility, larval mass and survival, and sex ratio. This study confirmed resistance of B. fusca to the Cry1Ab toxin (MON810) and that larvae collected from refugia at Vaalharts were resistant and survived on Bt maize. Compliance to refugia requirements was low especially during the initial 5 – 7 years after release. An alarmingly high number of farmers applied insecticides as preventative sprays on Bt maize and refugia. Except for moth longevity and LT50– values, no other fitness costs were observed to be associated with the resistance trait in the highly resistant B. fusca population used in this study. The LT50 may indicate some degree of fitness cost but does not translate into observable costs in terms of fecundity, larval mass and survival. The absence of fitness costs may promote the use of alternative Bt–resistance management strategies, such as the introduction of a multigene strategy. The introduction of a stacked event such as MON89034 which produces more than one protein with activity active against the resistant target pest, together with compliance to the refuge strategy, is most likely the only solution to managing Btresistant stem borer populations in South Africa. / Thesis (Ph.D. (Environmental Science))--North-West University, Potchefstroom Campus, 2011.

Host recognition behaviour of two braconid larval parasitoids Cotesia sesamiae and Cotesia flavipes was studied using suitable stemborer hosts [i.e. Busseola fusca for C. sesamiae, and Chilo partellus for C. flavipes] and one non-host [Eldana saccharina]. The wasps displayed similar sequences of behavioural steps when locating their hosts largely depending on their antennae for host recognition and both antennae and tarsi for final host acceptance and oviposition. Tactile and contact chemoreception stimuli from the hosts seem to play a major role in oviposition decision by the parasitoids. In addition, the external morphology and distribution pattern of sensilla present on antennae, tarsi and ovipositor of the parasitoids were examined by scanning electron and optic microscopy after staining with silver nitrate. Three sensillar types were identified on the distal antennomeres: (i) non-porous sensilla trichoidea most probably involved in mechanoreception, (ii) uniporous sensilla chaetica likely to be gustatory and, (iii) multiporous sensilla placodea likely to be olfactory. The tarsi possess a few sensilla chaetica which could be gustatory while the manubrium is likely to be used in detection of vibrations. The distal end of the ovipositor bears numerous multiporous dome-shaped sensilla. Additionally, the ability of the wasps to discriminate between contact cues was studied. When host larvae were washed in distilled water the wasps did not insert their ovipositors. However, ovipositor insertion resumed when washed host or non-host larvae were painted with water extracts of their respective host larvae. The water extracts of the suitable hosts were more attractive to the wasps than those of non-hosts. Similarly, the frass is important in host recognition during short-range examination as those of respective hosts are more intensely antennated than of non-hosts. The parasitoids were able to discriminate the regurgitant of E. saccharina by not antennating the cotton wool ball of this host; while the regurgitant of B. fusca and C. partellus appeared not useful in discriminating between the two species for both parasitoid species. Further analysis suggests the presence of a protein(s) component(s) in the regurgitant possibly responsible for host recognition and oviposition by C. flavipes. / Thesis (Ph.D. (Environmental Science))--North-West University, Potchefstroom Campus, 2009.

Based on surface area, South Africa is currently ranked 8th in planting genetically modified (GM) crops in the world. The stem borer, Busseola fusca (Fuller) (Lepidoptera: Noctuidae) is of economic importance throughout sub–Saharan Africa. Bt maize (MON810) has been grown to control lepidopterous stem borers in South Africa since its first release in 1998. The first report of resistance to Bt maize was made in the Christiana area of South Africa in 2007. The objectives of this study were to evaluate the status of resistance of populations of B. fusca to Bt maize; to evaluate farmers' perceptions of the regulatory aspects guiding the planting of Bt maize and refugia and how the field situation developed between 1998 and 2010; to compare the fitness of the fertility, fecundity and longevity of Bt–resistant and susceptible B. fusca populations and to determine if there are fitness costs associated with resistance of B. fusca to Bt maize. Questionnaire surveys were conducted amongst 185 farmers in seven districts throughout the maize production region. The questionnaire addressed signing of contracts upon purchasing GM seed, refuge compliance, pest management practices, perceived benefits and risks relating to Bt maize. In order to study fitness and fitness costs that may be associated with resistance development, the life history parameters of known Bt–resistant and susceptible populations were compared in the laboratory using a diapauses–as well as second–generation populations collected in maize fields. The following parameters were compared between different stem borers populations and treatments: pupal mass, moth longevity, fecundity, fertility, larval mass and survival, and sex ratio. This study confirmed resistance of B. fusca to the Cry1Ab toxin (MON810) and that larvae collected from refugia at Vaalharts were resistant and survived on Bt maize. Compliance to refugia requirements was low especially during the initial 5 – 7 years after release. An alarmingly high number of farmers applied insecticides as preventative sprays on Bt maize and refugia. Except for moth longevity and LT50– values, no other fitness costs were observed to be associated with the resistance trait in the highly resistant B. fusca population used in this study. The LT50 may indicate some degree of fitness cost but does not translate into observable costs in terms of fecundity, larval mass and survival. The absence of fitness costs may promote the use of alternative Bt–resistance management strategies, such as the introduction of a multigene strategy. The introduction of a stacked event such as MON89034 which produces more than one protein with activity active against the resistant target pest, together with compliance to the refuge strategy, is most likely the only solution to managing Btresistant stem borer populations in South Africa. / Thesis (Ph.D. (Environmental Science))--North-West University, Potchefstroom Campus, 2011.

