Bt: Mode of Action and Use

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1 200 Whalon and Wingerd Bt: Mode of Action and Use Archives of Insect Biochemistry and Physiology 54: (2003) Mark E. Whalon* and Byron A. Wingerd The insecticidal toxins from Bacillus thuringiensis (Bt) represent a class of biopesticides that are attractive alternatives to broad-spectrum hard chemistries. The U.S. Food Quality Protection Act and the European Economic Council directives aimed at reducing the use of carbamate and organophosphate insecticides were expected to increase the use of narrowly targeted, soft compounds like Bt. Here we summarize the unique mode of action of Bt, which contributes to pest selectivity. We also review the patterns of Bt use in general agriculture and in specific niche markets. Despite continued predictions of dramatic growth for biopesticides due to US Food Quality Protection Act induced cancellations of older insecticides, Bt use has remained relatively constant, even in niche markets where Bt has traditionally been relatively high. Arch. Insect Biochem. Physiol. 54: , Wiley-Liss, Inc. KEYWORDS: Bacillus thuringiensis; insecticidal protein; biopesticide; Bt use; molecular mechanism INTRODUCTION In the recent past, both the United States and Europe underwent radical changes in pesticide regulatory policy. In the United States, the Food Quality Protection Act ( mandated a number of changes from more protection for infants, children, pregnant women, and the elderly to aggregate and cumulative analysis of pesticide residues for compounds exhibiting a common mode of action. The Europe Economic Council (Directive 91/414) similarly has enacted several farreaching changes in pesticide regulation. These policy actions have significantly enhanced the potential for biopesticides in the market place by mandating the cancellation or mitigation of more than 50% of the registered uses of insecticide chemistries like the organophosphate and carbamate insecticides that have been the mainstay of crop and human and animal health protections since the mid-1960s. These policy changes have resulted in reductions in the number of older, broad-spectrum insecticides in favor of softer, more narrowly targeted, newer chemistries. Although this transition has been difficult for many crop producers and agrochemical companies, it has also been a very significant opportunity for biopesticide companies to exploit those markets vacated by cancelled or withdrawn chemistries. Have these niches been filled with biopesticides products like those derived from the soil microorganism, Bacillus thuringiensis (Bt)? A recent study by the US National Academy of Sciences, 2000 suggests a rapid growth of Bt-based biopesticides is occurring as replacements of competitive chemical products that are being banned or phased out presumably as a result of passage of the Food Quality Protection Act (Committee on the Future Role of Pesticides in US Agriculture, 2000). Yet has this proffered increase in Bt-based biopesticides actually occurred? Our purpose in this report was to explore this question as well as Department of Entomology, Michigan State University, East Lansing, Michigan Abbreviations used: Bt = Bacillus thuringiensis; Cry = insecticidal crystal toxin; RNAi = ribonucleic acid interference; NASS = National Agriculture Statistics Survey; USDA = United States Department of Agriculture; US FQPA = United States Food Quality Protection Act. *Correspondence to: Mark Whalon, Michigan State University, Department of Entomology, Wilson Dr, CIPS, B-11, East Lansing, MI whalon@msu.edu Presented at the National Meeting of the Entomological Society of America, Symposium: Biorational Insecticides Mechanism and Application, November Wiley-Liss, Inc. DOI: /arch Published online in Wiley InterScience ( Archives of Insect Biochemistry and Physiology

