H. armigera larvae are a key stage for pest management in conmercial irrigated cotton crops in South Africa. Effective survey methods for detecting larval populations in the field require an understanding of the biology of the larvae, particularly their feeding habits. Their feeding is central to the development of pest threshold levels for the implementation of integrated control programmes. This applies to routine surveys for the larvae as well as to the damage they cause. Biological characteristics of the larvae are described with the emphasis on the identification of the larval instars, which were consistently five in number in both field and laboratory populations. The distribution of H. armigera larvae on cotton plants in the field was examined, but was found to more or less random; had there been a clear preference for any height zones or compass direction this would have been an obvious avenue for improving the survey methods currently in use. Details of field and laboratory investigations of the selection of feeding sites by the larvae are given. The study confirmed a clear preference by the larvae for cotton buds, flowers and bolls (in the thesis collectively called "fruiting forms"), over leaves. There were indications that the larvae selected flowers more readily than buds or bolls. This "preference", however, is shown to be of no practical value for refining survey methods. Damage levels to cotton due to B. armigera are discussed. Both direct losses and indirect losses due to the abortion of fruiting forms are considered. These criteria are inadequate since they do not take into account the ability of cotton plants to compensate for these losses. It is concluded that this compensation by cotton plants should be taken into account in further studies of the pest status of B. armigera.

The Tachinid fly, Palexorista laxa (Curran) is an important parasitoid of Heliothis armigera (Hubner) in Africa and India. The biological characteristics of the parasitoid were studied with an emphasis on its host instar preference, host range, seasonality and percentage parasitism. The developmental stages were also studied and described. A high host mortality (48%-100%) due to parasite attack occurred when small larvae were parasitised (2nd & 3rd instars). A preference for the 4th and 5th host instars was shown by the flies. P. laxa was found to be a non-specific parasitoid and parasitised a number of lepidopterous larvae in the laboratory including Busseola fusca (Fuller), Chilo partellus (Swinhoe) and Eldana saccharina Walker, but preferred Ii. armigera. The percentage parasitism on H. armigera in the field was variable peaking at 48% in 1988/89. Of the various parasitoids recorded from H. armigera on sunflower P. laxa was by far the most abundant. Mating occurred immediately after emergence with the males emerging a day earlier than the females. Males were capable of multiple matings whereas females mated only once in a lifetime. P. laxa females produced incubated macrotype eggs which hatched almost immediately after oviposition. The duration of development of the incubated egg and larval stage was 6,7 days, the pupa 10,4 days for females and 9,4 days for males, and the adult lived up to 50 days at 25°C. A reduction in pupal weight, an increase in the duration of the pupal stage and higher pupal mortality accompanied an increase in density of parasitoids per host. A linear relationship was shown between adult mass and number of ovarioles and eggs in P. laxa females. The oviposition period was 26,9 days with a high variation in daily progeny production. The total fertility per female was 126,3 puparia. "Inexperienced" females deposited incubated eggs on all parts of the host body while "experienced" females confined oviposition to the head and thorax. P. laxa females did not discriminate between previously parasitised and non-parasitised hosts. The effect of temperature on larval and pupal survival and development time was studied at 20°, 22°, 25°, 28°and 30°C. As expected, duration of development decreased with an increase in temperature, and there was a reduction in mortality.

