Submitted by (edna.saturno@ufrpe.br) on 2016-11-28T12:40:36Z No. of bitstreams: 1 Luziani Rezende Bestete.pdf: 403712 bytes, checksum: 4a5246d287630547b7bb138f6fc4cd2f (MD5) / Made available in DSpace on 2016-11-28T12:40:36Z (GMT). No. of bitstreams: 1 Luziani Rezende Bestete.pdf: 403712 bytes, checksum: 4a5246d287630547b7bb138f6fc4cd2f (MD5) Previous issue date: 2011-02-01 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPES / The tomato fruitworm (TFW), Helicoverpa zea (Boddie) (Lep.: Noctuidae) is considered an important pest in several crops. The purpose of the study was to evaluate the use of castor bean oil, garlic and tobacco extracts to control H. zea larvae, and the influence of these products on the egg parasitoid Trichogramma pretiosum Riley (Hym: Trichogrammatidae). Larval mortality of TFW was investigated when treated with the alternative products at different concentrations through ingestion and contact aplication. Tests of selectivity and susceptibility of T. pretiosum were performed by spraying TFW eggs prior and post parasitism. Castor bean oil at 3% (v/v) through ingestion and contact treatments of TFW larvae caused mortality of 44 and 20%, respectively. Garlic extract at all concentrations and type of application resulted in TFW mortality equal or greater than the tobacco extract. The number of eggs parasitized by T. pretiosum was reduced by castor bean oil, while other biological parameters were not affected. Garlic extract positively interacted with T. pretiosum resulting in greater number of eggs parasitized, although it was observed a lower number of individuals emerging per parasitized egg in selectivity test. Castor bean oil was toxic to TFW larvae in both types of application. Garlic extract produced better results when ingested by TFW larvae and it was superior when compared to tobacco extract to control TFW larvae. Based on the results, castor bean oil and garlic extract are compatible with T. pretiosum, but the former one should be applied after the parasitoid release. Therefore, castor bean oil and garlic extract are promising as phytosanitary tools to control H. zea larvae. / A broca-grande (BRG), Helicoverpa zea (Boddie) (Lep.: Noctuidae) é considerada uma praga importante em diversas culturas. Assim, neste trabalho foi avaliado o uso do óleo de mamona e os extratos de alho e fumo para o controle de H. zea, e a influência desses sobre o parasitóide de ovos Trichogramma pretiosum Riley (Hym.: Trichogrammatidae). Foram estudadas as vias de aplicação por ingestão e contato, em diferentes concentrações, sobre a mortalidade larval da BRG. Testes de seletividade e susceptibilidade de T. pretiosum foram realizados através da pulverização dos ovos de BRG antes e após o parasitismo. O óleo de mamona a 3% (v/v) via ingestão e contato ocasionou 44 e 20% de mortalidade de larvas, respectivamente. O extrato de alho em todas as concentrações e em ambas as vias de aplicação foi sempre igual ou superior ao extrato de fumo, ocasionando as maiores mortalidades de larvas. O número de ovos da BRG parasitados por T. pretiosum foi afetado pelo óleo de mamona, porém os demais parâmetros não foram influenciados. O extrato de alho interagiu positivamente com T. pretiosum obtendo maior número de ovos parasitados, porém no teste de seletividade reduziu o número de indivíduos emergidos por ovo. O óleo de mamona mostrou toxicidade em ambas às vias de aplicação para a BRG. O extrato de alho foi mais eficiente para o controle da BRG quando comparado ao extrato de fumo, atuando principalmente por ingestão. Com base nos resultados, o óleo de mamona e o extrato de alho são compatíveis com a utilização de T. pretisoum, porém o óleo de mamona deve ser utilizado após a liberação do parasitóide. A utilização do óleo de mamona e extratos de alho é viável para o emprego em programas de manejo fitossanitário de H. zea.

