Water Deprivation Induces Biochemical Changes Without Reduction in the Insecticidal Activity of Maize and Soybean Transgenic Plants.

Like conventional crops, transgenic plants expressing insecticidal toxins from Bacillus thuringiensis (Bt) are subjected to water deprivation. However, the effects of water deprivation over the insecticidal activity of Bt plants are not well understood. We submitted Bt maize and Bt soybean to water deprivation and evaluated biochemical stress markers and the insecticidal activity of plants against target insects. Bt maize (DAS-Ø15Ø7-1 × MON-89Ø34-3 × MON-ØØ6Ø3-6 × SYN-IR162-4) containing the PowerCore Ultra traits, Bt soybean (DAS-444Ø6-6 × DAS-81419-2) with the Conkesta E3 traits, and commercial non-Bt cultivars were cultivated and exposed to water deprivation in the greenhouse. Leaves were harvested for quantification of hydrogen peroxide, malondialdeyde (MDA), and total phenolics and insecticidal activity. Maize or soybean leaf disks were used to evaluate the insecticidal activity against, respectively, Spodoptera frugiperda (J.E Smith) and Chrysodeixis includens (Walker) neonates. Except for Bt soybean, water deprivation increased hydrogen peroxide and MDA contents in Bt and non-Bt plants. Both biochemical markers of water deficit were observed in lower concentrations in Bt plants than in non-Bt commercial cultivars. Water deprivation did not result in changes of phenolic contents in Bt and non-Bt maize. For Bt or non-Bt soybean, phenolic contents were similar despite plants being exposed or not to water deprivation. Water deprivation did not alter substantially insect survival in non-Bt maize or non-Bt soybean. Despite water deprivation-induced biochemical changes in plants, both Bt plants maintained their insecticidal activity (100% mortality) against the target species.

Ecological Modelling of Insect Movement in Cropping Systems.

The spatio-temporal dynamics of insect pests in agricultural landscapes involves the potential of species to move, invade, colonise, and establish in different areas. This study revised the dispersal of the important crop pests Diabrotica speciosa Germar and Spodoptera frugiperda (J.E. Smith) by using computational modelling to represent the movement of these polyphagous pests in agricultural mosaics. The findings raise significant questions regarding the dispersal of pests through crops and refuge areas, indicating that understanding pest movement is essential for developing strategies to predict critical infestation levels to assist in pest-management decisions. In addition, our modelling approach can be adapted for other insect species and other cropping systems despite discussing two specific species in the current manuscript. We present an overview of studies, combining experimentation and ecological modelling, discussing the methods used and the importance of studying insect movement as well as the implications for agricultural landscapes in Brazil.

Assessing the effects of an acute exposure to worst-case concentration of Cry proteins on zebrafish using the embryotoxicity test and proteomics analysis.

Cry1C, Cry1F and Cry1Ab are insecticidal proteins from Bacillus thuringiensis (Bt) which are expressed in transgenic crops. Given the entry of these proteins into aquatic environments, it is relevant to evaluate their impacts on aquatic organisms. In this work, we sought to evaluate the effects of Cry1C, Cry1F and Cry1Ab on zebrafish embryos and larvae of a predicted worst-case scenario concentration of these proteins (set to 1.1 mg/L). For that, we coupled a traditional toxicity approach (the zebrafish embryotoxicity test and dosage of enzymatic biomarkers) to gel free proteomics analysis. At the concentration tested, these proteins did not cause adverse effects in the zebrafish early life stages, either by verifying phenotypic endpoints of toxicity or alterations in representative enzymatic biomarkers (catalase, glutathione-S-tranferase and lactate-dehydrogenase). At the molecular level, the Cry proteins tested lead to very small changes in the proteome of zebrafish larvae. In a global way, these proteins upregulated the expression of vitellogenins. Besides that, Cry1C e Cry1F deregulated heterogeneous nuclear ribonucleoproteins (Hnrnpa0l and Hnrnpaba, respectively), implicated in mRNA processing and gene regulation. Overall, these data lead to the conclusion that Cry1C, Cry1F and Cry1Ab proteins, even at a very high concentration, have limited effects in the early stages of zebrafish life.

