Bacillus thuringiensis

Bacillus thuringiensis (or Bt) is a Gram-positive, soil-dwelling bacterium, commonly used as a biological pesticide. B. thuringiensis also occurs naturally in the gut of caterpillars of various types of moths and butterflies, as well on leaf surfaces, aquatic environments, animal feces, insect-rich environments, and flour mills and grain-storage facilities.[1][2] It has also been observed to parasitize other moths such as Cadra calidella—in laboratory experiments working with C. calidella, many of the moths were diseased due to this parasite.[3]

Bacillus thuringiensis
Spores and bipyramidal crystals of Bacillus thuringiensis morrisoni strain T08025
Scientific classification
Domain: Bacteria
Phylum: Firmicutes
Class: Bacilli
Order: Bacillales
Family: Bacillaceae
Genus: Bacillus
Species:
B. thuringiensis
Binomial name
Bacillus thuringiensis
Berliner 1915
Gram stain of Bacillus thuringiensis under 1000 × magnification

During sporulation, many Bt strains produce crystal proteins (proteinaceous inclusions), called δ-endotoxins, that have insecticidal action. This has led to their use as insecticides, and more recently to genetically modified crops using Bt genes, such as Bt corn.[4] Many crystal-producing Bt strains, though, do not have insecticidal properties.[5] The subspecies israelensis is commonly used for control of mosquitoes[6] and of fungus gnats.[7]

Taxonomy and discovery

In 1902, B. thuringiensis was first discovered in silkworms by Japanese sericultural engineer Ishiwatari Shigetane (石渡 繁胤). He named it B. sotto,[8] using the Japanese word sottō (卒倒, 'collapse'), here referring to bacillary paralysis.[9] In 1911, German microbiologist Ernst Berliner rediscovered it when he isolated it as the cause of a disease called Schlaffsucht in flour moth caterpillars in Thuringia (hence the specific name thuringiensis, "Thuringian").[10] B. sotto would later be reassigned as B. thuringiensis var. sotto.[11]

In 1976, Robert A. Zakharyan reported the presence of a plasmid in a strain of B. thuringiensis and suggested the plasmid's involvement in endospore and crystal formation.[12][13] B. thuringiensis is closely related to B. cereus, a soil bacterium, and B. anthracis, the cause of anthrax; the three organisms differ mainly in their plasmids.[14]:34–35 Like other members of the genus, all three are aerobes capable of producing endospores.[1]

Tubulin was long thought to be specific to eukaryotes. More recently, however, several prokaryotic proteins have been shown to be related to tubulin.[15][16][17][18]

Subspecies

There are several dozen recognized subspecies of B. thuringiensis. Subspecies commonly used as insecticides include B. thuringiensis subspecies kurstaki (Btk), subspecies israelensis (Bti) and subspecies aizawa.

Mechanism of insecticidal action

Upon sporulation, B. thuringiensis forms crystals of proteinaceous insecticidal δ-endotoxins (called crystal proteins or Cry proteins), which are encoded by cry genes.[19] In most strains of B. thuringiensis, the cry genes are located on a plasmid (cry is not a chromosomal gene in most strains).[20][21][22]

Cry toxins have specific activities against insect species of the orders Lepidoptera (moths and butterflies), Diptera (flies and mosquitoes), Coleoptera (beetles) and Hymenoptera (wasps, bees, ants and sawflies), as well as against nematodes.[23][24] Thus, B. thuringiensis serves as an important reservoir of Cry toxins for production of biological insecticides and insect-resistant genetically modified crops. When insects ingest toxin crystals, their alkaline digestive tracts denature the insoluble crystals, making them soluble and thus amenable to being cut with proteases found in the insect gut, which liberate the toxin from the crystal.[20] The Cry toxin is then inserted into the insect gut cell membrane, paralyzing the digestive tract and forming a pore.[25] The insect stops eating and starves to death; live Bt bacteria may also colonize the insect, which can contribute to death.[20][25][26] The midgut bacteria of susceptible larvae may be required for B. thuringiensis insecticidal activity.[27]

