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Since World War II, synthetic organic insecticides have been used extensively throughout the world for controlling insects and mites that attack crops. Hundreds of millions of pounds of these chemicals are still used annually and have enabled production of a bountiful food supply in most countries. Despite continuing use, the detrimental effects of these chemicals on nontarget vertebrate and invertebrate populations have been recognized for decades. Moreover, the public is now more concerned than ever about the effects of chemical insecticides on their health, as is evident from the continuing growth in sales of organic foods.

New chemical insecticides developed over the past 20 years are more specific as well as more biodegradable, yet many, such as imidocloprid and spinosad, still have a broad spectrum of activity, causing high rates of mortality in many nontarget insect populations. The increased specificity of these insecticides provides environmental benefits, but by far the most significant advance of the last half of the twentieth century for decreasing the use and adverse effects of chemical insecticides is the development of insecticidal transgenic crops based on the Cry proteins of Bacillus thuringiensis (Bt). Since initial plantings in 1996, annual acreage of these crops, referred to as Bt crops, has grown to more than 40 million acres in the United States.1 This acreage consists mainly of Bt corn and Bt cotton used to control caterpillar pests such as the European corn borer (Ostrinia nubilalis), the pink bollworm (Pectinophora gossypiella), and species of budworms and bollworms belonging to the genera Heliothis and Helicoverpa. Additionally, within the last few years Bt corn developed for control of corn rootworms (Diabrotica species) has been released and will likely lead to further increases in Bt crop acreage and decreases in chemical insecticide uses in the United States.

Initial reluctance to plant Bt crops in other countries, owing to the use of recombinant DNA technology used to create these crops, has diminished over the past decade due to results obtained in the United States demonstrating significant economic and environmental benefits, especially reductions in chemical insecticide usage and concomitant nontarget effects, along with a corresponding increase in worker safety. The absence of any negative effects on human health has led to the recent adoption of Bt crops in several other countries, including Argentina, China, India, South Africa, and more recently, Spain. As evidence for the safety of Bt crops to nontarget vertebrates and invertebrates continues to mount, it is probable that these crops will be adopted in many other countries, including most of those in the European Union. As recently as 2006, less than 100,000 acres in Europe were planted with Bt crops due to governmental restrictions, largely due to public opinion against the planting of any kind of genetically engineered crop.1

The development of Bt crops should have been viewed as a positive development owing to their high degree of target specificity2,3 and their remarkable long-term safety record (extending for more than 40 years) of insecticides based on this bacterium.4-6 In contrast to chemical insecticides, no human deaths6 or even significant illnesses have been attributed to the use of Bt insecticides. However, a few studies highly publicized in the popular press, especially a study showing that Bt corn pollen could kill larvae of the Monarch butterfly in the laboratory, quickly led to widespread concern by the public and minor segments of the scientific community about the safety of these crops to nontarget organisms. Fortunately, the U.S. Environmental Protection Agency (EPA) and other governmental agencies stood by their standards of using the results of experimental studies and risk-assessment procedures, balancing benefits against risks rather than uninformed public opinion, to determine the safety of Bt crops to nontarget organisms, including humans. Based on these studies, the EPA has allowed existing registrations to remain in effect except where they were withdrawn voluntarily, as in the case of Starlink™ corn, and continues to proceed with evaluations of petitions to register new insecticidal transgenic crops based on Bt proteins. Nevertheless, although an overwhelming majority of the scientists7 who have examined the data on the safety of Bt insecticides and Bt crops concur that they are safe for humans and most nontarget organisms, significant concerns about safety remain for some scientists, as well as on the part of the poorly informed public.