2 Bt: Mode of Action and Use 201 to summarize the unique mode of action that Bt affords the market place. BT MODE OF ACTION There have been over 150 insecticidal crystal (Cry) proteins discovered in Bacillus thuringiensis and Bacillus cereus (Schnepf et al., 1998). While there is considerable genetic sequence variation between the classes of Cry toxins, there is a high level of conservation within a set of five functional blocks, and comparisons of the three-dimensional structures from Cry1, Cry2, and Cry3 reveal that they share a high degree of structural similarity (de Maagd et al., 2001). Because of the similarity in conserved domains and structure, it is also likely that they also share a high degree of functional similarity. The differences in sequence undoubtedly are responsible for insect order and binding site specificity. The general model for the function of Cry proteins is based on Cry1 because more work has been done on it than any other member of this class of protein (Aronson and Shai, 2001; Gringorten, 2001). Before describing the mechanism of Cry toxicity, it is important to have in mind the general anatomy of the insect gut and the normal physiology of the midgut where toxicity occurs. Plant material entering the gut first passes through the foregut where ingested material is further broken down into small pieces. Small spines that extend into the lumen of the foregut act as a sieve to prevent large particles from passing into the midgut. A generalized lepidopteran midgut includes a peritrophic membrane that lines the midgut epithelium. The peritrophic membrane is a matrix of intertwined chitin fibrils covered with a proteoglycan gel made of proteins and mucopolysaccharides. This membrane separates lumen of the midgut into two compartments: the ectoperitrophic space that contains the ingested particles in the center of the gut and the endoperitrophic space between the peritrophic membrane and the cellular wall of the midgut. The peritrophic membrane acts as a mechanical barrier to prevent physical damage to the epithelial cell layer of the midgut and as a screen through which nutrient material diffuse. The membrane has a small pore size and typically is not large enough to permit the diffusion of bacteria and very large toxic molecules from plants, such as tannins, into the endoperitrophic space (Chapman, 1998). Initial enzymatic digestion of food occurs in the ectoperitrophic space, and smaller digested molecules pass into the endoperitrophic space where they are further digested and absorbed by the microvilli covered cells of the midgut epithelium. Some of the digestive enzymes are compartmentalized, and others are attached to the peritrophic membrane. For example, the digestive enzymes trypsin and amylase that are needed for the initial digestion of food are found in the endoperitrophic space while the ectoperitrophic space contains aminopeptidase and trehalase where amino acids and sugars are transported through the membrane. A thorough review of insect gut physiology can be found in Chapman s 1998 The Insects. Phytophagous insects require an extremely alkaline midgut; the ph in lepidopteran larvae is commonly found to be in the range of 10 to 11. The high ph prevents tannins from complexing with and inactivating digestive enzymes. By dissociating tannins from leaf proteins, the digestibility of the leaf tissue is enhanced. Goblet cells in the midgut epithelium play a critical role in the maintenance of ph by secreting potassium carbonate into the lumen of the midgut. These cells are also central to maintaining high potassium concentrations with an energy-dependent potassium pump that pushes K+ from the hemolymph and columnar epithelial cells back into the gut lumen. The two gradients, high ph and K + concentration, in the lumen are used by amino acid symporters for the absorption of nutrients into columnar cells of the midgut epithelium (Gringorten, 2001). For Bt to successfully attack and colonize a larva, it must defeat each of the insect s defenses as it progresses through the steps of its pathology. After ingestion of the Bt crystal inclusion, toxicity is dependent on a complex process that requires multiple steps These include solubilization of the crystal proteins, proteolytic processing of the protoxin to the active form, high affinity binding with the December 2003

3 202 Whalon and Wingerd midgut receptor, and the irreversible insertion of the toxin into the membrane (Jenkins et al., 2000). The progression of Bt toxicity is summarized in Figure 1. There is a suite of specific characteristics that allow Bt to be a particularly effective pathogen. In order to pass through the foregut, Bt must be present as a very small spore rather than in its larger vegetative state, which would be more susceptible to damage and exclusion from the midgut. In the midgut, the ph is far too alkaline for the spore to germinate, but the insecticidal crystal proteins circumvent this aspect of the insect defense. Bt species contain a variety of endotoxins and helperfactors (Kumar and Venkateswerlu, 1998; Estruch et al., 