Desde a década de 50, os cientistas tem utilizado radiação ionizante para esterilizar insetos, que são liberados na natureza para acasalar, mas sem nenhuma descendência. Conhecida como a técnica do inseto estéril (TIE), este método de controle de inseto vem tradicionalmente utilizando radiações ionizantes para esterilizar os insetos, sendo uma técnica que não gera resíduo, e pode agir em sinergia com as demais técnicas dentro do manejo integrado de pragas. O Brasil por vários anos vem lutando contra o aumento de pragas, introduzindo novas táticas e técnicas dentro dos programas de MIP, para driblar a resistência dos produtos químicos, como: diminuir os resíduos dos agrotóxicos; para algumas culturas importantes do o nosso país, temos um largo espectro de pragas ocorrendo do inicio ao fim da colheita, uma delas é a cultura do algodoeiro e dentre as pragas chave dessa cultura, temos algumas lagartas extremamente importantes, entre elas Heliothis virescens e Helicoverpa armigera. Essas espécies são parecidas morfologicamente, sendo a segunda identificada a poucos anos aqui no Brasil. Ainda não há trabalhos no Brasil utilizando TIE como ferramenta adicional para lepidópteros, portanto objetivou-se com esse estudo avaliar o efeito de doses de radiação gama nas diferentes fases do ciclo evolutivo de Heliothis virescens e Helicoverpa armigera, bem como avaliar a esterilidade na geração P e a capacidade de competição dos insetos irradiados com os não irradiados. A fase pupa foi a que apresentou um melhor resultado, pois com 75 Gy atingiu a esterilidade em Heliothis virecens e 100 Gy esterilizou Helicoverpa armigera, portanto contemplou a fase e a dose escolhida para avaliar a competição entre os insetos irradiados e os insetos normais de ambas as espécies. E tanto Heliothis virecens como Helicoverpa armigera apresentaram um resultado satisfatório, pois os insetos irradiados conseguiram reduzir significativamente a viabilidade dos ovos na proporção de 9: 1: 1 / Since the 1950s, scientists have used ionizing radiation to sterilize insects, which are released in nature to mate, but without any progeny. Known as the sterile insect technique (TIE), this method of insect control has traditionally used ionizing radiation to sterilize insects, a technique that does not generate residues, and can act in synergy with the other techniques within integrated pest management. For several years, Brazil has been fighting against the increase of pests, introducing new tactics and techniques within the IPM programs, to overcome the resistance of chemical products, such as: reducing the residues of agrochemicals; For some important crops of our country, we have a wide spectrum of pests occurring from the beginning to the end of the harvest, one of them is the cotton crop and among the key pests of this crop, we have some extremely important caterpillars, among them Heliothis virescens and Helicoverpa armigera These species are morphologically similar, the second being identified a few years ago in Brazil. There are still no studies in Brazil using TIE as an additional tool for Lepidoptera, therefore the purpose of this study was to evaluate the effect of doses of gamma radiation in the different phases of the evolutionary cycle of Heliothis virescens and Helicoverpa armigera, as well as to evaluate the sterility in generation P And the ability of insects to irradiate with non-irradiated insects. The pupal phase presented the best result because 75 Gy achieved sterility in Heliothis virecens and 100 Gy sterilized Helicoverpa armigera, therefore it contemplated the phase and dose chosen to evaluate the competition between the irradiated insects and the normal insects of both species. Both Heliothis virecens and Helicoverpa armigera presented a satisfactory result, as the irradiated insects managed to significantly reduce the viability of the eggs in a ratio of 9: 1: 1

