A estratégia de pirâmide de genes tem sido explorada para retardar a evolução da resistência de insetos a plantas geneticamente modificadas que expressam proteínas inseticidas de Bacillus thuringiensis Berliner (Bt). No Brasil, às tecnologias de milho YieldGard VT PRO™ (VT PRO) e PowerCore™ (PW) que expressam as proteínas Cry1A.105/Cry2Ab2 e Cry1A.105/Cry2Ab2/Cry1F, respectivamente, foram liberadas para uso comercial em 2009. Para subsidiar programas de Manejo da Resistência de Insetos (MRI) foram conduzidos trabalhos para avaliar o risco de evolução da resistência de Spodoptera frugiperda (J. E. Smith) ao milho VT PRO e PW. Inicialmente foram realizados estudos para avaliar a atividade biológica de proteínas Bt expressas em diferentes estruturas da planta de milho VT PRO e PW sobre S. frugiperda e monitorar a suscetibilidade a Cry1A.105 e Cry2Ab2 em populações da praga coletadas em diferentes regiões geográficas do Brasil durante as safras de 2011 a 2014. Houve 100% de mortalidade de neonatas de S. frugiperda quando expostas ao tecido foliar de milho VT PRO e PW. No entanto, em estilo-estigmas e grãos, a mortalidade foi inferior a 50 e 6% respectivamente. Variabilidade geográfica na suscetibilidade de populações S. frugiperda a Cry1A.105 e Cry2Ab2 foi detectada, com reduções significativas na suscetibilidade a essas proteínas para algumas populações de 2011 a 2014. A técnica de \"F2 screen\" foi utilizada para a caracterização da resistência de S. frugiperda ao milho VT PRO e PW a partir de populações coletadas na safra de 2012. Verificou-se uma alta variabilidade na frequência fenotípica de isofamílias resistentes ao milho VT PRO e PW, sendo que as maiores frequências foram observadas em populações coletadas na região Central do Brasil. Com a técnica de \"F2 screen\" foi possível selecionar linhagens resistentes ao milho VT PRO e PW, denominadas de RR-2 e RR-3 respectivamente. Tanto a linhagem RR-2 quanto a RR-3 que foram criadas por 18 gerações consecutivas nos respectivos eventos de milho Bt apresentaram razões de resistência superiores a 3300, 2700 e ≈ 10 vezes a Cry1A.105, Cry1F e Cry2Ab2, respectivamente. Cruzamentos recíprocos das linhagens RR-2 e RR-3 com uma linhagem suscetível de referência revelaram que o padrão da herança da resistência é autossômica recessiva. A recessividade genética da resistência também foi confirmada pela mortalidade completa de indivíduos heterozigotos (descendentes provenientes de cruzamentos entre as linhagens RR-2 ou RR-3 com a linhagem suscetível) em tecidos de milho VT PRO e PW, demonstrando que esses eventos atendem ao conceito de alta dose para o MRI. Em retrocruzamentos da progênie F1 dos cruzamentos recíprocos com as linhagens resistentes confirmou-se a hipótese de que a resistência é poligênica. A presença de custo adaptativo associado à resistência foi verificada para as linhagens RR-2 e RR-3, porém ausente para os indivíduos heterozigotos, baseado nos parâmetros biológicos avaliados. Neste estudo fornecemos a primeira evidência do potencial de evolução da resistência de S. frugiperda a eventos de milho Bt piramidados e informações para o refinamento das estratégias de MRI para preservar a vida útil das tecnologias de milho Bt para o controle de S. frugiperda no Brasil. / The strategy of pyramid of genes has been exploited to delay the evolution of insect resistance to genetically modified crops expressing insecticidal proteins from from Bacillus thuringiensis Berliner (Bt). In Brazil, YieldGard VT Pro™ (VT PRO) and PowerCore™ (PW) corn technologies expressing Cry1A.105/Cry2Ab2 and Cry1A.105/Cry2Ab2/Cry1F proteins respectively were released for commercial use in 2009. Resistance risk assessment were conducted to support an Insect Resistance Management (IRM) program of Spodoptera frugiperda (J. E. Smith) (Lepidoptera: Noctuidae) to VT PRO and PW corn. Initially, studies were conducted to evaluate the biological activity of Bt proteins expressed in different plant structures of VT PRO and PW corn on S. frugiperda and to monitor the susceptibility to Cry1A.105 and Cry2Ab2 in pest populations collected from different geographical regions in Brazil from 2011 to 2014 