1996; Agaisse et al., 1999), of these, the Cry d-endotoxins play the most critical role in Bt mediated toxicity. The Cry proteins solubilized from crystal inclusions of the Bt spore are inactive in their pro-toxin form (Choma et al., 1990, 1991). Before toxicity can occur, the pro-toxin must be proteolytically processed. This requires the high ph found in the midgut as well as digestive enzymes from the insect. Activation involves the removal of both the carboxyl terminal and the amino terminal ends of the protein (Gringorten, 2001). Once activated, the Cry toxin diffuses from the lumen through the periplasmic membrane into the endoperiplasmic space. The fully processed and active Cry toxin now has access to the surface of the columnar epithelial cells (Hill and Pinnock, 1998). At the cell surface, a critical handshake occurs as the Cry protein binds to its receptor (Ferre and Van Rie, 2002). Aminopeptidases (Luo et al. 1997), which are involved in digestion, and cell adhesion molecules similar to cadherins (Vadlamudi et al. 1995) function as receptors for the cry proteins (Jenkins and Dean, 2001). Binding of the Cry proteins is thought to occur at a membrane proximal region of these membrane bound proteins (Dorsch et al., 2002). Evidence from receptor binding studies has demonstrated that some Cry proteins bind to more than one site on the cell surface (Jurat- Fuentes et al., 2002). In addition, glycosylation is critical for some of the interactions. As a result, resistance to one Cry toxin does not guarantee universal resistance to all Cry proteins, although the presence of multiple resistance alleles in one individual has been shown to contribute to cross-resistance to multiple Cry toxins (Sayyed et al., 2000; Herrero et al., 2001; Jurat-Fuentes et al., 2002). The Cry protein is composed of three distinct domains. The N-terminal domain (domain I) contains seven a-helical domains that are arranged in three pairs around a central helix. It is involved in membrane insertion. Domain II consists of three symmetrically folded b-sheets and plays a role in receptor recognition and binding. The C-terminal (Domain III) consists of two b-sheets in a jellyroll conformation and is involved in binding and recognition as well as pore formation and channel specificity. The three domains form an upsidedown L shape with domain III stacked on domain II and domain I hanging off the side (Schnepf et al. 1998; de Maagd et al. 2001). After binding, domain I goes through a rearrangement similar to that of opening an umbrella. The three pairs of a-helices in domain I open and insert into the membrane, placing domain III at the membrane surface over the inserted helices. Insertion of the cry protein appears to be irreversible (Li et al., 2001). Next, aggregation of inserted Cry proteins occurs, resulting in the formation of pores. The pores are most likely tetramers and form a K + selective ion channel (Gringorten, 2001). The formation of this channel immediately leads to two very significant and detrimental physiological changes in the insect. First, the K + gradient in the epithelial cells is disrupted, which leads to an increase in hemolymph K + concentrations. Second, the ph gradient is disrupted leading to a decrease in the ph of the midgut lumen and an increase in the hemolymph ph. Ultimately, the affected cells are destroyed by the high ph of the midgut and osmotic lysis. As a result of the lysis of cells in the midgut epithelium, the spore is allowed to germinate in a nearly neutral environment, bathed in the nutrients from ruptured cells. Most insects are not killed directly by the effects of the toxin but die as a result of rapidly induced gut paralysis and feeding inhibition, Archives of Insect Biochemistry and Physiology

4 Bt: Mode of Action and Use 203 Fig. 1. Mechanism of Cry protein toxicity. A: Ingestion of spores or recombinant protein by phytophagous larva. B: In the midgut, endotoxins are solubilized from Bt spores (s) and inclusions of crystallized protein. (cp). C: Cry toxins are proteolytically processed to active toxins in the midgut. Active toxin binds receptors on the surface of columnar epithelial cells. Bound toxin inserts into the cellular membrane. D: Cry toxins aggregate to form pores in the membrane. E: Pore formation leads to osmotic lysis. F: Heavy damage to midgut membranes leads to starvation or septicemia. December 2003