Desde a década de 50, os cientistas tem utilizado radiação ionizante para esterilizar insetos, que são liberados na natureza para acasalar, mas sem nenhuma descendência. Conhecida como a técnica do inseto estéril (TIE), este método de controle de inseto vem tradicionalmente utilizando radiações ionizantes para esterilizar os insetos, sendo uma técnica que não gera resíduo, e pode agir em sinergia com as demais técnicas dentro do manejo integrado de pragas. O Brasil por vários anos vem lutando contra o aumento de pragas, introduzindo novas táticas e técnicas dentro dos programas de MIP, para driblar a resistência dos produtos químicos, como: diminuir os resíduos dos agrotóxicos; para algumas culturas importantes do o nosso país, temos um largo espectro de pragas ocorrendo do inicio ao fim da colheita, uma delas é a cultura do algodoeiro e dentre as pragas chave dessa cultura, temos algumas lagartas extremamente importantes, entre elas Heliothis virescens e Helicoverpa armigera. Essas espécies são parecidas morfologicamente, sendo a segunda identificada a poucos anos aqui no Brasil. Ainda não há trabalhos no Brasil utilizando TIE como ferramenta adicional para lepidópteros, portanto objetivou-se com esse estudo avaliar o efeito de doses de radiação gama nas diferentes fases do ciclo evolutivo de Heliothis virescens e Helicoverpa armigera, bem como avaliar a esterilidade na geração P e a capacidade de competição dos insetos irradiados com os não irradiados. A fase pupa foi a que apresentou um melhor resultado, pois com 75 Gy atingiu a esterilidade em Heliothis virecens e 100 Gy esterilizou Helicoverpa armigera, portanto contemplou a fase e a dose escolhida para avaliar a competição entre os insetos irradiados e os insetos normais de ambas as espécies. E tanto Heliothis virecens como Helicoverpa armigera apresentaram um resultado satisfatório, pois os insetos irradiados conseguiram reduzir significativamente a viabilidade dos ovos na proporção de 9: 1: 1 / Since the 1950s, scientists have used ionizing radiation to sterilize insects, which are released in nature to mate, but without any progeny. Known as the sterile insect technique (TIE), this method of insect control has traditionally used ionizing radiation to sterilize insects, a technique that does not generate residues, and can act in synergy with the other techniques within integrated pest management. For several years, Brazil has been fighting against the increase of pests, introducing new tactics and techniques within the IPM programs, to overcome the resistance of chemical products, such as: reducing the residues of agrochemicals; For some important crops of our country, we have a wide spectrum of pests occurring from the beginning to the end of the harvest, one of them is the cotton crop and among the key pests of this crop, we have some extremely important caterpillars, among them Heliothis virescens and Helicoverpa armigera These species are morphologically similar, the second being identified a few years ago in Brazil. There are still no studies in Brazil using TIE as an additional tool for Lepidoptera, therefore the purpose of this study was to evaluate the effect of doses of gamma radiation in the different phases of the evolutionary cycle of Heliothis virescens and Helicoverpa armigera, as well as to evaluate the sterility in generation P And the ability of insects to irradiate with non-irradiated insects. The pupal phase presented the best result because 75 Gy achieved sterility in Heliothis virecens and 100 Gy sterilized Helicoverpa armigera, therefore it contemplated the phase and dose chosen to evaluate the competition between the irradiated insects and the normal insects of both species. Both Heliothis virecens and Helicoverpa armigera presented a satisfactory result, as the irradiated insects managed to significantly reduce the viability of the eggs in a ratio of 9: 1: 1

Helicoverpa armigera (cotton bollworm) is a major agricultural pest in Australia, Asia and Africa. Analysis of a midgut cDNA library revealed a surprising diversity of genes encoding lipases, enzymes that metabolise lipids. Prior to and during this project, 13 neural lipases and three acidic ones were discovered. These were classified and compared to other insect lipases using sequence alignments and phylogenetic analysis. Novel non-catalytic subfamilies were discovered, including one containing an H. armigera lipase. At least three other subfamilies were found to contain H. armigera lipases. Lipidopteran lipases were found to be almost entirely distinct from Dipteran ones.

Helicoverpa armigera Hübner (Lepidoptera: Noctuidae) (African bollworm) is a typical noctuid with a very catholic taste in food plants and whose larvae feed on a wide range of cultivated and wild plants. It has been identified as the most polyphagous and injurious pest in South Africa. Helicoverpa armigera is also a key pest of cotton in many parts of the world. This key pest requires extensive control as it adversely effects yield and has built up resistance to synthetic pyrethroid insecticides. Cotton is an important crop produced by commercial and small-scale farmers in South Africa. The local demand for cotton has not been exceeded yet, but to satisfy a demanding market, pest control costs play an important role in cotton production. The threat of an insect pest that has already shown resistance prompted the present study to investigate the possibility of resistance to Bt-cotton. Genetically engineered or Bt-cotton was introduced commercially in 1996 in South Africa. All Bt-cotton plants contain one or more foreign genes derived from the soil-dwelling bacterium, Bacillus thuringiensis (Berliner), which produces protein crystals. These crystals were isolated and transferred into the genome of a cotton plant resulting in the plant producing it’s own protein insecticide. In 1998, Monsanto (Pty) Ltd requested research into the geographic susceptibility of H. armigera to the insecticidal proteins in Bt-cotton in SA. Laboratory reared and field sampled populations of H. armigera were exposed to a diet mixed with various baseline concentrations of the Bt-gene Cry1Ac freeze dried protein. This study also determined the performance of H. armigera and Spodoptera littoralis (Boisduval) on different Bt-cotton field cultivars containing different Cry-protein genes. Results obtained indicated a significant difference in susceptibility in two field populations of H. armigera to the Bt-protein Cry1Ac, even though the LD50,s in the 2003 season did not indicate resistance. Bt-cotton cultivar 15985 BX controlled H. armigera and S. littoralis larvae, the best followed in descending order by cultivar 15985 X, 15985 B and DP50 B. Results on H. armigera also indicated that the Cry-proteins in the plant parts of the different cultivars did not diminish as the season progressed. The Bt-cotton cultivars induced retarded growth of larvae, due to either a repellent effect or lack of feeding by larvae. Widespread adoption of Bt-cotton by South African farmers led to regional declines in bollworm populations, reduced insecticide use, and increased yields. Genetically modified crops therefore contribute to a cost effective, sustainable, productive and efficient form of agriculture, with a resultant positive impact on the environment. As the market for commercial Bt-cotton in South Africa expands, it is recommended that a monitoring programme for potential resistant genes in H. armigera should be implemented at least every 2 - 3 years. This will ensure that effective resistance management strategies are utilised. Coupled with this are the Biosafety Risks regarding the effect of new proteins expressed in transgenic plants, which require further studies.