growing seasons. The mortality of neonate larvae of S. frugiperda was 100% when fed on leaf tissue of VT PRO and PW corn. However, the larval mortality when fed on silks and grains was less than 50 and 6% respectively. A geographical variation in the susceptibility of S. frugiperda to Cry1A.105 and Cry2Ab2 proteins was detected among populations, with significant reduction in susceptibility to these proteins in some populations from 2011 to 2014. The F2 screen technique was used to characterize the resistance of S. frugiperda to VT PRO and PW corn from populations sampled in 2012 growing season. High variability in the frequency of resistant phenotypic isofamilies to VT PRO and PW corn was obtained with higher frequencies in S. frugiperda populations from Midwestern region of Brazil. Resistant populations to VT PRO and PW corn were selected by using F2 screen which were designated as RR-2 and RR-3 strains respectively. Both RR-2 and RR-3 strains reared on respective Bt maize events for 18 consecutive generations showed resistance ratios greater than 3,300; 2,700 and ≈ 10-fold to Cry1A.105, Cry1F and Cry2Ab2 respectively. Reciprocal crosses of RR-2 and RR-3 strains with a susceptible reference strain revealed that the inheritance of resistance is autosomal recessive. The genetic recessiveness of the resistance was also confirmed by the complete mortality of heterozygous individuals (offspring from the crosses between RR-2 or RR-3 strains with susceptible strain) on VT PRO and PW corn leaf tissues, indicating that these events meet the concept of high-dose for IRM strategies. Backcrosses of F1 progenies with both resistant strains revealed that resistance is polygenic. Fitness costs associated with resistance were found in RR-2 e RR-3 strains but not in heterozygous individuals, based on life history traits. In this study, we reported the first evidence of the potential of S. frugiperda to evolve resistance to pyramided Bt corn events, as well as provide valuable information to support the current IRM strategies to preserve the useful life of Bt corn technologies for S. frugiperda control in Brazil.

Plantas transgênicas que expressam toxinas de Bacillus thuringiensis Berliner (Bt) têm sido amplamente utilizadas para o controle de Spodoptera frugiperda (J. E. Smith) no Brasil. Entretanto, a evolução da resistência é um dos maiores entraves para a continuidade do uso desta tecnologia. Para subsidiar programas de Manejo da Resistência de Insetos (MRI), foram conduzidos estudos para o aprimoramento dos programas de manejo da resistência de S. frugiperda a tecnologias Bt. Foram realizadas estudos para determinar a dominância funcional da resistência de S. frugiperda a tecnologias Bt mediante a avaliação da sobrevivência de larvas neonatas provenientes das linhagens de S. frugiperda resistentes ao milho Herculex® que expressa a proteína Cry1F (HX-R), ao milho YieldGard VT PRO™ que expressa as proteínas Cry1A.105 e Cry2Ab2 (VT-R), ao milho PowerCore™ que expressa as proteínas Cry1A.105, Cry2Ab2 e Cry1F (PW-R), e ao milho Agrisure Viptera™ que expressa a proteína Vip3Aa20 (Vip-R), além da linhagem suscetível (Sus) e de suas respectivas linhagens heterozigotas em diversas tecnologias de milho e algodão Bt. Posteriormente, um método prático para o monitoramento fenotípico da suscetibilidade a diferentes tecnologias de milho e algodão Bt foi testado a partir da avaliação da sobrevivência de larvas neonatas em folhas de plantas Bt em populações de S. frugiperda provenientes dos Estados do Rio Grande do Sul, Paraná, São Paulo, Goiás e Bahia na safra agrícola 2014/15. E por último, a estimativa da frequência de alelos de resistência de S. frugiperda a Vip3Aa20 foi validada pelo método de F1 screen. Em geral, observou-se alta mortalidade dos heterozigotos nas tecnologias Bt testadas, comprovando que a resistência de S. frugiperda a proteínas Bt é funcionalmente recessiva o que suporta a estratégia de refúgio em programas de MRI. Verificou-se também que linhagens resistentes a eventos que expressam proteínas Cry não sobrevivem em tecnologias que expressam proteína Vip. No monitoramento prático da suscetibilidade a