5 204 Whalon and Wingerd and subsequent starvation or septicemia (Gringorten, 2001). MECHANISMS OF RESISTANCE The biochemical mechanisms of resistance to Bt in insects fall into three categories. The predominant and most characterized mechanism of resistance is the altered binding of Cry toxins to receptors in the midgut. This is manifested either as decreased binding affinity or as a reduction in binding sites. The second mechanism of resistance is due to alterations in the proteolytic processing of the Cry toxin. These alterations result in decreased protoxin solubilization, decreased rates of activation, or increased rates of toxin degradation. A third mechanism has been suggested, where the rapid regeneration of the damaged midgut epithelium prevents septicemia (Ferre and Van Rie, 2002). Binding of Cry toxins to the membrane occurs in a two-step process. In the first reversible step, the Cry toxin associates with factors on the membrane. In the second step, irreversible binding occurs, presumably when the toxin is inserted into the membrane (Cooper et al., 1998). Pore formation can occur independently in vivo with high concentrations of Cry protein, but the addition of brush border membrane proteins drastically reduces the necessary concentration (English et al., 1991). The receptor proteins found to contribute to both Cry binding and pore formation belong to two classes of proteins. The first class of proteins identified as putative Cry receptors were found in Manduca sexta (Knight et al., 1994). These aminopeptidases are glycosylphosphatidylinositol anchored proteins that are ubiquitously expressed on the surface of microvilli that cover the apical surface of the columnar epithelial cells of the midgut. They function in the N- terminal degradation of polypeptide chains during digestion. Initial experiments demonstrated that binding of Cry1Ac1 and pore formation occurred with lipid vesicles that were reconstituted with partially purified aminopeptidase (APN). More recently, two lines of evidence have provided unequivocal evidence for the functioning of APN as a receptor. First, Gill and Ellar (2002) expressed a M. Sexta APN in the midgut of the fruit fly, Drosophila melanogaster. As a result, the transgenic Drosophila became highly susceptible to the lepidopteran specific active toxin Cry1Ac1. The second line of evidence that supports a functional role of APN as a Cry receptor was carried out in the cutworm, Spodoptera litura. Using RNAi, Rajaopal et al. (2002) silenced the expression of the APN-encoding gene slapn. This resulted in a significant decrease in the susceptibility of S. litura larvae to the Cry1C toxin. The second class of receptors are similar to a large family of transmembrane proteins that are thought to mediate calcium-dependent cell aggregation and sorting, as well as maintain cell-to-cell contacts. These cadherin-like receptors were first identified in M. sexta as receptors for Cry1A toxins (Vadlamudi et al., 1995). This cadherin-like receptor (BT-R1), when expressed in heterologus cell lines, is found to confer high affinity binding to several Cry toxins (Keeton and Bulla, 1997). In addition, epitope mapping of the receptor has specifically located specific domains that are required for toxin binding (Keeton and Bulla, 1997; Dorsch et al., 2002). Furthermore, Nagamatsu et al. (1999) demonstrated that BtR175 (cadherin-like receptor from Bombyx mori) is directly involved in cell lysis and expression of this receptor in a toxin in-susceptible cell line resulted in the formation of pores and cell swelling upon treatment with the Cry1Aa d- endotoxin. A laboratory selected strain of Heliothis virescens strain YHD2 (tobacco budworm) that is over 10,000 times more resistant to Cry1Ac than wild type strains was reported by Gould et al. (1995). Recently Gahan et al. (2001) showed that this resistance is due to a mutation in HevCaLP, a H. virescens homologue of the BtR175 cadherin receptor. They postulated that alleles with a molecular lesion somewhere in the HevCaLP gene, which prevents it from functioning as a lethal target for a Cry toxin, could complement another similar but not identical allele resulting in a heterozygous but resistant phenotype (Gould et al., 1995). This hypothesis seems to have been verified by Morin et Archives of Insect Biochemistry and Physiology