It has been shown that a small RNA called BtsR1 can silence the Cry toxin when outside the host by binding to the RBS site of the Cry5Ba toxin transcript and inhibiting its expression. The silencing results in increased ingestion by C. elegans and is relieved inside the host, resulting in host death.[28]

In 1996 another class of insecticidal proteins in Bt was discovered: the vegetative insecticidal proteins (Vip; InterPro: IPR022180).[29][30] Vip proteins do not share sequence homology with Cry proteins, in general do not compete for the same receptors, and some kill different insects than do Cry proteins.[29]

In 2000, a novel subgroup of Cry protein, designated parasporin, was discovered from non-insecticidal B. thuringiensis isolates.[31] The proteins of parasporin group are defined as B. thuringiensis and related bacterial parasporal proteins that are not hemolytic, but capable of preferentially killing cancer cells.[32] As of January 2013, parasporins comprise six subfamilies: PS1 to PS6.[33]

Use of spores and proteins in pest control

Spores and crystalline insecticidal proteins produced by B. thuringiensis have been used to control insect pests since the 1920s and are often applied as liquid sprays.[34] They are now used as specific insecticides under trade names such as DiPel and Thuricide. Because of their specificity, these pesticides are regarded as environmentally friendly, with little or no effect on humans, wildlife, pollinators, and most other beneficial insects, and are used in organic farming;[24] however, the manuals for these products do contain many environmental and human health warnings,[35][36] and a 2012 European regulatory peer review of five approved strains found, while data exist to support some claims of low toxicity to humans and the environment, the data are insufficient to justify many of these claims.[37]

New strains of Bt are developed and introduced over time[38] as insects develop resistance to Bt,[39] or the desire occurs to force mutations to modify organism characteristics[40] or to use homologous recombinant genetic engineering to improve crystal size and increase pesticidal activity[41] or broaden the host range of Bt and obtain more effective formulations.[42] Each new strain is given a unique number and registered with the U.S. EPA[43] and allowances may be given for genetic modification depending on "its parental strains, the proposed pesticide use pattern, and the manner and extent to which the organism has been genetically modified".[44] Formulations of Bt that are approved for organic farming in the US are listed at the website of the Organic Materials Review Institute (OMRI)[45] and several university extension websites offer advice on how to use Bt spore or protein preparations in organic farming.[46][47]

Use of Bt genes in genetic engineering of plants for pest control

The Belgian company Plant Genetic Systems (now part of Bayer CropScience) was the first company (in 1985) to develop genetically modified crops (tobacco) with insect tolerance by expressing cry genes from B. thuringiensis; the resulting crops contain delta endotoxin.[48][49] The Bt tobacco was never commercialized; tobacco plants are used to test genetic modifications since they are easy to manipulate genetically and are not part of the food supply.[50][51]

Bt toxins present in peanut leaves (bottom dish) protect it from extensive damage caused to unprotected peanut leaves by lesser cornstalk borer larvae (top dish).[52]

Usage

In 1995, potato plants producing CRY 3A Bt toxin were approved safe by the Environmental Protection Agency, making it the first human-modified pesticide-producing crop to be approved in the US,[53][54] though many plants produce pesticides naturally, including tobacco, coffee plants, cocoa, and black walnut. This was the 'New Leaf' potato, and it was removed from the market in 2001 due to lack of interest.[55] For current crops and their acreage under cultivation, see genetically modified crops.