Thus, the purpose of this paper is to review and summarize the studies that support the safety of Bt insecticides and Bt crops based on insecticidal Bt proteins. To do this, we first provide an overview of the biology of B. thuringiensis, including what is known about the mechanisms by which this species causes insect death. This section provides the scientific basis for understanding why Bt insecticides and the Cry proteins used in Bt crops are so much more specific, and thus safer, than chemical insecticides. Next we summarize the safety studies on bacterial insecticides for nontarget organisms, including vertebrates, which support their continued registration for insect control. This section includes analysis of reports that claim Bt can cause infection or food poisoning in humans, as well as summaries of recent epidemiological studies of human populations exposed to aerial applications of Bt insecticides in residential areas to control insect pests in Canada and New Zealand. These studies are important for understanding the potential effects of Bt crops because the complexity, i.e., the type and number of insecticidal components in products that use B. thuringiensis as the active ingredient, are much greater and more variable than the Cry proteins used in Bt crops. Current Bt crops typically contain only one or two Cry proteins, whereas, as we show, Bt insecticides used in agriculture, forestry, and vector control contain a multiplicity of insecticidal proteins, along with the spore and other insecticidal components. This reduction in toxin complexity by itself suggests that Bt crops should be more specific and thus safer to nontarget invertebrates, other animals, and humans.

Finally, we review recent long-term, multiyear field studies carried out under operational growing conditions in the United States and Australia on Bt cotton and Bt corn, where the effects of these crops on nontarget invertebrate communities were extensively evaluated. Taken together, this combination of studies evaluating the effects of Bt insecticides and Bt crops shows that this technology is remarkably safe for humans and nontarget organisms — unparalleled among pest control technologies developed over the past century that can be adopted for use ranging from small- to large-scale agriculture. These studies suggest that whenever and wherever it is agronomically possible and economically feasible, Bt crops should be incorporated into biological control and integrated pest management programs to improve crop protection, protect the environment, and yield a safer food supply.

3.2 BIoloGY of Bacillus thuringiensis

The insecticidal bacterium Bacillus thuringiensis (Bt) is a common Gram-positive, spore-forming aerobic bacterium that can be readily cultured on simple media such as nutrient agar from a variety of environmental sources including soil, water, plant surfaces, grain dust, dead insects, and insect feces.8 Its life cycle is simple. When nutrients and environmental conditions are sufficient for growth, the spore germinates, producing a vegetative cell that grows and reproduces by binary fission. Cells continue to multiply until one or more nutrients, such as sugars, amino acids, or oxygen, become insufficient for continued vegetative growth. Under these conditions, the bacterium sporulates, producing a spore and parasporal body, the latter composed primarily of one or more proteins (most of which are insecticidal, in the form of crystalline inclusions) (Figure 3.1). These are commonly referred to in the literature as insecticidal crystal proteins or endotoxins (formally, 8-endotoxins),4 and can comprise as much as 40% of the dry weight of a sporulated culture. These proteins are actually protoxins that must be activated by proteolytic cleavage to be toxic,2 which we discuss in more detail later.

There are two major types of insecticidal crystal proteins, Cry (for crystal) and Cyt (for cytolytic) proteins,2 and variations of each of these types. Genes encoding more than 120 Cry proteins and 12 Cyt proteins have been cloned and sequenced.3 Most Cry proteins are active against lepidopteran insects, with a few being toxic to

FIGURE 3.1 Spores and parasporal insecticidal crystals produced by Bacillus thuringiensis. The crystals contain Cry and Cyt proteins responsible for the acute intoxication effects of this insecticidal bacterium. (A) Sporulating cells of B. thuringiensis. The arrowheads point to the crystalline parasporal body adjacent to the spore formed in each cell. (B) Crystals produced by the HD1 isolate of B. thuringiensis subsp. kurstaki (Btk). The three CrylA proteins co-crystallize during synthesis to form the bipyramidal crystal, whereas the Cry2A protein crystallizes separately, forming a quasi-cuboidal crystal. (C) Surface structure of a single bipyramidal crystal revealing the packing arrangement of Cry1 molecules. (D) Transmission electron micrograph through a Btk parasporal body. Note the Cry2A (P2) crystal is typically embedded within the CrylA (Pl) crystal. This arrangement apparently evolved to enhance activity of this isolate and others with a similar arrangement of insecticidal inclusions.

