Bacillus thuringiensis (Bt) is a valuable source of insecticidal proteins for use in conventional sprayable formulations and in transgenic crops, and it is the most promising alternative to synthetic insecticides. However, evolution of resistance in insect populations is a serious threat to this technology. So far, only one insect species has evolved significant levels of resistance in the field, but laboratory selection experiments have shown the high potential of other species to evolve resistance against Bt. We have reviewed the current knowledge on the biochemical mechanisms and genetics of resistance to Bt products and insecticidal crystal proteins. The understanding of the biochemical and genetic basis of resistance to Bt can help design appropriate management tactics to delay or reduce the evolution of resistance in insect populations.

Current knowledge of biochemical mechanisms of insect resistance to Bacillus thuringiensis is reviewed. Available information on resistance inheritance and on patterns of cross-resistance is included. Modification of the binding sites for B. thuringiensis insecticidal crystal proteins has been found in different populations of three insect species. This resistance mechanism seems to be inherited as a single recessive or partially recessive major gene, and the resistance levels reached are high. Altered proteolytic processing of B. thuringiensis crystal proteins has been suggested to be involved in one case of resistance. From the available data it seems that binding site modification is the most significant resistance mechanism under field conditions. ' We use the word 'toxin' to refer to the. activated fom of the crystal proteins, and tbe expression 'insecticidal crystal protein (ICP)' to refer to either the protoxin or its activated form. 0378-1097/95/$09.50 8 1995 Federation of European Microbiological Societies. All rights reserved SSDI 0378-1097(95)00271-5 ' Bfe = B. thuringiensis var. entamocidus. Bf = commercial formulations of B. thuringiemis. Dipel and Javelin are tradenames of formulations of B. thuringiensis var. kursraki. * LC,,, (or LD,,,) of resistant strain divided by LC,, (or LD,,) of susceptible control strain. J. Ferri et al. / FEMS Microbiology Letters 132 (1995) l-7 3

Resistance to the Bacillus thuringiensis bioinsecticide in a field population of Plutella xylostella is due to a change in a midgut membrane receptor (insecticidal crystal ABSTRACTThe biochemical mechanism for resistance to Bacillus thuringiensis crystal proteins was studied in a field population of diamondback moths (Plutella xylostella) with a reduced susceptibility to the bioinsecticidal spray. The toxicity and binding characteristics of three crystal proteins [CryIA(b), CryIB, and CryICI were compared between the field population and a laboratory strain. The field population proved resistant (>200-fold compared with the laboratory strain) to CryIA(b), one of the crystal proteins in the insecticidal formulation. Binding studies showed that the two strains differ in a membrane receptor that recognizes CryIA(b). This crystal protein did not bind to the brush-border membrane of the midgut epithelial cells of the field population, either because of strongly reduced binding affinity or because of the complete absence of the receptor molecule. Both strains proved fully susceptible to the CryIB and CryIC crystal proteins, which were not present in the B. thuringiensis formulation used in the field. Characteristics of CryIB and CryIC binding to brushborder membranes of midgut epithelial cells were virtually identical in the laboratory and the field population.

SUMMARYEntomopathogenic bacteria produce insecticidal proteins that accumulate in inclusion bodies or parasporal crystals (such as the Cry and Cyt proteins) as well as insecticidal proteins that are secreted into the culture medium. Among the latter are the Vip proteins, which are divided into four families according to their amino acid identity. The Vip1 and Vip2 proteins act as binary toxins and are toxic to some members of the Coleoptera and Hemiptera. The Vip1 component is thought to bind to receptors in the membrane of the insect midgut, and the Vip2 component enters the cell, where it displays its ADP-ribosyltransferase activity against actin, preventing microfilament formation. Vip3 has no sequence similarity to Vip1 or Vip2 and is toxic to a wide variety of members of the Lepidoptera. Its mode of action has been shown to resemble that of the Cry proteins in terms of proteolytic activation, binding to the midgut epithelial membrane, and pore formation, although Vip3A proteins do not share binding sites with Cry proteins. The latter property makes them good candidates to be combined with Cry proteins in transgenic plants (Bacillus thuringiensis-treated crops [Bt crops]) to prevent or delay insect resistance and to broaden the insecticidal spectrum. There are commercially grown varieties of Bt cotton and Bt maize that express the Vip3Aa protein in combination with Cry proteins. For the most recently reported Vip4 family, no target insects have been found yet.