Insect herbivores often secrete glucose oxidase (GOX) onto plants to counteract plant defenses and potential pathogens. Whether generalist herbivores always have significantly higher GOX activities than their specialist counterparts at any comparable stage or conditions and how this is realized remain unknown. To address these two general questions, we subjected larvae of a pair of sister species differed mainly in host range, the generalist Helicoverpa armigera and its specialist counterpart Helicoverpa assulta, to the same sets of stage, protein to digestible carbohydrate (P:C) ratio, allelochemical or host plant treatments for simultaneous analyses of GOX transcripts and activities in their labial glands. GOX activity and transcripts are upregulated concurrently with food ingestion and body growth, downregulated with stopping ingestion and wandering for pupation in both species. The three tested host plants upregulated GOX transcripts, and to a lesser extent, GOX activity in both species. There were significant differences in both GOX transcripts and activity elicited by allelochemicals, but only in GOX transcripts by P:C ratios in both species. GOX activities were higher in H. armigera than H. assulta in all the comparable treatments, but GOX transcripts were significantly higher either in generalists or in specialists, depending on the developmental stages, host plants, P:C ratio and allelochemicals they encounter. These data indicate that the greater GOX activity in generalist herbivores is not achieved by greater transcription rate, but by greater transcript stability, greater translation rate, better enzyme stability and/or their combination.

The Tetraviridae are a family of ss (+) RNA viruses that specifically infect lepidopteran insects. Their icosahedral capsids are non-enveloped and approximately 40 nm in diameter with T=4 quasi-equivalent symmetry. The omegatetraviruses, which are structurally the best characterised in the family, include Helicoverpa armigera stunt virus (HaSV) and Nudaurelia capensis omega virus (NwV). The omegatetravirus procapsid is composed of 240 identical copies of the capsid precursor proteins, which undergo autoproteolytic cleavage at its carboxyl-terminus generating the mature capsid protein (b) and γ-peptide. This process occurs in vitro following a shift from pH 7.6 to pH 6.0. The viral capsid encapsidates two ss genomic RNAs: The larger RNA1 encodes the viral replicase as well as three small ORFs while RNA2 encodes the capsid precursor protein together with an overlapping ORF designated P17. While a wealth of structural data pertaining to the assembly and maturation of omegatetraviruses is available, little is known about how this relates to their lifecycle. The principle aim of the research described in this thesis was to use an experimental system developed in the yeast, Saccharomyces cerevisiae, to investigate the assembly of HaSV and NwV virus-like particles (VLPs) in terms of maturation and encapsidation of viral RNAs, in vivo. The yeast expression system used two promoter systems for expression of capsid precursor protein: in the first, a hybrid promoter (PGADH) was used for high-level expression, while the second, PGAL1, produced substantially lower levels of the virus capsid protein precursors. An increase in the level of HaSV capsid protein precursor (p71) via the PGADH promoter resulted in a dramatic increase in VLP assembly as compared with the PGAL system. A protein equivalent to the mature capsid protein (p64) appeared at later time intervals following induction of transcription. Transmission electron microscopic studies showed that p64 correlated with the presence of mature VLPs as opposed to procapsids in cells containing p71. This confirmed that the presence of p64 denoted maturation of VLPs in vivo. Further investigation indicated that maturation correlated with cell aging and the onset of apoptosis. It was shown that induction of apoptosis resulted in VLP maturation while inhibition of apoptosis prevented maturation. These results suggested that the process of apoptosis might be the trigger for maturation of virus procapsids in their host cells. The increase in the efficiency of VLP assembly observed in the high-level expression system was proposed to be due to an increase in the cellular concentrations of viral RNA. To test this hypothesis, HaSV P71 was co-expressed with either P71 mRNA or full length RNA2. An increase in the solubility of p71 was observed in cells expressing increased levels of both RNAs, but there was no increase in the efficiency of VLP assembly. Northern analysis of encapsidated RNAs revealed that there was no selective encapsidation of either P71 mRNA or viral RNA2. This data indicated that the increase in viral RNA was not the reason for increased efficiency of VLP assembly, but most likely resulted from higher concentrations of p71 itself. It was decided to determine whether a highly efficient nodavirus replication system developed in yeast for heterologous production of proteins, could be used as a method for expressing the capsid protein precursor. The aim of using this system was to determine if VLPs assembled in a replication system specifically encapsidated viral RNA. Transcripts encoding the NwV capsid protein precursor (p70) were generated in yeast cells by replication of a hybrid RNA template by the Nodamura virus (NoV) replicase. Western analysis confirmed the presence of p70 as well as a protein of 62 kDa corresponding to the mature NwV capsid protein. Northern analysis of purified VLPs showed that NoV RNA1 and RNA3 were encapsidated, but no RNA2 was detected. Taken together, the data lead to the conclusion that specific encapsidation of tetraviral RNAs required more than close proximity of the viral RNAs and assembling virus-like particles. Encapsidation specificity in the omegatetraviruses may require additional viral proteins such as p17 during encapsidation or specific viral RNA encapsidation was replication-dependent. Replication-dependent assembly has been shown in the nodaviruses.