FIGURE 3.1 Spores and parasporal insecticidal crystals produced by Bacillus thuringiensis. The crystals contain Cry and Cyt proteins responsible for the acute intoxication effects of this insecticidal bacterium. (A) Sporulating cells of B. thuringiensis. The arrowheads point to the crystalline parasporal body adjacent to the spore formed in each cell. (B) Crystals produced by the HD1 isolate of B. thuringiensis subsp. kurstaki (Btk). The three CrylA proteins co-crystallize during synthesis to form the bipyramidal crystal, whereas the Cry2A protein crystallizes separately, forming a quasi-cuboidal crystal. (C) Surface structure of a single bipyramidal crystal revealing the packing arrangement of Cry1 molecules. (D) Transmission electron micrograph through a Btk parasporal body. Note the Cry2A (P2) crystal is typically embedded within the CrylA (Pl) crystal. This arrangement apparently evolved to enhance activity of this isolate and others with a similar arrangement of insecticidal inclusions.

dipteran (flies) or coleopteran (beetles) insects, or nematodes. Cyt proteins are toxic to mosquito and black fly larvae, and a few beetle species, and occur typically in what are referred to as mosquitocidal subspecies, such as B. thuringiensis subsp. israelensis (Bti). In addition, Bt can also produce other types of insecticidal proteins during vegetative growth, referred to as vegetative insecticidal proteins (VIPs). At present, most commercial Bt crops are based on Cry proteins, although VIPs are now being used in combination with these to construct "stacked" crops, i.e., crops that contain multiple insecticidal and other proteins. No Cyt proteins are currently used in Bt crops.

The role of these insecticidal proteins in the biology of B. thuringiensis is to paralyze certain types of insects after crystals and spores have been ingested so that the latter can germinate and colonize the insect body, which provides and excellent source of nutrients for reproduction. As with most pathogens, Bt has optimal hosts, such as the larvae of many species of grain-feeding moths of the lepidopteran family (Pyralidae). In these, the bacterium invades the body and proliferates extensively, yielding millions of spores per larva. In less-than-optimal hosts, even though the insecticidal proteins can paralyze and often kill larvae — providing that appropriate Cry receptors are present on midgut epithelial cells — reproduction is less extensive.

3.2.1 Systematics, Nomenclature, and Insecticidal Protein Diversity

The insecticidal crystals formed by Cry and Cyt proteins are the principal characteristic that differentiates B. thuringiensis from B. cereus as well as other species of the B. cereus group. As far as is known, most if not all Cry and Cyt proteins are encoded on plasmids present in Bt, i.e., not on the bacterial chromosome.3 Thus, if these plasmids are lost from a strain or are deliberately eliminated by plasmid curing, the resulting strain would be identified as B. cereus. Several earlier as well as recent studies of the phenotypic and genomic properties of B. thuringiensis and B. cereus provide strong evidence that the former is essentially the latter species bearing plasmids encoding endotoxins.9-11 Despite this, B. thuringiensis is still considered a valid species due to a combination of tradition and practical value, and this is unlikely to change (at least in the near future).

In some studies, it has been suggested that B. cereus, B. thuringiensis, and B. anthracis are all members of the same species.12 Although there is ample evidence that B. cereus and B. thuringiensis are members of the same species, the idea that B. anthracis is a member of this same species is not supported by the evidence. Among other features, though, it has been shown that Bt plasmids can be transmitted to and replicate in B. cereus; the two plasmids that encode the toxins of B. anthracis do not occur naturally in Bt or B. cereus and do not have parasporal bodies containing Bt Cry proteins that have been found in B. anthracis. This implies that there are probably natural barriers, currently not understood, to plasmid mobilization and transmission that exist among these species, and probably that "cross-talk" between their different toxin-encoding plasmids and chromosomal genes of their normal host species controls toxin production. At present, this supports considering B. anthracis as a species different from B. cereus and B. thuringiensis.

