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.

Trichogramma limit pest damage to Ord River Irrigation Area (ORIA) cotton crops by killing the developing embryo of their insect host at the egg stage, effectively reducing the number of emergent pests ingesting transgenic tissue. Their impact on the potentially resistant species, Helicoverpa armigera (Hübner), is considered integral to the Insect Resistance Management (IRM) strategy for transgenic cotton production in the ORIA. This thesis examines aspects of Trichogramma ecology pertinent to this strategy. The dominant species of egg parasitoid in ORIA cotton crops is the introduced Trichogramma pretiosum Riley. Other species make up less than one percent of collected specimens. Surveys revealed T. pretiosum has been introduced or adventitiously dispersed to all developed agricultural regions of northern Australia. Several new species were discovered during surveys in more pristine habitats. Based on field collected eggs, T. pretiosum appears to prefer H. armigera over H. punctigera Wallengren as a host in ORIA cotton. Measured as percent parasitism, Trichogramma activity appears highly variable and does not necessarily coincide with periods of peak insect pest density. Host abundance alone does not define conditions suitable for Trichogramma activity. Environmental constraints on wasp survival, such as the impact of temperature, humidity and insecticide applications, limit their effectiveness in biological control. Despite consistently high rates of percentage egg parasitism (60-99%), acceptable pest control is not readily achieved in ORIA cotton without the aid of insecticides as Helicoverpa numbers exceed damage thresholds. Insecticides inhibit Trichogramma considerably, hence the conundrum regarding initiation of insecticidal control. Trichogramma activity is relatively high early season (May to July), significantly limiting the buildup of pests. Trichogramma effectively stifle Helicoverpa population increase following initial pest egg lay at least during high density years. The impact of farming practices, especially insecticide applications, should be avoided early season to ensure pest mortality attributed to Trichogramma egg parasitisation is maximised. Pre-season habitat manipulation to establish large populations of Trichogramma in alternative hosts is advised. Despite being rare in surrounding habitats and suffering near 50% immature mortality in the field, Trichogramma effectively disperse into young crops attractive to ovipositing hosts and display a high intrinsic rate of increase. Spatial patterns of parasitism tend toward heterogeneity and do not necessarily coincide with host spatio-temporal dynamics. Both host abundance patterns and mean rates of parasitism are not good indicators of parasitoid patchiness. Parasitism rates are highest within the middle strata of the plant canopy prior to complete canopy closure despite a similar number of host eggs being available elsewhere in the plant. Pest density declines as the season progresses. However, insecticide applications become necessary if Bt expression in cotton plants wanes and larval damage increases. Measuring parasitism during periods of declining and low host density is prone to inaccuracy due to small sample size, but can be overcome with the use of egg cards. A better indication of parasitoid activity is achieved using egg cards during periods when insecticide applications are possibly required. As Trichogramma are most active in ORIA cotton from morning to early afternoon, insecticide applications if needed should occur outside of these periods. Trichogramma survival is constrained by environmental influences. Adult female T. pretiosum were exposed to ambient conditions in dialysis tubing sleeve cages to test survival and fecundity in cotton fields. Peak survival and fecundity occurs mid season with both life history variants displaying an inverse relationship to temperature. Adult female T. pretiosum survive longest in the field when provided with sustenance however mortality attributed to handling was considerable. Sleeve cages are not effective for survival measurement of small parasitoids if the cages are frequently moved. Trichogramma effectively reduce pest abundance but are clearly hindered by insecticides and hot dry conditions in ORIA cotton crops. The decision to initiate insecticide applications is best delayed unless absolutely necessary to avoid disruption of Trichogramma impact on pests. Parasitoid activity must be carefully monitored if chemical control becomes imminent. The impact of Trichogramma on pest species can then be optimally exploited.

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.

The bollworm, Helicoverpa zea (Boddie), is an economically important pest of cotton and is becoming more difficult to control in the United States. This project was designed to develop a better understanding of the effects of resistance development and plant structures on the efficacy of Bt cotton and foliar insecticides. Experiments examined larval behavior and damage in current cotton varieties expressing proteins from Bacillus thuringiensis. Other experiments examined the effects of floral structures on the control of bollworm using foliar insecticides. Results suggest that larval feeding and avoidance behavior is dependent on point of eclosion. Fruiting form damage increased in two-gene cotton compared to earlier research but was low in cotton expressing the Vip3A protein. Increases in damage can lead to more frequent applications of foliar insecticides. Floral structures can hinder insecticide efficacy by reducing exposure. Results from these experiments will be important for refining management recommendations for bollworm in Bt cotton.

Helicoverpa zea, commonly referred to as corn earworm, has been a major pest of corn and cotton, along with many major crops grown in United States. Migration of this pest plays a major role in its distribution and successful survival. Part of the current resistance management strategy for transgenic crops, using non-Bt crops as refuges, is totally based on the movement of the adult populations between the Bt and non-Bt fields, and successful exchange of genetic material between the rare resistant and susceptible populations. To study the movement and migration patterns, and their implications in resistance management, suitable molecular genetic markers were comparatively selected, followed by a study of temporal variations in populations from north Mississippi. The dominant marker system Inter Simple Sequence Repeats (ISSR) was selected for the study based on higher polymorphism (5.0) and PIC (0.34) values compared to Simple Sequence Repeats (SSR) and Sequence Related Amplification Polymorphism (SRAP) marker systems. 53 ISSR loci were used in understanding the temporal variations in H. zea. Percent polymorphism and heterozygosity levels showed variation among the twelve collections tested. Early spring putative migrants were distinct from the rest of the generations. Population differentiation was higher in the beginning of the season and then declined by end of the season (pairwise FST = 0.341, early in March and 0.025, late in August). Average heterozygosity levels recorded were 0.11, coinciding with 1993 and 2002 data. The result of this study supports the existence of spring migration of adults and their contribution to the local gene pool. The extent of genetic exchange between the putative migrants and the local populations varied among three years. There is variation in population densities from migrants and local emergence in one of the three years. The results of this study indicate a need for continuous monitoring for genetic changes and their possible implications for resistance management in transgenic crops in Mississippi.

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.