tecnologias Bt, sobrevivência larval superior a 70% foi observada para populações de campo do Paraná, Goiás e Bahia no milho Herculex®. Em tecnologias de milho PowerCore™ e YieldGard VT PRO™ houve sobrevivência larval variando de 1,1 a 17,9%. Em contraste, não houve sobreviventes em tecnologias de milho Viptera™. Em algodão WideStrike® que expressa as proteínas Cry1Ac e Cry1F, sobrevivência acima de 41% foi observada para populações de campo de S. frugiperda. A sobrevivência larval em Bollgard II® que expressa as proteínas Cry1Ac e Cry2Ab2 variou de 14 a 40%. No algodão TwinLink® que expressa as proteínas Cry1Ab e Cry2Ae, a sobrevivência larval das populações foi menor que 20%. O método de F1 screen foi eficiente na detecção de alelos de resistência a Vip3Aa20 em populações de S. frugiperda provenientes de diferentes regiões produtoras de milho no Brasil na safra 2014/2015. De 263 isofamílias testadas, foram detectadas três isofamílias positivas oriundas do Paraná, Mato Grosso e Goiás. A frequência de resistência estimada a Vip3Aa20 variou de 0,0140 a 0,0367 nas populações avaliadas, sendo que a frequência total foi de 0,0076. Neste estudo, fornecemos informações para refinar as estratégias de MRI, além de introduzir novas técnicas para monitorar a resistência de S. frugiperda a tecnologias Bt no Brasil. / Transgenic plants expressing toxins from Bacillus thuringiensis Berliner (Bt) have been widely used to the control of Spodoptera frugiperda (J. E. Smith) in Brazil. However, the resistance evolution is one of the major threats to the continuous use of this technology. To subsidize Insect Resistance Management (IRM), studies were conducted to improve S. frugiperda resistance management programs to Bt technologies. Studies to determine functional dominance of resistance of S. frugiperda to Bt technologies were conducted by evaluating neonate larval survival of S. frugiperda strains resistant to Herculex® maize expressing Cry1F protein (HX-R), to YieldGard VT PRO™ maize expressing Cry1A.105 and Cry2Ab2 proteins (VT-R), to PowerCore™ maize expressing Cry1A.105, Cry2Ab2 and Cry1F proteins (PW-R) and to Agrisure Viptera™ maize expressing Vip3Aa20 protein (Vip- R), in addition to susceptible strain (Sus) and the respective heterozygous strains in several Bt maize and cotton technologies cultivated in Brazil. Then, a practical method for phenotypic resistance monitoring of several Bt maize and cotton were tested, based on neonate larval survival on Bt leaf tissue in S. frugiperda populations collected from Rio Grande do Sul, Paraná, São Paulo, Goiás and Bahia States. Finally, the F1 screen method was validated to estimate the frequency of Vip3Aa20 resistance alleles in S. frugiperda. In general, high mortality of heterozygous individuals was observed on Bt technologies, confirming that resistance of S. frugiperda to Bt proteins is functionally recessive and supporting the importance of refuge areas in IRM programs. No larval survival on Vip expressing maize was found with strains of S. frugiperda resistant to maize expressing Cry toxins. In the practical resistance monitoring, more than 70% of larval survival in field populations of S. frugiperda from Paraná, Goiás and Bahia was detected in Herculex® maize. Larval survival on PowerCore™ and YieldGard VT PRO™ maize technologies ranged from 1.1 to 17.9%. In contrast, no larval survival of field populations was observed on Viptera™ maize technologies. On WideStrike® cotton, more than 41% larval survival was observed in field populations of S. frugiperda. The larval survival was on Bollgard II® ranged from 14 to 40%. In TwinLink® the larval survival was lower than 20%. The F1 screen method was efficient in detecting Vip3Aa20 resistance alleles in field populations of S. frugiperda. From a total of 263 isofamily lines tested, three positive isofamily lines from Paraná, Mato Grosso and Goiás were found. The frequency of Vip3Aa20 resistance alleles ranged from 0.0140 to 0.0367, with overall frequency of 0.0076. In this study, we provide valuable information to improve IRM strategies and propose new methods to monitor resistance of S. frugiperda to Bt technologies in Brazil.