As a species, Bt is subdivided into more than 70 subspecies, which are not based on insecticidal protein complements or target spectrum but, rather, on the antigenic properties of the flagellar (H) antigen.13 Each new isolate that bears a flagellar antigen type that differs detectably from the others in immunological assays is assigned a new H antigen serovariety number and subspecific name. Thus, for example, of those used commonly in bacterial insecticides, there are four main subspecies (Table 3.1): Bacillus thuringiensis subsp. kurstaki (H 3a3b3c) and B. thuringeinsis subsp. aiza-wai (H 7) used against lepidopteran pests; B. thuringiensis subsp. israelensis (H 14) used against mosquitoes and black fly larvae; and B. thuringiensis subsp. morrisoni strain tenebrionis (H 8a8b), used against certain coleopteran pests, such as the Colorado potato beetle (Leptinotarsa decemlineata).

Target spectrum is frequently correlated with flagellar serovariety (also referred to as serotype). However, the correlation is far from absolute because this identification is not based on insecticidal protein complements, which can vary markedly even within the same subspecies/serovariety. For example, within the subspecies/ serovariety B. thuringiensis subsp. morrisoni (H 8a8b), isolates exist that are toxic to lepidopteran, dipteran, or coleopteran larvae. Because the plasmid complements, and therefore the insecticidal protein complements, can vary within a subspecies/ serovariety, isolates that have distinctive target spectra and/or toxicity are typically given specific designations.

The most widely used Bt isolate in agriculture and forestry, for example, is the HD1 isolate of B. thuringiensis subsp. kurstaki (H 3a3b3c), which is toxic to many different important lepidopteran pests of field and vegetable crops, as well as many forest pests. This isolate, the active ingredient of commercial products such as DiPel

TABLE 3.1

Important Subspecies of Bacillus thuringiensis Used in Bacterial Insecticides

Subspecies/ Serovarietya kurstaki

H-Antigen

3a3b3c

Major Endotoxin Proteins (Mass in kDa)

mornsomb israelensis

Insect Spectrum (Target Croup)

Lepidoptera

Cry1Aa (133), Cry1Ab (131)e Cry1Ac (133)e, Cry2Aa (72)c

7 Cry1Aa (133), Cry1Ab (131) Lepidoptera

Cry1Ca (135), Cry1D (133) 8a8b Cry3Aa (73)e Coleoptera

14 Cry4Aa (134), Cry4Ab (128) Dipterad

Cry11Aa (72), Cyt1Aa (27) a Data from Lecadet et al., Updating the H-antigen classification of Bacillus thuringiensis, J. Appl.

Microbiol, 86, 660, 1999. b Strain tenebrionis, commonly referred to as B. t. subsp. tenebrionis or san diego. c Also toxic to larvae of nematoceran dipterans (e.g., mosquitoes and black flies). d Only toxic to species of the dipteran suborder Nematocera (e.g., mosquitoes and black flies). e Used to construct insect-resistant transgenic crops.

and Foray 48B, produces four major endotoxin proteins (CrylAa, CrylAb, CrylAc, and Cry2Aa), which together account for its broad target spectrum. Of relevance to the safety of transgenic crops, this isolate has served as the genetic source of the Cry proteins used most extensively in Bt crops to control lepidopteran pests, specifically, CrylAc used in Bt cotton and CrylAb used in certain types of Bt corn. However, there are numerous other isolates of this subspecies that produce fewer Cry proteins, for example, HD73, which has a plasmid complement that only produces a single Cry protein, CrylAc. As a result, HD73 has a very limited target spectrum. Alternatively, the ONR 60A isolate of B. thuringeinsis subsp. israelensis and the PGl4 isolate of B. thuringiensis subsp. morrisoni both bear a large, l28-kb plasmid (pBtoxis) that encodes a different set of insecticidal proteins, namely Cry4Aa, Cry4Ba, CryllAa, and CytlA, responsible for the mosquitocidal activity of these isolates."

Regardless of the subspecies/serovariety, the only way to be certain of the target spectrum of a new isolate is to conduct bioassays against a range of insect species and combine this information with the cloning, sequencing, and analysis of genes encoding the insecticidal proteins. In general, each subspecies/serovariety has the capability of encoding a range of Cry genes and, correspondingly, many of these genes occur in different subspecies/serovarieties.