6 Bt: Mode of Action and Use 205 al. (2003). They used molecular markers for field resistance of pink bollworm to Cry1Ac transgenic cotton, and identified three separate alleles that resulted in deleted or truncated cadherin receptors. Individuals inheriting two different cadherin-defect alleles exhibited high levels of resistance (Morin et al., 2003). Receptor-mediated resistance has not yet been directly linked to the aminopeptidases; however, other posttranslational modifications to these receptors may play a significant role in resistance. Marroquin et al. (2000) used a genetic screen to identify mutations in Caenorhabditis elegans that would allow the nematode to survive in the presence of Cry5B. Characterization of a mutant strain revealed a loss of function mutation in BRE-5, a putative b-1,3-galactosyltransferase that transfers galactose onto proteins and lipids. A number of studies have underscored the importance of carbohydrates in binding of Cry toxins (Ferre and Van Rie, 2002; Jenkins et al., 1999; Burton et al., 1999). More recently Jurat-Fuantes et al. (2002) discovered an additional mechanism behind the high resistance in the H. virescens YHD2 strain. In their studies, they found that reduced toxin binding and pore formation were correlated with altered glycosylation of specific proteins from brush border membrane vesicles. Domain II of several Cry toxins are very similar to lectin domains found in plants. These lectins bind galactose b-1,3-n-acetylgalactoseamine, which is the same structure made by BRE-5 from C. elegans. If toxin binding requires this carbohydrate structure, then this could explain how some Cry toxins can bind more than one receptor, and could also explain cases of cross resistance where inheritance is linked to only one allele. Mechanisms of resistance that are not receptor related involve the processing of the Cry toxin in the gut. A protease-mediated mechanism was identified in a Bt resistant strain of Plodia interpunctella (Indianmeal moth). Slower protoxin hydrolysis in gut extracts due to the absence of a major gut proteinase resulted in the increased resistance to the toxin (Oppert et al., 1997). A similar effect is seen in Plutella xylostella (Diamondback moth) where it contributes as a minor mechanism of resistance (Jin et al., 2000). In addition to decreased activation of protoxin, an increased rate of active toxin degradation may also contribute resistance. This mechanism of increased protease activity is also seen in the Colorado Potato Beetle (CPB). In a strain selected for its resistance to Cry3Aa (Rahardja and Whalon, 1995; Whalon et al., 1993), distinct protease species were found to be elevated (Loseva et al., 2002). This work also suggested that increases in protease expression levels may be related to an innate defense response since increased protease activity profiles are correlated with increased resistance. While altered binding and processing of toxins represent the majority of resistance mechanisms, other less well-represented mechanisms may also contribute to resistance. In some cases, the level of damage sustained in the midgut of sensitive and resistant larvae is almost indistinguishable. Using cultured midgut cells from H. virescens, Loeb et al. (2001) found that toxin-treated cultures exhibited a profound expansion of stem and differentiating cell numbers and that this increase in cell growth rate was the result of the induced synthesis of peptide growth factors. Such growth would mitigate continual low dose responses and may help to explain how chronic exposure to sublethal Bt levels results in increased tolerance of feeding larvae in other species (Costa et al., 2000). TRENDS IN BT USE Currently, there are over 250 biopesticide manufactures worldwide. Since 1972, however, over 72% of biopesticide business ventures failed (PAN, 2002). Bt has consistently been one of the most consistent and significant biopesticides in the market place. Bt products have essentially carried several companies that achieved a market place presence through uncertain economic times. Currently, biopesticides represent approximately 1% of the world pesticide market, and Bt products represent ~80% of all biopesticides sold. The Organization for Economic Cooperation and Development predicts that biopesticide may grow to 20% of the world s pesticide market by Other organiza- December 2003