Food safety knowledge on the Bt mutant protein Cry8Ka5 employed in the development of coleopteran-resistant transgenic cotton plants.

Insecticidal Cry proteins from Bacillus thuringiensis (Bt) have been exploited in the development of genetically modified (GM) crops for pest control. However, several pests are still difficult to control such as the coleopteran boll weevil Anthonomus grandis. By applying in vitro molecular evolution to the cry8Ka1 gene sequence, variants were generated with improved activity against A. grandis. Among them, Cry8Ka5 mutant protein showed coleoptericidal activity 3-fold higher (LC50 2.83 µg/mL) than that of the original protein (Cry8Ka1). Cry8Ka5 has been used in breeding programs in order to obtain coleopteran-resistant cotton plants. Nevertheless, there is some concern in relation to the food safety of transgenic crops, especially to the heterologously expressed proteins. In this context, our research group has performed risk assessment studies on Cry8Ka5, using the tests recommended by Codex as well as tests that we proposed as alternative and/or complementary approaches. Our results on the risk analysis of Cry8Ka5 taken together with those of other Cry proteins, point out that there is a high degree of certainty on their food safety. It is reasonable to emphasize that most safety studies on Cry proteins have essentially used the Codex approach. However, other methodologies would potentially provide additional information such as studies on the effects of Cry proteins and derived peptides on the indigenous gastrointestinal microbiota and on intestinal epithelial cells of humans. Additionally, emerging technologies such as toxicogenomics potentially will offer sensitive alternatives for some current approaches or methods.

Probabilistic tools for assessment of pest resistance risk associated to insecticidal transgenic crops

One of the main risks associated to transgenic crops expressing Bacillus thuringiensis (Bt) toxins is the evolution of pest resistance. The adoption of Bt crops requires environmental risk assessment that includes resistance risk estimation, useful for definition of resistance management strategies aiming to delay resistance evolution. In this context, resistance risk is defined as the probability of the Bt toxin resistance allele frequency (RFreq) exceeding a critical value (CriticalFreq). Mathematical simulation models have been used to estimate (RFreq) over pest generations. In 1998, Caprio developed a deterministic simulation model with few parameters that can be used to obtain RFreq point estimates from point information about model parameters and decision variables involved in that process. In this work, the resistance risk was estimated using Caprio´s model, by incorporating uncertainty to the resistance allele initial frequency (InitialFreq). The main objective was to evaluate the influence of different probability distribution functions on the risk estimates. The simulation results showed that the influence of InitialFreq input distributions on the risk estimates changes along pest generations. The risk estimates considering input Normal distribution for InitialFreq are similar to those ones obtained considering Triangular distribution if their variances are equal. The use of Uniform distribution instead the Normal or Triangular due to the lack of information about InitialFreq leads to an overestimation of risk estimates for the initial generations and sub estimation for the generations after the one for which the critical frequency is achieved.