This brief background demonstrates how the insecticidal protein complexity can vary within and among various isolates and subspecies of B. thuringiensis. Suffice it to say that there is enormous variation among the plasmids and insecticidal protein complements that occur among the collections of Bt isolates, now estimated to be about l00,000, grouped together under the more than 70 subspecies of B. thuringiensis. As noted above, more than l20 different types of genes encoding Cry proteins, and at least l2 different types of genes encoding Cyt proteins, have been cloned and sequenced.

As a group, the Cry protein family contains considerable diversity, enabling Bt strains to kill different hosts under appropriate conditions (Table 3.2). Most Cry proteins are of the Cryl type, a class of molecules in which the overwhelming majority are toxic to lepidopteran insects.2,3 These molecules are typically in the range of l33-l50 kDa in mass. Cry2 molecules, depending on the specific protein, are also toxic to lepidopterans, but some, such as Cry2Aa, are toxic to both lepidopterans and dipterans (mosquito larvae, in this case). Cry2 molecules are generally about half the mass, i.e., 65 kDa, of Cryl proteins, and in essence are naturally truncated molecules consisting of the N-terminal half of the latter (the portion of the molecule that contains the active protein). Cry3 proteins are similar in mass to Cry2 proteins, but they are only insecticidal to coleopteran insects. The other major Cry type used in bacterial insecticides, the Cry4 proteins, are, like Cryl molecules, in the l35-kDa range but are toxic to nematoceran dipterans, the suborder that contains the mosquitoes and black flies. Phylogenetic studies indicate that all of the above Cry types evolved over millions of years from the same ancestral molecule, the diversity in host spectra being selected for when mutant strains wound up in the midguts of insect species belonging to different orders.

Although each type of Cry protein has a limited target spectrum — typically lepidopteran, dipteran, or coleopteran insects, or nematodes — the target spectrum of a specific protein (e.g., CrylAc) is always much narrower than the type as a whole.

TABLE 3.2

Toxicity of Bt Cry Proteins to First Instars of Various Pest Insect Species3

LC50 in ng/cm2 of diet or waterbc

TABLE 3.2

Toxicity of Bt Cry Proteins to First Instars of Various Pest Insect Species3

LC50 in ng/cm2 of diet or waterbc

Cry

Tobacco

Tobacco

cotton

Yellow Fever

Colorado Potato

Protein11

Hornworm

Budworm

Leafworm

Mosquito

Beetle

CrylAa

5.2

90

> l350

> 5000

> 5000

CrylAb

8.6

l0

> l350

> 5000

> 5000

CrylAc

5.3

l.6

> l350

> 5000

> 5000

CrylC

> l28

> 256

l04

> 5000

> 5000

CryllA

> 5000

> 5000

> 5000

60

> 5000

Cry3A

> 5000

> 5000

> 5000

> 5000

< 200

a Tobacco hornworm (Manduca sexta), tobacco budworm (Heliothis virescens), cotton leafworm (Spodoptera littoralis), yellow fever mosquito (Aedes aegypti), Colorado potato beetle (Leptinotarsa decimlineata). Modified from Hofte, H. and Whitely, H.R., Insecticidal crystal proteins of Bacillus thuringiensis, Microbiol. Rev., 53, 242, 1989.133 b Values > 5000 indicate a lack of toxicity at high doses; doses equivalent to field applications rates that would not be economical. Lack of toxicity at these rates illustrates the high degree of insect specificity characteristic of Cry proteins. c For insecticidal activity of other Cry proteins, see www.glfc.cfs.nrcan.gc.ca/bacillus. d For updates of Cry taxonomy, see www.lifesci.sussex.ac.uk/home/Neil_Crickmore/Bt/.

a Tobacco hornworm (Manduca sexta), tobacco budworm (Heliothis virescens), cotton leafworm (Spodoptera littoralis), yellow fever mosquito (Aedes aegypti), Colorado potato beetle (Leptinotarsa decimlineata). Modified from Hofte, H. and Whitely, H.R., Insecticidal crystal proteins of Bacillus thuringiensis, Microbiol. Rev., 53, 242, 1989.133 b Values > 5000 indicate a lack of toxicity at high doses; doses equivalent to field applications rates that would not be economical. Lack of toxicity at these rates illustrates the high degree of insect specificity characteristic of Cry proteins. c For insecticidal activity of other Cry proteins, see www.glfc.cfs.nrcan.gc.ca/bacillus. d For updates of Cry taxonomy, see www.lifesci.sussex.ac.uk/home/Neil_Crickmore/Bt/.