7 206 Whalon and Wingerd tions like the World Health Organization, Food and Agriculture Organization, and European Commission are also predicting dramatic growth for biopesticides over the next years. Sprayable Bt formulations have penetrated cotton, fruit and vegetable, aquatic, and other insecticide markets (USDA). New Bt formulations have consistently made gains in a limited number of fruit and specialty vegetable markets over the last number of years. For instance, from 1989 to 2001, Bt remained the mainstay for Lepidoptera control in organic production throughout the world. Organic producer dependence on Bt for cabbage production was approximately three times that of conventional cabbage producers, 7.7 ± 1.3 and 2.1 ± 0.51 kg/ha, respectively (USDA/NASS ). These data support a general assertion made by many organic workers that Bt is more important to the organic community than to other producers because conventional producers have a greater array of management tools to choose from. In the near future, however, an organic formulation for Spinosad, a fermentation macrocyclic lactone product, may change Bt s organic market dominance. Bt s rapid environmental degradation leading to short field-residuals, narrow selectivity (primarily lepidoptera and a few coleopteran and dipteran spp.), and a mode of action requiring ingestion rather than contact toxicity make Bt a very safe product, but hamper its effectiveness as a broad spectrum, long-acting insecticide. These attributes require pest managers to carefully time spray applications and apply sprays more frequently than many moderate to long residual sprays. Further, Bt is at a disadvantage in crops that are attacked by a complex of sensitive and non-sensitive species, because other competing products with a broader spectrum of insecticidal activity may yield control across the whole complex yielding a more efficient, less frequent spray program. These attributes and a competitive marketplace with the introduction of many new low-risk compounds may explain why Bt sales have been flat. Market outlet can also have a significant impact on Bt use. For instance, the frequency of Bt applications on tomatoes produced for fresh consumption was significantly higher than sprays applied to tomatoes for processing between 1990 and 2000, 3.7 ± 1.54 and 1.28 ± 1.16 applications, respectively (USDA/NASS, ). How has Bt fared in the post FQPA era in the United States? There is considerable year-to-year and site-specific variation in most insecticide use statistics depending on pest pressure, environmental conditions, operational practices, marketing decisions, etc. Nevertheless, Bt use data collected in sound statistical format can yield some insight into use trends. For instance, recent data from USDA/ NASS indicate that Bt products are holding their market position in many crops or their position is slightly eroding, but there is no dramatic increase in arguably the most significant insecticide biopesticide despite the US FQPA induced extinction for older insecticides like the organophosphates in US specialty crops. For instance, strawberry, brambles, and blueberry producers in the United States use Bt sprays to control loopers, leafrollers, and fruitworms. From 1990 to 2001, the US berry acreage treated with Bt declined (Fig. 2A). Percent berry crops treated in 1991 was over 50%, but by 2001 it had declined to around 20%. Yet these berry data reflect an overall steady decline trend. We could not effectively test our hypothesis with actual active ingredient data in any crop from NASS, however, since very little proprietary data is available publicly for confidential business reasons. Given that NASS data result from actual producer/user surveys, we believe they accurately reflect aggregated use by crop and are sufficient to discern use and frequency of spray applications for Bt in specialty crops. In addition, they are essentially the only data available for this study. In contrast to berries, Grapes (Fig. 2B) over the same period experienced an increase in percent acres treated with Bt. For instance, in 1993 less than 5% of the grape crop was treated, yet by 2000, an exceptionally high Bt use year, over 30% of the crop was treated. This anomaly could be a reaction to FQPA-induced changes but probably reflects a specific pest-induced anomaly. The overall trend may Archives of Insect Biochemistry and Physiology

