Screening for Bacillus thuringiensis Crystal Proteins Active against the Cabbage Looper, Trichoplusia ni Marı ´a M. Iracheta,* Benito Pereyra-Alfe ´rez,* Luis Gala ´ n-Wong,* and Juan Ferre ´† ,1 *Departamento de Microbiologı´a e Inmunologı´a, Facultad de Ciencias Biolo ´gicas/UANL, 66450 Monterrey, N. L., Me ´xico; and †Departament de Gene `tica, Universitat de Vale `ncia, 46100 Burjassot, Valencia, Spain Received March 30, 2000; accepted April 18, 2000 Toxicity tests were performed to find among Cry1 and Cry2 Bacillus thuringiensis crystal proteins those with high activity against the cabbage looper. Tests were performed with neonate larvae on surface-con- taminated artificial diet. The crystal proteins found to be toxic were, from higher to lower toxicity: Cry1Ac, Cry1Ab, Cry1C, Cry2Aa, Cry1J, and Cry1F (LC 50 of 1.1– 4.1, 3.4 – 4.4, 12, 34, 87, and 250 ng/cm 2 , respectively). Cry1B, Cry1D, and Cry1E can be considered nontoxic (LC 50 higher than 2500 ng/cm 2 ). Cry1Aa was moder- ately toxic to nontoxic, depending on the source (LC 50 of 420 ng/cm 2 from PGS and 8100 ng/cm 2 from Ecogen). In vitro binding assays with trypsin-activated 125 I-la- beled Cry1Aa, Cry1Ab, and Cry1Ac crystal proteins and brush border membrane vesicles from midgut lar- vae showed a direct correlation between toxicity and binding affinity. Heterologous competition experi- ments indicated that Cry1Aa and Cry1F bind, though only at very high concentrations, to the Cry1Ab/ Cry1Ac shared high-affinity binding site. © 2000 Academic Press Key Words: Bacillus thuringiensis; Trichoplusia ni; -endotoxins; crystal proteins; biological control; cab- bage looper; membrane receptors; resistance manage- ment. INTRODUCTION Bioinsecticides based on Bacillus thuringiensis are the most used environment-friendly alternative to syn- thetic pesticides for the biological control of agricul- tural and forest pests and vectors of human diseases (Lambert and Peferoen, 1992; Schnepf et al., 1998). B. thuringiensis synthesizes a crystalline parasporal in- clusion composed of one or several proteins known as insecticidal crystal proteins (ICP) or -endotoxins, with high level of both specificity and toxicity against sev- eral insect orders (Aronson et al., 1986; Ho ¨fte and Whiteley, 1989) and certain nematodes and protozoan pathogens (Feitelson et al., 1992). Upon ingestion by susceptible insect larvae, crystalline inclusions are sol- ubilized in the midgut lumen and converted to active toxins by trypsin-like proteases. The activated toxins bind to specific receptors on the brush border apical membrane of midgut columnar cells and insert into the membrane, where they form pores. As a consequence, epithelial midgut cells swell and lyse, causing the lar- vae to stop feeding and eventually die by septicemia or starvation (Knowles, 1994; Peyronet et al., 1997). For the optimal use of B. thuringiensis crystal pro- tein mixtures either in sprays or in transgenic plants, it is important to know which of them show the highest toxicity for a particular pest and which of them share the same midgut receptors to avoid some types of cross- resistance (Lee et al., 1995; Ballester et al., 1999). Trichoplusia ni is a polyphagous lepidopteran insect that is considered among the most important pests of corn and cotton in Me ´xico. This insect has also been chosen as one of the reference insects in the determi- nation of lepidopteran-active B. thuringiensis formula- tions. To generate information from which to chose appropriate strategies of use of B. thuringiensis in IPM and biological control programs of this pest, we tested 10 individual crystal proteins for its toxicity against this insect. Variability among Cry1A crystal proteins from different sources was also assessed. In vitro bind- ing assays with 125 I-labeled crystal proteins and brush border membrane vesicles (BBMV) from midgut larvae were used to determine plausible patterns of cross- resistance. MATERIALS AND METHODS Insects. Insects were obtained from the Departa- mento de Microbiologı ´a e Inmunologı ´a, FCB/UANL, Me ´xico. This colony had been reared in the laboratory for more than 8 years. Insects were reared on a lepi- 1 To whom correspondence should be addressed at: Departament de Gene `tica, Facultat de CC. Biolo `giques, Dr. Moliner 50, 46100- Bursjassot (Vale `ncia), Spain. Fax: (34) 96 398 3029. E-mail: Juan.Ferre@uv.es. Journal of Invertebrate Pathology 76, 70 –75 (2000) doi:10.1006/jipa.2000.4946, available online at http://www.idealibrary.com on 70 0022-2011/00 $35.00 Copyright © 2000 by Academic Press All rights of reproduction in any form reserved.