In addition to the spectrum, the toxicity of each Cry protein within a type can vary significantly from one insect species to another, even in cases where insect species are closely related. For example, two different lepidopteran species of the family Noctuidae can differ markedly in their sensitivity to CrylAc, from being highly sensitive (Heliothis virescens) to being essentially nonsensitive (Spodoptera exigua). For this reason, different Cry proteins are used in different Bt crops for insect resistance, i.e., to provide a high level of control for different insect pest species, or two different Cry proteins would be used in the same crop to control different pest species. Examples of the latter are new corn varieties that produce both CrylA proteins for control of lepidopteran larvae and Cry3 proteins for control of corn rootworms, which are coleopteran insects.

During the last decade, the number of Cry protein types has expanded dramatically as a result of the search for new proteins with novel target spectra. The current list of Cry proteins includes more than 50 different holotypes (see http://www.lifesci. sussex.ac.uk/home/Neil_Crickmore/Bt/), Cry1 through Cry 50; most, but not all, of which are related phylogenetically, i.e., appear to have evolved from the same molecule. In addition to Cry protein types, there are nine holotypes of Cyt proteins. These proteins have a mass in the range of 26-28 kDa and are phylogenetically unrelated to Cry proteins, i.e., they share no significant degree of amino acid identity/similarity and have a spectrum of activity limited to certain dipteran and coleopteran species. Data on the toxicity of the most important Cry and Cyt proteins can be found at http://www.glfc.cfs.nrcan.gc.ca/bacillus, a web site maintained by the Canadian Forest Service.

3.2.2 Toxicity and Mode of Action

Knowing the precise complement of insecticidal proteins produced by a specific isolate of B. thuringiensis can go along way toward explaining its toxicity and lethality to a particular insect or nematode species. However, several Bt components other than endotoxins contribute to the activity of a particular isolate against a specific insect species (Table 3.3). Owing to the overwhelming interest in Cry proteins, most of these other factors have received relatively little attention. Among the most important of these are the spore, P-exotoxin, antibiotics such as zwittermicin, vegetative insecticidal proteins (VIPs), phospholipases, chitinases, and various proteases. In some target insects, Cry proteins alone are sufficient to intoxicate larvae by destroying enough midgut epithelial cells to allow the alkaline midgut juices to flow into the hemolymph and raise the blood pH, which causes paralysis and cessation of feeding.15 This is typically followed by death in a few days due to either the toxicity of the insecticidal protein(s) alone, as in the case of mosquitoes and black flies, or a combination of these and infection and colonization of the larva by B. thuringiensis, the latter being the typical cause of death in most lepidopteran species.15

For example, in highly susceptible species such as grain-feeding lepidopteran larvae of the family Pyralidae, as paralysis sets in due to intoxication by Cry proteins, Bt spores germinate in the midgut as the alkaline pH (8-10) drops to around 7. The resulting vegetative cells invade the larva, colonize the hemolymph and other tissues, and reproduce to an extent that the cadaver becomes virtually a pure culture of Bt (Figure 3.2). In other species, such as most Spodoptera species, death appears to depend on a combination of factors. These include Cry proteins, VIPs, P-exotoxin (a competitive inhibitor of mRNA polymerase, which is not allowed in bacterial insecticides in the United States and Europe because it is teratogenic at high levels), and various enzymes that help break down midgut barriers to infection by Bt and other bacteria present in the midgut lumen. In some species, such as larvae of the gypsy moth (Lymantria dispar), naturally occurring midgut bacteria may also be the cause of death,16 but this appears to be an exception to the rule. Pests like these are not natural hosts for Bt, as there is no benefit to intoxicating such insects if there is no tissue colonization and reproduction for this bacterium. These species are sensitive to Cry proteins because their midgut characteristics, including pH and toxin receptors, are the same as or similar to those of bona fide Bt hosts.

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