8 Bt: Mode of Action and Use 207 Fig. 2. Percent crop treated with Bacillus thuringiensis (Bt) on berries (A), grapes (B), and tree fruits (C). The number of applications of Bt on apples is presented in D. All data are from those reported in USDA/NASS pesticide use statistics. be attributable to increased applications to control grape berry moth (Endopiza viteana) and other Lepidoptera species in regions where these pest s populations have increased as a result of the FQPA cancellation of broad-spectrum insecticides. This trend may also reflect increased conversion of winegrape growers throughout the United States, but particularly in California, to organic practices where there are few alternatives other than Bt for Lepidoptera control. Bt use in tree fruit targets an array of Lepidoptera including the green fruit worm complex, cutworms, loopers, leafrollers and fruitworms. Since Bt products are not very effective against internal feeding larvae infesting apples, these products are rarely targeted at codling moth, Cydia pomenella, or oriental fruitmoth, Grapholitha molesta, except in organic orchards. The percent of tree fruit treated with Bt was constant from 1990 to 2001 (Fig. 2C). All tree fruit crops averaged 18.1% of their acreage treated with Bt, in contrast to almost 25% of the apples acreage that was treated with Bt sprays. For instance, pears averaged 8.6, citrus 13.3, and cherries 19.1% of the crop treated. This trend probably reflects the larger Lepidopteran pest complex attacking apples in comparison to other tree fruits and because there is more organic apple acreage in the United States than almost all other tree fruits combined. Although treated apple acreage remained unchanged from , the number of Bt applications declined (Fig. 2D) over the same period. Perhaps this reduction indicates less reliance on Bt or change(s) in product formulation/performance, increased usage rate per spray, or a combination of these factors. December 2003

9 208 Whalon and Wingerd Bt use in vegetables ( ) either demonstrated a slight decline in use as in crucifers (Fig. 3A) and cucumbers (Fig. 3B) or no change as in all greens (Fig. 3C) including spinach, lettuce of all types, and chard. In addition, melons (Fig. 3D) were no exception to this no significant change trend for Bt percentage crop treated or number of applications applied. After examining data from several other vegetables, we believe that these trends are consistent across vegetables for Bt use. Therefore, the vegetable Bt use data also support the assertion that Bt use has not dramatically increased since the passage of the FQPA. CONCLUSIONS The advantages of Bt as an insecticide including low mammalian and non-target impacts, rapid breakdown in UV light, and narrow spectrum of primarily leaf-feeding Lepidopteran targets apparently were not sufficient to entice pest managers to shift to BT sprays as an alternative to fill the insect management vacuum created when USEPA began to enforce FQPA-mandated cancellation and mitigation of broad spectrum insecticides like the organophosphate insecticides. Perhaps these advantages may not be sufficient to warrant increased use by hard-pressed producers. Many of these producers may realize a lower return on investment per Bt spray as compared to other, conventional broader-spectrum FQPA alternatives. Obviously, Bt s rather specific mode of action limits its utility to insects with appropriate Bt midgut receptors and those that graze plant surfaces easily treated with Bt sprays. More recently, an array of low-risk, organophosphate alternatives have Fig. 3. Percent crop treated with Bacillus thuringiensis (Bt) on vegetable crops including crucifers (A), cucumbers (B), all leafy greens (C), and melons (D) as reported in USDA/ NASS vegetable use statistics. Archives of Insect Biochemistry and Physiology

10 Bt: Mode of Action and Use 209 been registered by the USEPA, many through the efforts of Interregional Project 4 (IR-4), particularly in specialty or minor crops. These classes of compounds include pheromones, Spinosyns, neonicotinoids, neem, Baculoviruses, and others. Various strategies have been deployed to increase ingestion or to protect Bt products in the environment including consumption stimulants or feeding additives, UV protectants, spreader/stickers and microbial-encapsulation (see current or historical active ingredient specifications on Bt product labels). Resistance is important, but probably not an Achilles heal for Bt sprays since many crops are not attacked by the diamond back moth, Plutella xylostella, the only insect to consistently develop resistance to Bt products in the field. In fact, many of the disadvantages of Bt (short residual activity, narrow spectrum of activity, etc.) may actually reduce resistance selection. Perhaps Bt s limited species activity spectrum and short