Field corn, Zea mays L., has been introduced to the market with pyramided Bacillus thuringiensis (Bt) corn technologies. These technologies reduce kernel damage from ear feeding caterpillar pests, including corn earworm, Helicoverpa zea (Boddie). The first generation Bt traits in field corn demonstrated limited activity on corn earworm feeding on grain in ears. The pyramided corn technologies have greater cumulative protein concentrations and improved expression throughout the plant, so these corn traits should provide an effective management tool against these pests. In addition, reduced kernel injury may also have a direct effect on physical grain quality. The results for this trial showed no relationship between number of damaged kernels and yield was observed for corn hybrid expressing the Herculex®, YieldGard®, or Genuity VT Triple Pro® technologies. A significant relationship between both damaged kernels and test weight and damaged kernels and aflatoxin concentrations was observed in two technologies.

Transgenic corn hybrids expressing Bt toxins are widely deployed to control pests such as Helicoverpa zea (Boddie). However, Bt resistance can reduce the efficacy of Bt corn hybrids. Volunteer corn expressing Bt toxins may impact Bt resistance management of pests. Surveys in Mississippi revealed the occurrence of fall season volunteer corn in high densities. Helicoverpa zea larvae were found feeding during both vegetative and reproductive stages. However, Bt parentage and low to moderate water and nitrogen stresses did not significantly influence H. zea larval growth and development on Bt positive volunteer plants. Additionally, pollen mediated Bt gene flow to non-Bt refuge ears from Bt hybrids significantly reduced H. zea larval growth on cross-pollinated refuge ears. The implications of these findings are that volunteer corn in most Mississippi corn production regions will have little impact on H. zea Bt resistance management, but resistance management could be compromised in more southern regions.

Uridine diphosphate glycosyltransferase (UGT) is a multigene family of enzymes responsible for catalyzing glycosylation of small hydrophobic molecules. Recently, a genomic analysis of the corn earworm (Helicoverpa zea) identified 45 different UGT genes. We discovered a UGT gene (UGT34) showing high levels of expression exclusively in the silk gland tissue. The expression levels of UGT34 were analyzed in different developmental stages and silk gland sub-segments, revealing that UGT34 is generally expressed at all larval instar stages and largely expressed in the middle and posterior subsegments of the silk glands. The soybean looper (Chrysodeixis includens), another noctuid moth species, was analyzed and found to have similar gene expression patterns. To determine UGT34 function RNA interference (RNAi) was used, but it revealed to be unsuccessful. Taken together, the present study implies that UGT34 plays an important role in silk glands, yet its molecular and physiological function needs to be determined by further study.

HearNPV was applied to a soybean field and evaluated for residual efficacy at different time intervals (0, 12, 24, 36, 48, and 72 hours) after application in laboratory bioassays with treated leaves. Larval mortality was rated at five, seven and twelve days after larval infestation. Larval mortality increased as the number of days after infestation increased. To evaluate the feeding habits of Helicoverpa zea at the 2nd and 3rd instar, HearNPV exposed larvae and untreated larvae were weighed at zero (before diet exposure), four, and seven days. At four days after exposure, larval weight gain of specimens exposed to HearNPV was reduced by 80% and 45% for 2nd and 3rd instar larvae, respectively. At seven days after exposure, larval weight gain of 3rd instar specimens exposed to HearNPV was reduced by 60%.