Sunflower production in South Africa has increased four fold since the 1970's. This study was done to elucidate the pest status of Heliothis armigera (Hiibner) on sunflower. Field studies were undertaken at Warmbaths, Brits and Delmas during the summer seasons of 1988/89 to 1990/91. The infestations at the latter two areas were negligible throughout the study period and their statistics have not been included in this thesis. Plant development and oviposition by natural H. armigera populations were found to be correlated as, regardless of planting date, oviposition started at six to seven weeks after planting; when the flowering stage began. A peak in egg numbers was reached by the tenth week with an average for the three seasons at two eggs per plant. The eggs were laid singly on the bracts and bases of the flower buds. A peak in larval numbers was reached at the thirteenth week after planting with the average for the three seasons of 0,4 larvae per plant. The preferred feeding sites were on, between and under the bracts from where the larvae burrow into the pithy tissue of the receptacle. Only six percent of the larvae were found feeding directly on the achenes. Eggs and larvae collected were reared to determine the degree of parasitism. Overall, 19% of the eggs were parasitised; 18% by Telenomus ullyetti Nixon (Scelionidae) and one percent by Trichogrammatoidea lutea (Trichogrammatidae). Larval parasitism at Warmbaths was 23% in 1988/89, 27% in 1989/90 and 34% in 1990/91. Of the parasitised larvae, 44% succumbed to Palexorista prob. laxa (Tachinidae). The remainder were unidentified Braconidae and Ichneumonidae. Predators, such as chrysopids, were observed during the study but their effect on egg and larvae numbers was not studied in detail. A polyhedral virus occurred late in the seasons and caused mortality of the larvae. The low numbers of H. armigera on sunflower, the slight damage to the crop and the reasonably high rate of parasitism, all seem to indicate that H. armigera is not an economica1ly important pest of sunflower and that additional control methods are not justified.

Tetraviruses are a family of small non-enveloped positive sense RNA viruses that exclusively infect members of the order Lepidoptera. Their replication biology is poorly studied because, with the exception of Providence virus (PrV), tetraviruses are unable to replicate in tissue culture cells. The overall aim of the research described in this thesis was to develop a fundamental understanding of the replication of tetraviruses, focussing on the site of replication within host cells and in particular, the subcellular localisation of the viral replicase. Helicoverpa armigera stunt virus (HaSV, Genus: Omegatetravirus) was chosen for this study because it is the only tetravirus for which the cDNAs have been shown to be infectious. In the absence of tissue culture cell lines susceptible to HaSV infection, the approach was to use confocal fluorescence microscopy to examine the subcellular localisation of the HaSV replicase fused to enhanced green fluorescent protein (EGFP) in mammalian and insect tissue culture cells. The replicase (with EGFP fused at its C-terminus) localised to punctate structures throughout the cytoplasm of transfected HeLa and Sf9 cells. These structures were then shown – using live cell imaging and time lapse photography – to behave similarly to cellular endocytic organelles and fluorescence partially overlapped with membranes containing the late endosomal marker protein CD63. Biochemical fractionation of Sf9 cells expressing the replicase via a recombinant baculovirus (as well as transfected HeLa and Sf9 cells expressing EGFP-replicase fusion proteins) demonstrated that the replicase was strongly associated with detergentresistant membranes (DRMs) in these cells. Deletion analysis of the replicase coding sequence revealed two regions involved in the generation of the punctuate structures. Firstly, the C-terminal half of the replicase RNAdependant RNA polymerase domain was found to be essential for targeting and the tight association with DRMs while the second region, within the Nterminal 44 amino acids, enhanced localisation through a combination of secondary structural elements and sequence-specific functions. A comparative immunofluorescence study on PrV, which replicates as a persistent infection in an insect midgut cell line, showed that the PrV replicase also localised to punctate structures in the cytoplasm. Biochemical fractionation showed that the replicase was also strongly associated with DRMs. This thesis describes the development of new experimental systems for the study of tetravirus replication biology and the data lead to the conclusion that the HaSV replicase associates with DRMs derived from alternate endocytic pathway organelles.

