Second generation Bt crops (insect resistant crops carrying Bacillus thuringiensis genes) combine more than one gene that codes for insecticidal proteins in the same plant to provide better control of agricultural pests. Some of the new combinations involve co-expression of cry and vip genes. Because Cry and Vip proteins have different midgut targets and possibly different mechanisms of toxicity, it is important to evaluate possible synergistic or antagonistic interactions between these two classes of toxins. Three members of the Cry1 class of proteins and three from the Vip3A class were tested against Heliothis virescens for possible interactions. At the level of LC50, Cry1Ac was the most active protein, whereas the rest of proteins tested were similarly active. However, at the level of LC90, Cry1Aa and Cry1Ca were the least active proteins, and Cry1Ac and Vip3A proteins were not significantly different. Under the experimental conditions used in this study, we found an antagonistic effect of Cry1Ca with the three Vip3A proteins. The interaction between Cry1Ca and Vip3Aa was also tested on two other species of Lepidoptera. Whereas antagonism was observed in Spodoptera frugiperda, synergism was found in Diatraea saccharalis. In all cases, the interaction between Vip3A and Cry1 proteins was more evident at the LC90 level than at the LC50 level. The fact that the same combination of proteins may result in a synergistic or an antagonistic interaction may be an indication that there are different types of interactions within the host, depending on the insect species tested.

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The biological potential of Vip and Cry proteins from Bacillus is well known and widely established. Thus, it is important to look for new genes showing different modes of action, selecting those with differentiated entomotoxic activity against Diatraea flavipennella and Elasmopalpus lignosellus, which are secondary pests of sugarcane. Therefore, Cry1 and Vip3 proteins were expressed in Escherichia coli, and their toxicities were evaluated based on bioassays using neonate larvae. Of those, the most toxic were Cry1Ac and Vip3Aa considering the LC50 values. Toxins from E. coli were purified, solubilized, trypsinized, and biotinylated. Brush Border Membrane Vesicles (BBMVs) were prepared from intestines of the two species to perform homologous and heterologous competition assays. The binding assays demonstrated interactions between Cry1Aa, Cry1Ac, and Vip3Aa toxins and proteins from the BBMV of D. flavipennella and E. lignosellus. Homologous competition assays demonstrated that binding to one of the BBMV proteins was specific for each toxin. Heterologous competition assays indicated that Vip3Aa was unable to compete for Cry1Ac toxin binding. Our results suggest that Cry1Ac and Vip3Aa may have potential in future production of transgenic sugarcane for control of D. flavipennella and E. lignosellus, but more research is needed on the potential antagonism or synergism of the toxins in these pests.

Resumo -O objetivo deste trabalho foi avaliar a suscetibilidade das lagartas Anticarsia gemmatalis (Lepidoptera: Erebidae) e Chrysodeixis includens (Lepidoptera: Noctuidae) às proteínas Cry1 e Vip3A, bem como determinar se há a interação entre essas proteínas no controle das duas espécies. Bioensaios com as proteínas isoladas e em combinações foram realizados, e as concentrações letais CL 50 e CL 90 foram estimadas para cada condição. As proteínas Cry1Aa, Cry1Ac e Vip3Af foram as mais efetivas no controle de A. gemmatalis, enquanto Cry1Ac, Vip3Aa e Vip3Af foram mais efetivas no de C. includens. As proteínas Cry1Ac e Cry1Ca causaram maior inibição do desenvolvimento das larvas sobreviventes à CL 50 , em ambas as espécies. Combinações entre Vip3A e Cry1 apresentam efeito sinérgico no controle das espécies e a combinação Vip3Aa+Cry1Ea destaca-se no controle de A. gemmatalis e C. includens. Essas proteínas combinadas são promissoras na construção de plantas piramidadas, para o controle simultâneo das pragas.Termos para indexação: Anticarsia gemmatalis, Chrysodeixis includens, manejo da resistência, piramidação de genes, sinergismo, soja transgênica. Interaction of Cry1 and Vip3A proteins of Bacillus thuringiensis for the control of lepidopteran insect pestsAbstract -The objective of this work was to evaluate the susceptibility of Anticarsia gemmatalis (Lepidoptera: Erebidae) and Chrysodeixis includens (Lepidoptera: Noctuidae) caterpillars to Cry1 and Vip3A proteins, as well as to determine if there is any interaction between these proteins on the control of the two species. Bioassays with both isolated and combined proteins were carried out, and lethal concentrations LC 50 and LC 90 were estimated for each condition. Cry1Aa, Cry1Ac, and Vip3Af were the more effective proteins for the control of A. gemmatalis, while Cry1Ac, Vip3Aa, and Vip3Af were more effective for the control of C. includens. Cry1Ac and Cry1Ca proteins caused the highest inhibition to the development of larvae that survived the LC 50 dose in both species. Different combinations of Vip3A and Cry1 have synergistic effect in the control of both species, and the combination Vip3Aa + Cry1Ea showed an outstanding control of A. gemmatalis and C. includens. These proteins are promising for building pyramided plants for the simultaneous control of the pests.

Resumo -O objetivo deste trabalho foi avaliar a toxicidade e a capacidade de ligação das proteínas Cry1Aa, Cry1Ab, Cry1Ac e Cry1Ca, de Bacillus thuringiensis, a receptores intestinais de Helicoverpa armigera. Realizou-se análise de ligação das proteínas ativadas às vesículas de membrana da microvilosidade apical (VMMA) do intestino médio de H. armigera, além de ensaios de competição heteróloga para avaliar sua capacidade de ligação. Cry1Ac destacou-se como a proteína mais tóxica, seguida por Cry1Ab e Cry1Aa. A proteína Cry1Ca não foi tóxica às lagartas e, portanto, não foi possível determinar os seus parâmetros de toxicidade CL 50 e CL 90 . As proteínas Cry1Aa, Cry1Ab e Cry1Ac são capazes de se ligar a um mesmo receptor nas membranas intestinais, o que aumenta o risco do desenvolvimento de resistência cruzada. Portanto, a utilização conjunta dessas proteínas deve ser evitada.Termos para indexação: Bacillus thuringiensis, manejo da resistência, manejo integrado de pragas, piramidação de genes, proteínas inseticidas, vesículas de membrana. Toxicity and binding capacity of Cry1 proteins to Helicoverpa armigera (Lepidoptera: Noctuidae) intestine receptorsAbstract -The objective of this work was to evaluate the toxicity and the binding capacity of the Cry1Aa, Cry1Ab, Cry1Ac, and Cry1Ca proteins, from Bacillus thuringiensis, to Helicoverpa armigera intestine receptors. Binding analysis of the activated proteins to the brush-border membrane vesicles (BBMV) in the midgut of H. armigera, besides heterologous competition assays to evaluate their binding capacity, was performed. Cry1Ac stood out as the most toxic protein, followed by Cry1Ab and Cry1Aa. The Cry1Ca protein had no toxicity to the caterpillars and, therefore, it was not possible to evaluate its LC 50 and LC 90 toxicity parameters. The Cry1Aa, Cry1Ab, and Cry1Ac proteins are able to bind themselves to the same receptor in the midgut membrane, which increases the risk of developing cross-resistance. Therefore, the use of these proteins together should be avoided.