field life is still this biopesticide s greatest challenge in the marketplace despite FQPA-induced opportunities. LITERATURE CITED Agaisse H, Gominet M, Okstad OA, Kolsto AB, Lereclus D PlcR is a pleiotropic regulator of extracellular virulence factor gene expression in Bacillus thuringiensis. Mol Microbiol 32: Aronson AI, Shai Y Why Bacillus thuringiensis insecticidal toxins are so effective: unique features of their mode of action. Fems Microbiol Lett 195:1 8. Burton SL, Ellar DJ, Li J, Derbyshire DJ N-acetylgalactosamine on the putative insect receptor aminopeptidase N is recognised by a site on the domain III lectin-like fold of a Bacillus thuringiensis insecticidal toxin. J Mol Biol 287: Chapman, R. F The insects. Cambridge: Cambridge University Press. Choma CT, Surewicz WK, Carey PR, Pozsgay M, Raynor T, Kaplan H Unusual proteolysis of the protoxin and toxin from Bacillus thuringiensis: structural implications. Eur J Biochem 189: Choma CT, Surewicz WK, Kaplan H The toxic moiety of the Bacillus thuringiensis protoxin undergoes a conformational change upon activation. Biochem Biophys Res Commun 179: Committee on the Future Role of Pesticides in US Agriculture, B.o.A.a.N.R., Board on Environmental Studies and Toxicology, National Research Council Technological and biological changes and the future of pest management. The future role of pesticides in US Agriculture. Washington, DC: National Academy Press. p Cooper MA, Carroll J, Travis ER, Williams DH, Ellar DJ Bacillus thuringiensis Cry1Ac toxin interaction with Manduca sexta aminopeptidase N in a model membrane environment (vol 333, p 677, 1998). Biochem J 335: Costa SD, Barbercheck ME, Kennedy GG Sublethal acute and chronic exposure of Colorado potato beetle (Coleoptera:Chrysomelidae) to the delta-endotoxin of Bacillus thuringiensis. J Econ Entomol 93: de Maagd RA, Bravo A, Crickmore N How Bacillus thuringiensis has evolved specific toxins to colonize the insect world. Trends Genet 17: Dorsch JA, Candas M, Griko NB, Maaty WSA, Midboe EG, Vadlamudi RK, Bulla LA Cry1A toxins of Bacillus thuringiensis bind specifically to a region adjacent to the membrane-proximal extracellular domain of BT-R-1 in Manduca sexta: involvement of a cadherin in the entomopathogenicity of Bacillus thuringiensis. Insect Biochem Mol Biol 32: English LH, Readdy TL, Bastian AE Delta-endotoxin-induced leakage of rb-86+-k+ and H2O from phospholipidvesicles is catalyzed by reconstituted midgut membrane. Insect Biochem 21: Estruch JJ, Warren GW, Mullins MA, Nye GJ, Craig JA, Koziel MG Vip3A, a novel Bacillus thuringiensis vegetative insecticidal protein with a wide spectrum of activities against lepidopteran insects. Proc Natl Acad Sci USA 93: Ferre J, Van Rie J Biochemistry and genetics of insect resistance to Bacillus thuringiensis. Annu Rev Entomol 47: Gahan LJ, Gould F, Heckel DG Identification of a gene associated with bit resistance in Heliothis virescens. Science 293: Gill M, Ellar D Transgenic Drosophila reveals a functional in vivo receptor for the Bacillus thuringiensis toxin Cry1Ac1. Insect Mol Biol 11(6): December 2003

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12 Bt: Mode of Action and Use 211 Rahardja U, Whalon ME Inheritance of resistance to Bacillus thuringiensis Subsp Tenebrionis Cryiiia delta-endotoxin in Colorado potato beetle (Coleoptera, Chrysomelidae). J Econ Entomol 88: Rajagopal R, Sivakumar S, Agrawal N, Malhotra P, Bhatnagar RK Silencing of midgut aminopeptidase N of Spodoptera litura by double-stranded RNA establishes its role as Bacillus thuringiensis toxin receptor. J Biol Chem 277: Sayyed AH, Haward R, Herrero S, Ferre J, Wright DJ Genetic and biochemical approach for characterization of resistance to Bacillus thuringiensis toxin Cry1Ac in a field population of the diamondback moth, Plutella xylostella. Appl Environ Microbiol 66: Schnepf E, Crickmore N, Van Rie J, Lereclus D, Baum J, Feitelson J, Zeigler DR, Dean DH Bacillus thuringiensis and its pesticidal crystal proteins. Microbiol Mol Biol Rev 62: USDA National Agriculture Statistics Service: fruit and vegetable Bt use and number of applications data ( Vadlamudi RK, Weber E, Ji IH, Ji TH, Bulla LA Cloning and expression of a receptor for an insecticidal toxin of Bacillus thuringiensis. J Biol Chem 270: Whalon ME, Miller DL, Hollingworth RM, Grafius EJ, Miller JR Selection of a Colorado potato beetle (Coleoptera, Chrysomelidae) strain resistant to Bacillus thuringiensis. J Econ Entomol 86: December 2003