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The environmental impacts of genetically modified (GM) crop plants such as Bt (Bacillus thuringiensis) maize have not yet been fully assessed in South Africa. Bt maize designed to express Bt endotoxin for control of Busseola fusca (Fuller) (Lepidoptera: Noctuidae) and Chilo partellus (Swinhoe) (Lepidoptera: Crambidae) is planted on approximately 1.103 million hectares in South Africa. The monitoring of GM crops after release is important in order to assess and evaluate possible environmental effects. No risk assessment for Bt maize was done in South Africa before its release in 1998 and no targeted post-release monitoring of possible resistance development or impact on non-target species have been done. Awareness has risen in South Africa through research highlighting the possible effects GM crops may have. The aim of this study was to determine, through feeding experiments, the effects of Bt maize on selected non-target Lepidoptera, Coleoptera and Diptera species that occur in maize agro-ecosystems in South Africa. Results provide information for use in future risk assessment studies on Bt maize and indicate which species could possibly be of importance in post-release monitoring of Bt maize. Priority insect species were identified and laboratory- and semifield experiments were conducted to evaluate the effect of Bt maize on these species. In the light of the reportedly lower toxicity of Bt maize to certain noctuid borers, the effect of Bt maize was evaluated on Sesamia calamistis (Hampson), Agrotis segetum (Denis & Schiffermüller), and Helicoverpa armigera (Hubner). Feeding studies were also conducted to determine the effect of Bt maize on non-target Coleoptera, i.e. Heteronychus arator Fabricius (Coleoptera: Scarabaeidae) and Somaticus angulatus (Fahraeus) (Coleoptera: Tenebrionidae). The effect of indirect exposure of the stem borer parasitoid Sturmiopsis parasitica (Curran) (Diptera: Tachinidae) to Bt toxin was evaluated to determine if there is any effect when it parasitizes Bt-resistant B. fusca larvae that have fed on Bt maize. Results from the study conducted with S. calamistis indicated that Bt maize of both events (Bt11 and MON810) were highly toxic to S. calamistis. The behavioural characteristic of S. calamistis to feed behind leaf sheaths and to enter stems directly did not result in escape of exposure to the toxin. Larval feeding on leaf sheaths therefore resulted in the ingestion of sufficient toxin to kill larvae before they entered maize stems. Results showed that the effect of Cry1Ab toxin on the biology of A. segetum larvae and moths were largely insignificant. Whorl leaves were observed to be an unsuitable food source for H. armigera larvae and larval growth was poor. No larvae survived to the pupal stage on any of the Bt maize treatments. When feeding on maize ears H. armigera larval mass increased on non-Bt maize whereas no increase occurred on Bt maize. The feeding study conducted with Coleoptera showed that the effect of Bt maize on H. arator and S. angulatus was insignificant and no differences were observed in any of the parameters measured for the two species. Although not always significant, the percentage parasitism of Bt-consuming host larvae by S. parasitica was always higher compared to host larvae that fed on non-Bt maize. It could be that Bt toxin affects B. fusca fitness to such an extent that the immune systems of host larvae were less effective. The different parameters tested for S. parasitica indicated only one case where fly maggots originating from diapause host larvae feeding on non-Bt maize had a greater mass compared to host larvae that fed on Bt maize. The same applied to S. parasitica pupal length. For other parameters tested there were no significant differences. Sesamia calamistis is stenophagous and occurs in mixed populations with other borer species. It was therefore concluded that the ecological impact of local extinctions of S. calamistis caused by Bt maize is not expected to be great. Bt maize will most likely not have any significant effect on the control of A. segetum under field conditions. The feeding study conducted with H. armigera quantified the effects of Bt maize on this species and provided important information on the potential of Bt maize as protection against this polyphagous pest. However, the likelihood of H. armigera becoming an important secondary pest is high. It can be concluded that the Cry1Ab toxin targeting lepidopteran pests will not have adverse effects on H. arator or S. angulatus. Although some adverse effects were observed on S. parasitica mass and pupal length it is most likely that this will not contribute to adverse effects in the field, but that there rather be synergism between Bt maize and S. parasitica. An ecological approach was followed in which the potential effects of exposure of priority species to Bt toxin in maize was investigated. A series of selection matrixes were developed in which each of the above mentioned species was ranked for its maximum potential exposure to Bt toxin by assessing it occurrence, abundance, presence and linkage in the maize ecosystem. Through the use of these selection matrixes, knowledge gaps were identified for future research and to guide the design of ecologically realistic experiments. This study contributes to knowledge regarding the possible effects of Bt maize on the most economically important non-target pests in South Africa. There is, however, a need to evaluate other non-target species in feeding studies, as well as in field studies. From this study it can be concluded that some species can be eliminated from further testing since Bt maize had no adverse effect while more research have to be conducted on other species. / Thesis (Ph.D. (Environmental Science)--North-West University, Potchefstroom Campus, 2010.