The capsid protein of Helicoverpa armigera stunt virus (HaSV) a T=4 insect virus was expressed in Spodoptera frugiperda 9 cells using a baculovirus vector. When the insect cells were infected at a high MOl the expressed coat protein assembled into virus-like particles (VLPs) that spontaneously underwent maturation and were morphologically indistinguishable from wild-type HaSV. The VLPs were electron dense when viewed under EM and encapsidated their coat protein mRNA. When Sf9 cells were infected at a low multiplicity of infection (MOl) the expressed capsid protein assembled into procapsids that did not spontaneously undergo maturation. These procapsids underwent autoproteolytic maturation cleavage when they were treated with an acidic buffer. The procapsids were used in the encapsidation of a FITC labelled peptide. The peptide encapsidating VLPs showed an increase in their buoyant density that was not collaborated by an increase in the concentration of the FITC labelled peptide detected when these samples were compared to control samples with similar buoyant densities.

In this study, a sensitive high performance thin-layer chromatography method (HPTLC) was developed to detect lipids in microgram (ug) quantities of hemolymph. The effect of age, diurnal rhythms, and flight activity on hemolymph 1,2-diacylglyceride concentrations in male and female Heliothis zea (Boddie) (Lepidoptera: Noctuidae) were quantitated. The HPTLC method involved a two-stage, one dimensional solvent system, 1 ). benzene:ether(diethyl):ethanol:acetic acid, 50:40:2.0:0.2; 2). hexane:heptane: ether (diethyl): acetic acid, 63:18:18:1. The solvent system separated alkanes, steryl ester, mono-, di- and triacylglycerides, free fatty acids and cholesterol. Lipids were detected with a fluorescent reagent, 1 mM 6-p-toludino-2-napthalenesulfonic acid (TNS) in 95% methanol. Detection sensitivities were 50 ng qualitatively and 500 ng quantitatively. Most male and female Heliothis zea flight activity occurred between 17⁰⁰ and 3⁰⁰ EST each day. Male flight activity after the day of eclosion through 6 days was 0.8, 1.0, 7.0, 3.3, 3.8, 2.8 and 4.0 times the flight activity of the females. Females had a unimodal pattern of flight activity between 17⁰⁰ and 19⁰⁰ Males had a bimodal pattern between 17⁰⁰ and 19⁰⁰, but another higher peak of flight activity existed between 20⁰⁰ and 24⁰⁰. The hemolymph of Heliothis zea males and females prior to flight (17⁰⁰) was composed of mainly diacylglycerides (50% w/w), and triacylglycerides (35% w/w). In addition, cholesterol esters (2%) and less than 1% monoacylglycerides and cholesterol were present, but free fatty acids were not detectable ( <0.5 ug/ul). The lipid class, 1,2-diacylglycerides, apparently are used for flight energy. They occured in the highest concentrations in both males and females prior to flight (17⁰⁰) (approximately 32 ug/ul), then decreased steadily throughout flight to approximately 16 ug/ul at 2⁰⁰ when flight activity was reduced or ceased. Males and females appear to have similar 1,2-diacylglyceride concentrations prior to, during and after flight activity. / M.S.