A broca-da-cana Diatraeasaccharalis(Fabricius) é uma das pragas-alvo do milho geneticamente modificado que expressagene(s) que codifica(m) proteína(s) de BacillusthuringiensisBerliner(milho Bt) no Brasil. Para estabelecer estratégias proativas de manejo de resistência, os objetivos do presente trabalho foram (1)estabelecer linhas-básicas de suscetibilidade à proteína Cry1F de B. thuringiensisem populações de D. saccharalis coletadas nas principais regiões produtoras de milho no Brasil na safra de 2010/2011, (2) realizar monitoramentoda suscetibilidade de populações de D. saccharalis na safra 2011/2012 e (3) avaliar a atividade biológica do milho Bt (evento TC1507) em diferentes estádios fenológicos no controle de D. saccharalis em condições laboratoriais. Para a caracterização das linhas-básicas de suscetibilidade foram realizados bioensaios com a proteína purificada Cry1F aplicada superficialmente em dieta artificial.As CL50 de Cry1F estimadas variaram de 0,47 a 7,02 ng de Cry1F/ cm2 dieta artificial para as populações avaliadas (variação de ?15 vezes). Foi definida e validada a concentração diagnóstica de 112 ng de Cry1F/cm²,baseada na estimativa da CL99 das populações testadas para o monitoramento da suscetibilidade. A avaliação da atividade biológica de milho Bt (evento TC1507) foi realizada com dois híbridos (Pioneer 30F35 e Dow 2B688) nos estádios fenológicos V3, V6 e V9 e em duas épocas de plantio. Alta atividade de controle de D. saccharalis foi obtida para os dois híbridos de milho Bt nos diferentes estádios fenológicos e épocas de plantio. Portanto, verificou-se que a espécie D. saccharalis é altamente suscetível à proteína Cry1F. Contudo, a implementação de estratégias de manejo da resistência é de fundamental importância para preservar a vida útil da tecnologia de milho Bt como importante ferramenta em programas de MIP. / The sugarcane borer Diatraeasaccharalis (Fabricius) is one of the target pests of genetically modified maizeexpressinggenes that code for insecticidal proteins from Bacillus thuringiensis Berliner (Bt maize) in Brazil. To implement proactive resistance management strategies, we leaded studies (1) to establish baseline susceptibility to Cry1F protein from B. thuringiensis in populations of D. saccharalis collected from major maize production regions in Brazil during 2010/2011 growing season, (2) to leadsusceptibility monitoring in populations of D. saccharalis collected in 2011/2012 growing season, and (3) to assess the biological activity of Bt maize (event TC1507) at different growth stages forD. saccharaliscontrol under laboratory conditions. The baseline susceptibility wascharacterizedwith purified protein Cry1F applied superficially on artificial diet. The estimatesof LC50 of Cry1F ranged from 0.47 to 7.02 ng Cry1F/cm2of artificial diet for the populations evaluated (? 15-fold variation).A diagnostic concentration of 112 ng Cry1F/cm ² was defined and validated for monitoring the susceptibility based on estimation of CL99 of all tested populations. The biological activity of Bt maize (event TC1507) was assessed by using two hybrids (Pioneer 30F35 and Dow 2B688) at growth stages V3, V6 and V9 and two planting seasons. High level of D. saccharaliscontrol was obtained for the twoBt maize hybrids at different growth stages and planting seasons. Therefore, we conclude that D. saccharalis is highly susceptible to Cry1F protein. However, implementation of insect resistance management strategies is essentialin order to preserve the lifetime of Bttechnology as an important tool in IPM programs.