Evolution of pest resistance to al proteins produced by Baciuw thw'mgwensis (Bt) would dera our ability to control agricltural pests with genetically engineered crops designe to express genes coding for these proteins. One of the few microbes that has been used successfully in agricultural insect pest control is Bacillus thuringiensis (Bt). Liquid and powder formulations of this bacterium hold a small but growing share of the pest-control-agent market (1). Genes from Bt that code for production ofa set ofinsecticidal proteins are being cloned and transferred to a number of crop plants (2)(3)(4). Expression ofBt genes in crop plants is appealing because the toxicity of the proteins coded for by these genes is restricted to specific groups of insects (5) (9)(10)(11).Excitement over the potential of engineered plants with Bt genes has been tempered by laboratory and field work which indicates that pest insects have the capacity to adapt to Bt and its toxic proteins (34). However, studies of insect resistance to Bt toxins have found that resistance is toxin-specific.Indian meal moths that were >100-fold resistant to the Bt toxin CryIA(b) were not at all resistant to CryIC (12). Diamondback moths that were >200-fold resistant to Cry-IA(b) were not resistant to CryIB or CryIC (13). A strain of Heliothis virescens selected with CryIA(b) and a mixture of CryIA and CryIIA proteins in an HD-1 strain of Bt was significantly resistant to only some strains of Bt (14). Restriction of resistance to a single or highly related group of toxins may be explained by studies indicating that the biochemical basis for resistance includes changes in receptors in the insect midgut (12,13,15).While high levels of resistance are cause for concern, the specificity of resistance has led to a belief that as insects become resistant to one Bt toxin, that toxin could be successfully replaced by a different Bt toxin (16). Additionally, genetic analyses of Bt resistance have demonstrated that the resistance is usually inherited as a mostly recessive trait (14,15,(17)(18)(19)(20). The rate at which such recessive traits become established in a population can be significantly decreased by the proper use ofBt-toxin-producing plants, so these genetic results have prompted the development of specific "resistance management" strategies (21,22).We report here on Bt-toxin resistance in a strain of H. virescens, a pest of cotton, soybean, tobacco, tomato, and other agricultural crops. The resistance in our strain of H. virescens is not toxin-specific, does not appear to be related to changes in midgut receptors, and is not inherited as a recessive trait when larvae are exposed to high doses of Bt toxin. EXPERIMENTAL PROCEDURESDesign of Selection Experiment. As part of a larger study on insect resistance to Bt toxins, a strain of H. virescens was selected for survival on an artificial diet containing CryIA(c). The selected strain and the control strain were initiated from a sample of a field population collected in July 1988. Precautions, which are...

Insecticidal proteins from the soil bacterium Bacillus thuringiensis (Bt) are becoming a cornerstone of ecologically sound pest management. However, if pests quickly adapt, the benefits of environmentally benign Bt toxins in sprays and genetically engineered crops will be short-lived. The diamondback moth (Plutella xylostella) is the first insect to evolve resistance to Bt in open-field populations. Here we report that populations from Hawaii and Pennsylvania share a genetic locus at which a recessive mutation associated with reduced toxin binding confers extremely high resistance to four Bt toxins. In contrast, resistance in a population from the Philippines shows multilocus control, a narrower spectrum, and for some Bt toxins, inheritance that is not recessive and not associated with reduced binding. The observed variation in the genetic and biochemical basis of resistance to Bt, which is unlike patterns documented for some synthetic insecticides, profoundly affects the choice of strategies for combating resistance.