Um dos principais riscos associados às culturas inseticidas que expressam toxinas da bactéria Bacillus thuringiensis (Bt) é a evolução de resistência em pragas alvo. A adoção das culturas Bt requer avaliações prévias de impacto ambiental que incluem quantificação desse risco, informação útil para definição de estratégias de manejo para retardar o processo de evolução da resistência. O risco de resistência é definido como a probabilidade de a freqüência do alelo de resistência à toxina Bt (RFreq) na população da praga alvo ser superior a um valor crítico (CriticalFreq). Modelos matemáticos de simulação têm sido utilizados para estimar RFreq ao longo das gerações da praga. Em 1998, Caprio desenvolveu um modelo determinístico, com poucos parâmetros, que produz estimativas pontuais de RFreq a partir de informações também pontuais sobre os parâmetros e variáveis de decisão envolvidos no processo. Neste trabalho, o risco de resistência foi estimado utilizando o modelo de Caprio, incorporando-se incerteza ao parâmetro freqüência inicial do alelo de resistência (InitialFreq). Avaliou-se o efeito de diferentes distribuições de InitialFreq sobre as estimativas de risco. Observou-se que essas estimativas são afetadas pela distribuição de InitialFreq de modo diferenciado ao longo das gerações. As estimativas obtidas considerando a distribuição Normal são similares àquelas considerando a distribuição Triangular quando as referidas distribuições têm a mesma variância. O uso da distribuição Uniforme, em vez da Normal ou Triangular, leva à superestimação das estimativas de risco de resistência nas gerações iniciais e subestimação nas gerações subseqüentes àquela em que a CriticalFreq é atingida.

Probabilistic tools for assessment of pest resistance risk associated to insecticidal transgenic crops

One of the main risks associated to transgenic crops expressing Bacillus thuringiensis (Bt) toxins is the evolution of pest resistance. The adoption of Bt crops requires environmental risk assessment that includes resistance risk estimation, useful for definition of resistance management strategies aiming to delay resistance evolution. In this context, resistance risk is defined as the probability of the Bt toxin resistance allele frequency (RFreq) exceeding a critical value (CriticalFreq). Mathematical simulation models have been used to estimate (RFreq) over pest generations. In 1998, Caprio developed a deterministic simulation model with few parameters that can be used to obtain RFreq point estimates from point information about model parameters and decision variables involved in that process. In this work, the resistance risk was estimated using Caprio´s model, by incorporating uncertainty to the resistance allele initial frequency (InitialFreq). The main objective was to evaluate the influence of different probability distribution functions on the risk estimates. The simulation results showed that the influence of InitialFreq input distributions on the risk estimates changes along pest generations. The risk estimates considering input Normal distribution for InitialFreq are similar to those ones obtained considering Triangular distribution if their variances are equal. The use of Uniform distribution instead the Normal or Triangular due to the lack of information about InitialFreq leads to an overestimation of risk estimates for the initial generations and sub estimation for the generations after the one for which the critical frequency is achieved.

Um dos principais riscos associados às culturas inseticidas que expressam toxinas da bactéria Bacillus thuringiensis (Bt) é a evolução de resistência em pragas alvo. A adoção das culturas Bt requer avaliações prévias de impacto ambiental que incluem quantificação desse risco, informação útil para definição de estratégias de manejo para retardar o processo de evolução da resistência. O risco de resistência é definido como a probabilidade de a freqüência do alelo de resistência à toxina Bt (RFreq) na população da praga alvo ser superior a um valor crítico (CriticalFreq). Modelos matemáticos de simulação têm sido utilizados para estimar RFreq ao longo das gerações da praga. Em 1998, Caprio desenvolveu um modelo determinístico, com poucos parâmetros, que produz estimativas pontuais de RFreq a partir de informações também pontuais sobre os parâmetros e variáveis de decisão envolvidos no processo. Neste trabalho, o risco de resistência foi estimado utilizando o modelo de Caprio, incorporando-se incerteza ao parâmetro freqüência inicial do alelo de resistência (InitialFreq). Avaliou-se o efeito de diferentes distribuições de InitialFreq sobre as estimativas de risco. Observou-se que essas estimativas são afetadas pela distribuição de InitialFreq de modo diferenciado ao longo das gerações. As estimativas obtidas considerando a distribuição Normal são similares àquelas considerando a distribuição Triangular quando as referidas distribuições têm a mesma variância. O uso da distribuição Uniforme, em vez da Normal ou Triangular, leva à superestimação das estimativas de risco de resistência nas gerações iniciais e subestimação nas gerações subseqüentes àquela em que a CriticalFreq é atingida.