In den letzten 20 Jahren hat sich der Maiszünsler (<i>Ostrinia nubilalis HÜBNER</i>), aus der Schmetterlingsfamilie der Pyralidae oder Zünsler, zum bedeutendsten tierischen Schädling des Maises (<i>Zea mays</i>) entwickelt. Eine Möglichkeit den Befall des Maiszünslers abzuwenden, bietet der Anbau von <i>Bacillus thuringiensis</i>-Mais (Bt-Mais). Mit Hilfe der Gentechnik wurden Gene des Bakteriums <i>Bacillus thuringiensis</i> übertragen, die einen für Fraßinsekten giftigen Wirkstoff bilden, wodurch die Pflanzen während der kompletten Vegetation vor den Larven des Maiszünslers geschützt sind.<br><br> Ziel des vorliegenden Projektes war es, in einer 3-jährigen Studie die Auswirkungen des großflächigen Anbaus von Bt-Mais auf die ökologische Situation und den Handlungsrahmen des integrierten Pflanzenschutzes komplex zu untersuchen. Dazu wurden in Betrieben im Oderbruch, das als permanentes Befallsgebiet des Maiszünslers gilt, in den Jahren 2002 bis 2004 jährlich zwei Felder mit jeweils einer Bt-Sorte und einer konventionellen Sorte angelegt. Zusätzlich wurden biologische und chemische Maiszünsler-Bekämpfungsvarianten geprüft.<br><br> Durch verschiedene Methoden wie Bonituren, Ganzpflanzenernten, Bodenfallenfänge und Beobachtungen des Wahlverhaltens von (Flug-)insekten konnten Aussagen zum Vorkommen von Insekten und Spinnentieren getroffen werden, wobei hierfür Daten aus Untersuchungen der Jahre 2000 und 2001 im Oderbruch ergänzend herangezogen werden konnten. Durch Ertragsmessungen, Energie- und Qualitätsermittlungen, sowie Fusarium- und Mykotoxinanalysen konnte der Anbau von Bt-Mais als neue Alternative zur Bekämpfung des Maiszünslers bewertet werden.<br><br> Bezüglich des Auftretens von Insekten und Spinnentieren wurden im Mittel der fünfjährigen Datenerhebung beim Vergleich der Bt-Sorte zur konventionellen Sorte, mit Ausnahme der fast 100 %igen Bekämpfung des Maiszünslers, keine signifikanten Unterschiede festgestellt. Hierfür wurde ein besonderes Augenmerk auf Thripse, Wanzen, Blattläuse und deren Fraßfeinde, sowie mittels Bodenfallenfängen auf Laufkäfer und Spinnen gerichtet.<br><br> Die erwarteten ökonomischen Vorteile wie etwa Ertragsplus oder bessere Nährstoff- und Energiegehalte durch geringeren Schaden beim Anbau von Bt-Mais als Silomais blieben in den Untersuchungsjahren aus. Allerdings zeigten Fusarium- und Mykotoxinanalysen eine geringere Belastung des Bt-Maises, was möglicherweise auf den geringeren Schaden zurückzuführen ist, da beschädigte Pflanzen für Fusarium und Mykotoxine anfälliger sind.<br><br> Desweiteren konnten erste methodische Ansätze für ein auf EU-Ebene gefordertes, den Anbau von Bt-Mais begleitendes Monitoring, erarbeitet werden. So konnten Vorschläge für geeignete Methoden, deren Umfang sowie des Zeitpunktes der Durchführungen gemacht werden. / In the last 20 years the European corn borer (<i>Ostrinia nubilalis</i>, <i>Pyralidae</i>) has become the most important pest in maize (<i>Zea mays</i>). One of a couple of possibilities to reduce the infestation by the European corn borer is the cultivation of <i>Bacillus thuringiensis</i> maize (Bt maize). Genetic engineering transmitted genes from <i>Bacillus thuringiensis</i>, which produce a substance that is toxic to feeding insects and thus protect plants against the larvae of the European corn borer during the whole vegetation.<br><br> The present project is a 3-year study to identify the effects of Bt maize growing on the ecological situation and the possibilities of integrated plant protection. From 2002 to 2004, two fields in the Oderbruch region, where Ostrinia nubilalis occurs, were each planted with Bt maize and a conventional maize variety every year. Furthermore, a biological and a chemical strategy against the European corn borer were verified.