Host recognition behaviour of two braconid larval parasitoids Cotesia sesamiae and Cotesia flavipes was studied using suitable stemborer hosts [i.e. Busseola fusca for C. sesamiae, and Chilo partellus for C. flavipes] and one non-host [Eldana saccharina]. The wasps displayed similar sequences of behavioural steps when locating their hosts largely depending on their antennae for host recognition and both antennae and tarsi for final host acceptance and oviposition. Tactile and contact chemoreception stimuli from the hosts seem to play a major role in oviposition decision by the parasitoids. In addition, the external morphology and distribution pattern of sensilla present on antennae, tarsi and ovipositor of the parasitoids were examined by scanning electron and optic microscopy after staining with silver nitrate. Three sensillar types were identified on the distal antennomeres: (i) non-porous sensilla trichoidea most probably involved in mechanoreception, (ii) uniporous sensilla chaetica likely to be gustatory and, (iii) multiporous sensilla placodea likely to be olfactory. The tarsi possess a few sensilla chaetica which could be gustatory while the manubrium is likely to be used in detection of vibrations. The distal end of the ovipositor bears numerous multiporous dome-shaped sensilla. Additionally, the ability of the wasps to discriminate between contact cues was studied. When host larvae were washed in distilled water the wasps did not insert their ovipositors. However, ovipositor insertion resumed when washed host or non-host larvae were painted with water extracts of their respective host larvae. The water extracts of the suitable hosts were more attractive to the wasps than those of non-hosts. Similarly, the frass is important in host recognition during short-range examination as those of respective hosts are more intensely antennated than of non-hosts. The parasitoids were able to discriminate the regurgitant of E. saccharina by not antennating the cotton wool ball of this host; while the regurgitant of B. fusca and C. partellus appeared not useful in discriminating between the two species for both parasitoid species. Further analysis suggests the presence of a protein(s) component(s) in the regurgitant possibly responsible for host recognition and oviposition by C. flavipes. / Thesis (Ph.D. (Environmental Science))--North-West University, Potchefstroom Campus, 2009.

Organic insecticides play a big role in reducing the usage of chemical insecticides and their negative impact on the environment. Bacillus thuringiensis (Bt) spays are the only tool that organic farmers are allowed to use for the control of pests. Genetic engineering and modification of crops have been made possible with scientific advances in cell and molecular biology. These advances are used to transfer some of the Bt Cry toxins into crops for control of target species to reduce yield loss. Bt maize were commercialised for the first time in South Africa in 1998 and the economic important stem borers, Busseola fusca (Fuller) (Lepidoptera: Noctuidae), Chilo partellus (Swinhoe) (Lepidoptera: Crambidae) and Sesamia calamistis (Hampson) (Lepidoptera: Noctuidae) were exposed to the Cry1Ab toxin that is found in Bt maize. Busseola fusca developed resistance to Cry1Ab under field conditions within eight years after it had been released. Eldana saccharina (Walker) (Lepidoptera: Pyralidae) is a major pest on sugarcane in South Africa and although it has not been recorded on maize in this country, is it known as a major pest of maize in other African countries. African armyworm, Spodoptera exempta (Walker) (Lepidoptera: Noctuidae) has a very wide distribution in Africa and is known to be an occasional pest on maize. The aims of this dissertation were to determine the efficacy of Bt spray applications for control of four lepidopteran pests and whether development of Cry1Ab resistance by B. fusca caused a loss in susceptibility to other Bt toxins (i.e. cross-resistance). Susceptibility bioassays with 10 day old larvae were conducted under laboratory conditions. Treatments included application of various dosage rates of Dipel® and deltamethrin as well as exposure to MON810 (maize leaves). Stemborer populations of C. partellus, E. saccharina, and B. fusca (Venda) as well as the S. exempta were effectively controlled by the Bt spray, Dipel®. Care should be taken not be interpret the percentage C. partellus, E. saccharina and S. exempta larvae that survived after exposure to MON810 and Bt spray treatments as development of resistance without verification of these experiments with earlier instars that are known to be more susceptible. Spodoptera exempta is active throughout a year in temperate zones of Africa. If S. exempta develop resistance to Cry toxins and Bt maize events would be released for commercial planting in these areas, S. exempta pose a threat added to their injuriousness. Busseola fusca larvae were sampled from Venda (susceptible population), Ventersdorp and the Vaalharts Irrigation Scheme (resistant population). The Ventersdorp B. fusca population was controlled by MON810 and MON89034 and Bt sprays, but the percentage larvae that survived showed reduced susceptibility within the population. Dipel® treatments, MON810 and MON89034 did not provide effective control of the Vaalharts B. fusca population reported to be resistant to Cry1Ab, in two experiments. The high survival rates indicate a reduction in susceptibility to Cry toxins other than Cry1Ab and therefore development of cross resistance in the Vaalharts B. fusca population. / MSc (Environmental Sciences), North-West University, Potchefstroom Campus, 2014