Four subpopulations of a Plutella xylostella (L.) strain from Malaysia (F 4 to F 8 ) were selected with Bacillus thuringiensis subsp. kurstaki HD-1, Bacillus thuringiensis subsp. aizawai, Cry1Ab, and Cry1Ac, respectively, while a fifth subpopulation was left as unselected (UNSEL-MEL). Bioassays at F 9 found that selection with Cry1Ac, Cry1Ab, B. thuringiensis subsp. kurstaki, and B. thuringiensis subsp. aizawai gave resistance ratios of >95, 10, 7, and 3, respectively, compared with UNSEL-MEL (>10,500, 500, >100, and 26, respectively, compared with a susceptible population, ROTH). Resistance to Cry1Ac, Cry1Ab, B. thuringiensis subsp. kurstaki, and B. thuringiensis subsp. aizawai in UNSEL-MEL declined significantly by F 9 . The Cry1Ac-selected population showed very little cross-resistance to Cry1Ab, B. thuringiensis subsp. kurstaki, and B. thuringiensis subsp. aizawai (5-, 1-, and 4-fold compared with UNSEL-MEL), whereas the Cry1Ab-, B. thuringiensis subsp. kurstaki-, and B. thuringiensis subsp. aizawai-selected populations showed high cross-resistance to Cry1Ac (60-, 100-, and 70-fold). The Cry1Ac-selected population was reselected (F 9 to F 13 ) to give a resistance ratio of >2,400 compared with UNSEL-MEL. Binding studies with 125 I-labeled Cry1Ab and Cry1Ac revealed complete lack of binding to brush border membrane vesicles prepared from Cry1Ac-selected larvae (F 15 ). Binding was also reduced, although less drastically, in the revertant population, which indicates that a modification in the common binding site of these two toxins was involved in the resistance mechanism in the original population. Reciprocal genetic crosses between Cry1Ac-reselected and ROTH insects indicated that resistance was autosomal and showed incomplete dominance. At the highest dose of Cry1Ac tested, resistance was recessive while at the lowest dose it was almost completely dominant. The F 2 progeny from a backcross of F 1 progeny with ROTH was tested with a concentration of Cry1Ac which would kill 100% of ROTH moths. Eight of the 12 families tested had 60 to 90% mortality, which indicated that more than one allele on separate loci was responsible for resistance to Cry1Ac.

The cabbage looper, Trichoplusia ni, is one of only two insect species that have evolved resistance to Bacillus thuringiensis in agricultural situations. The trait of resistance to B. thuringiensis toxin Cry1Ac from a greenhouseevolved resistant population of T. ni was introgressed into a highly inbred susceptible laboratory strain. The resulting introgression strain, GLEN-Cry1Ac-BCS, and its nearly isogenic susceptible strain were subjected to comparative genetic and biochemical studies to determine the mechanism of resistance. Results showed that midgut proteases, hemolymph melanization activity, and midgut esterase were not altered in the GLEN-Cry1Ac-BCS strain. The pattern of cross-resistance of the GLEN-Cry1Ac-BCS strain to 11 B. thuringiensis Cry toxins showed a correlation of the resistance with the Cry1Ab/Cry1Ac binding site in T. ni. This cross-resistance pattern is different from that found in a previously reported laboratory-selected Cry1Ab-resistant T. ni strain, evidently indicating that the greenhouse-evolved resistance involves a mechanism different from the laboratory-selected resistance. Determination of specific binding of B. thuringiensis toxins Cry1Ab and Cry1Ac to the midgut brush border membranes confirmed the loss of midgut binding to Cry1Ab and Cry1Ac in the resistant larvae. The loss of midgut binding to Cry1Ab/Cry1Ac is inherited as a recessive trait, which is consistent with the recessive inheritance of Cry1Ab/Cry1Ac resistance in this greenhouse-derived T. ni population. Therefore, it is concluded that the mechanism for the greenhouse-evolved Cry1Ac resistance in T. ni is an alteration affecting the binding of Cry1Ab and Cry1Ac to the Cry1Ab/Cry1Ac binding site in the midgut.