<br><br> Different methods like counts, harvest of whole plants, pitfall traps and observation the landing behaviour of flying insects were used to determine the abundance of insects and spiders. Furthermore, we could use additional data from studies obtained in the Oderbruch region in 2000 and 2001. The determination of yield, quality and energy content of the crops as well as of the degree of Fusarium infection and contamination by mycotoxins led to the conclusion that the cultivation of Bt maize is a new alternative strategy to control the European corn borer.<br><br> The average occurrence of insects and spiders did not differ significantly between Bt maize and the conventional variety in the 5 years of data recording. The only exception is the almost total control of the European corn borer. Attention was especially paid to thrips, bugs, aphids and their feeding enemies and using ground traps to ground beetles and spiders.<br><br> The expected economic benefits like increased yield or nutrient and energy content of the crop as a result of a minimized damage to silage Bt maize were not achieved in the years under investigation. However, the analysis of Fusarium and mycotoxins indicated a lower exposure of Bt maize, which may result from a lower damage caused by Ostrinia nubilalis, and damaged plants are more susceptible to Fusarium and mycotoxins.<br><br> Furthermore, we developed a first methodological approach for the monitoring procedure of Bt maize growing required by the EU. We have made proposals on appropriate methods, their extent as well as the optimum time of their application.

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.

Resistance development and possible non–target effects have been of concern since the first deployment of genetically modified crops with insecticidal properties. It is especially at the third trophic level and with important predators such as lacewings (Chrysoperla spp.) (Neuroptera: Chrysopidae) where negative effects of Cry 1Ab protein could have adverse effects in agro–ecosystems. Monitoring of the effect of genetically modified Bt maize on non–target organisms is required by law in South–Africa. Neuroptera are excellent indicators of environmental and habitat transformation, and also include key species for signifying areas and faunas that require priority protection. Monitoring techniques, especially for insect eggs, are often labour intensive and time consuming. A study was conducted to determine the preferred oviposition site of Chrysoperla spp. on maize plants to facilitate time–effective searching for eggs of these beneficial insects. Furthermore we determined if the presence of aphids on plants influenced Chrysoperla spp. oviposition preference. Another study was conducted to evaluate the effect of indirect exposure of C. pudica to Cry 1Ab protein, through healthy Bt–maize feeding prey, on its biology. Daily flight activity patterns and the height at which chrysopid adults fly above the crop canopy were also determined, as well as the movement of adult Chrysoperla spp. between maize fields and adjacent headlands. A clear spatial oviposition pattern was observed on maize plants and oviposition was not random as reported in earlier studies. This data facilitates rapid monitoring of the presence of eggs in maize cropping systems and is also of use in general pest management. Choicetest data showed that females responded positively to host plants that were infested with aphids. Feeding studies in which C. pudica larvae were indirectly exposed to Bt–toxin at the 3rd trophic level, showed a limited effect of Bt–toxin on only a few of the parameters that were evaluated. The pupal period and percentage adult emergence of larvae exposed to an unusually high amount of Bt–toxin was significantly shorter and lower respectively than that of the control group. The overall result of this study, in which the possible effect of food quality (prey) was excluded, showed that Cry 1Ab protein had an adverse affect only on certain fitness components during the life cycle of C. pudica. However, since this study represented a worst–case scenario where diverse prey was not available to C. pudica, negligible effects is expected under field conditions where prey is more diverse. It was determined that chrysopids was most active between 16:00 – 23:00 and that they fly largely between 0.5 m – 2.5 m above ground level. An attempt was also made to quantify migration between different vegetations types. This part was terminated because of bad weather conditions at several occasions when the experiment was attempted. Chrysopids were never present in grassland vegetation, but an adjacent lucerne field maintained a large population. As the maize crop developed chrysopid population numbers increased inside the field, presumably originating from the lucerne field. / Thesis (M.Sc (Environmental Science))--North-West University, Potchefstroom Campus, 2011.

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.

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.

Resistance development and possible non–target effects have been of concern since the first deployment of genetically modified crops with insecticidal properties. It is especially at the third trophic level and with important predators such as lacewings (Chrysoperla spp.) (Neuroptera: Chrysopidae) where negative effects of Cry 1Ab protein could have adverse effects in agro–ecosystems. Monitoring of the effect of genetically modified Bt maize on non–target organisms is required by law in South–Africa. Neuroptera are excellent indicators of environmental and habitat transformation, and also include key species for signifying areas and faunas that require priority protection. Monitoring techniques, especially for insect eggs, are often labour intensive and time consuming. A study was conducted to determine the preferred oviposition site of Chrysoperla spp. on maize plants to facilitate time–effective searching for eggs of these beneficial insects. Furthermore we determined if the presence of aphids on plants influenced Chrysoperla spp. oviposition preference. Another study was conducted to evaluate the effect of indirect exposure of C. pudica to Cry 1Ab protein, through healthy Bt–maize feeding prey, on its biology. Daily flight activity patterns and the height at which chrysopid adults fly above the crop canopy were also determined, as well as the movement of adult Chrysoperla spp. between maize fields and adjacent headlands. A clear spatial oviposition pattern was observed on maize plants and oviposition was not random as reported in earlier studies. This data facilitates rapid monitoring of the presence of eggs in maize cropping systems and is also of use in general pest management. Choicetest data showed that females responded positively to host plants that were infested with aphids. Feeding studies in which C. pudica larvae were indirectly exposed to Bt–toxin at the 3rd trophic level, showed a limited effect of Bt–toxin on only a few of the parameters that were evaluated. The pupal period and percentage adult emergence of larvae exposed to an unusually high amount of Bt–toxin was significantly shorter and lower respectively than that of the control group. The overall result of this study, in which the possible effect of food quality (prey) was excluded, showed that Cry 1Ab protein had an adverse affect only on certain fitness components during the life cycle of C. pudica. However, since this study represented a worst–case scenario where diverse prey was not available to C. pudica, negligible effects is expected under field conditions where prey is more diverse. It was determined that chrysopids was most active between 16:00 – 23:00 and that they fly largely between 0.5 m – 2.5 m above ground level. An attempt was also made to quantify migration between different vegetations types. This part was terminated because of bad weather conditions at several occasions when the experiment was attempted. Chrysopids were never present in grassland vegetation, but an adjacent lucerne field maintained a large population. As the maize crop developed chrysopid population numbers increased inside the field, presumably originating from the lucerne field. / Thesis (M.Sc (Environmental Science))--North-West University, Potchefstroom Campus, 2011.

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.