Bt Research 2012, Vol.3, No.4, 20-28 http://bt.sophiapublisher.com 20 Research Report Open Access Cry10Aa Protein is Highly Toxic to Anthonomus grandis Boheman (Coleoptera: Curculionidae), an Important Insect Pest in Brazilian Cotton Crop Fields Raimundo Wagner de Souza Aguiar 1 , Érica Soares Martins 2,4 , Bergmann Morais Ribeiro 3 , Rose Gomes Monnerat 4 1. Fundação Universidade Federal do Tocantins, Campus Universitário de Gurupi, Gurupi, TO, Brazil; 2. Faculdades Promove de Brasília, Campus Águas Claras, Brasília, DF; 3. Universidade de Brasília, Departamento de Biologia Celular, Campus Universitário, Asa Norte, Brasília, DF, Brazil; 4. Embrapa Recursos Genéticos e Biotecnologia, Controle biológico, Asa Norte, Brasília 70770-900, Brazil Corresponding author email: rose.monnerat@cenargen.embrapa.br; Authors Bt Research, 2012, Vol.3, No.4 doi: 10.5376/bt.2012.03.0004 Received: 22 May, 2012 Accepted: 19 Jul., 2012 Published: 20 Jul., 2012 This is an open access article published under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Preferred citation for this article: de Souza Aguiar et al., 2012, Cry10Aa Protein is Highly Toxic to Anthonomus grandis Boheman (Coleoptera: Curculionidae), an Important Insect Pest in Brazilian Cotton Crop Fields, Bt Research, Vol.3, No.4 20-28 (doi: 10.5376/bt.2012.03.0004) Abstract The cry10Aa gene from the Brazilian Bacillus thuringiensis subsp israelensis S1804 strain was introduced into the genome of the baculovirus Autographa californica multiple nucleopolyhedrovirus (AcMNPV) in order to evaluate its expression in insect cells and its toxicity to A. grandis. The recombinant virus (vSyncry10Aa) was amplified in Trichoplusia ni (BTI-Tn5B1-4) cells and used to infect Spodoptera frugiperda larvae. Total extracts from S. frugiperda infected with the recombinant viruses were analyzed by SDS-PAGE and showed the presence of a polypeptide around 85 kD. Cuboid-shaped protein crystals were observed in insect extracts by light and scanning electron microscopy. Bioassays, using the recombinant virus infected-insect extracts, showed high toxicity to A. grandis larvae, with a LC 50 of 7.12 µg/mL. Furthermore, a competition binding assay with the recombinant biotin-labeled Cry10A protein and brush border membrane vesicles (BBMV) from A. grandis indicated that the toxin binds specifically to BBMVs. Therefore, the Cry10A protein has a potential to be used in transgenic cotton plants for the control of this important insect pest. Keywords Bacillus thuringiensis; Toxicity; Recombinant protein Introduction Bacillus thuringiensis (Bt) is a naturally occurring, Gram positive, spore forming soil bacterium. The entomocidal properties of Bt are mediated by Cry proteins (δ endotoxins), which form parasporal crystal inclusions during the bacterial stationary growth phase (Monnerat and Bravo, 2000; Schnepf et al., 1998). These crystal inclusions are produced by one or more insecticidal proteins, which can exhibit toxicity and specificity toward a select group of Lepidopteran, Coleopteran and Dipteran insect species (Schnepf et al., 1998). Different Cry toxins showed great potential for the control of several economically devastating insect-pests when bioengineered into crop plants (Betz et al., 2000; Chattopadhyay et al., 2004). To date, many plant species have been genetically modified with cry genes, resulting in transgenic plants with high level of resistance to insect pests (Hilder and Boulter, 1999; Christou et al., 2006; van Rie, 2000) and a viable alternative to chemical control. Cotton is one of the most important crops cultivated worldwide. The cotton productive chain is one of the most important in Brazil and in the world, as it generates thousands of direct and indirect jobs and annually only the Brazilian cotton industry generates around US $ 1.5 billion (Martins et al., 2007). The cotton boll weevil, Anthonomus grandis (Coleoptera: Curculionidae), is a devastating cotton pest responsible for more than 50% of insecticide costs in Brazilian cotton crop fields. Moreover, A. grandis larvae resides inside floral buds and results in the destruction of fiber quality and hampers chemical control, causing considerable yield losses (Martins et al., 2007). However, due to A. grandis economic importance, the search for Cry toxins specific to this species is as essential as the knowledge on the toxic processes involved. Bacillus thuringiensis subsp. israelensis (Bti) S1804, is a Brazilian soil strain serotype H-14, toxic to Aedes aegypti (Diptera: Culicidae). Previous work,