In 1996, genetically modified maize producing Bt Cry protein was approved, which killed the European corn borer and related species; subsequent Bt genes were introduced that killed corn rootworm larvae.[56]

The Bt genes engineered into crops and approved for release include, singly and stacked: Cry1A.105, CryIAb, CryIF, Cry2Ab, Cry3Bb1, Cry34Ab1, Cry35Ab1, mCry3A, and VIP, and the engineered crops include corn and cotton.[57][58]:285ff

Corn genetically modified to produce VIP was first approved in the US in 2010.[59]

In India, by 2014, more than seven million cotton farmers, occupying twenty-six million acres, had adopted Bt cotton.[60]

Monsanto developed a soybean expressing Cry1Ac and the glyphosate-resistance gene for the Brazilian market, which completed the Brazilian regulatory process in 2010.[61][62]

Kenyans examining insect-resistant transgenic Bt corn

Safety studies

The use of Bt toxins as plant-incorporated protectants prompted the need for extensive evaluation of their safety for use in foods and potential unintended impacts on the environment.

Dietary risk assessment

Concerns over the safety of consumption of genetically-modified plant materials that contain Cry proteins have been addressed in extensive dietary risk assessment studies. While the target pests are exposed to the toxins primarily through leaf and stalk material, Cry proteins are also expressed in other parts of the plant, including trace amounts in maize kernels which are ultimately consumed by both humans and animals.[63]

Toxicology studies

Animal models have been used to assess human health risk from consumption of products containing Cry proteins. The United States Environmental Protection Agency recognizes mouse acute oral feeding studies where doses as high as 5,000 mg/kg body weight resulted in no observed adverse effects.[64] Research on other known toxic proteins suggests that toxicity occurs at much lower doses, further suggesting that Bt toxins are not toxic to mammals.[65] The results of toxicology studies are further strengthened by the lack of observed toxicity from decades of use of B. thuringiensis and its crystalline proteins as an insecticidal spray.[66]

Allergenicity studies

Introduction of a new protein raised concerns regarding the potential for allergic responses in sensitive individuals. Bioinformatic analysis of known allergens has indicated there is no concern of allergic reactions as a result of consumption of Bt toxins.[67] Additionally, skin prick testing using purified Bt protein resulted in no detectable production of toxin-specific IgE antibodies, even in atopic patients.[68]

Digestibility studies

Studies have been conducted to evaluate the fate of Bt toxins that are ingested in foods. Bt toxin proteins have been shown to digest within minutes of exposure to simulated gastric fluids.[69] The instability of the proteins in digestive fluids is an additional indication that Cry proteins are unlikely to be allergenic, since most known food allergens resist degradation and are ultimately absorbed in the small intestine.[70]

Ecological risk assessment

Ecological risk assessment aims to ensure there is no unintended impact on non-target organisms and no contamination of natural resources as a result of the use of a new substance, such as the use of Bt in genetically-modified crops. The impact of Bt toxins on the environments where transgenic plants are grown has been evaluated to ensure no adverse effects outside of targeted crop pests.[71]

Persistence in environment

Concerns over possible environmental impact from accumulation of Bt toxins from plant tissues, pollen dispersal, and direct secretion from roots have been investigated. Bt toxins may persist in soil for over 200 days, with half-lives between 1.6 and 22 days. Much of the toxin is initially degraded rapidly by microorganisms in the environment, while some is adsorbed by organic matter and persists longer.[72] Some studies, in contrast, claim that the toxins do not persist in the soil.[72][73][74] Bt toxins are less likely to accumulate in bodies of water, but pollen shed or soil runoff may deposit them in an aquatic ecosystem. Fish species are not susceptible to Bt toxins if exposed.[75]

Impact on non-target organisms

The toxic nature of Bt proteins has an adverse impact on many major crop pests, but ecological risk assessments have been conducted to ensure safety of beneficial non-target organisms that may come into contact with the toxins. Widespread concerns over toxicity in non-target lepidopterans, such as the monarch butterfly, have been disproved through proper exposure characterization, where it was determined that non-target organisms are not exposed to high enough amounts of the Bt toxins to have an adverse effect on the population.[76] Soil-dwelling organisms, potentially exposed to Bt toxins through root exudates, are not impacted by the growth of Bt crops.[77]

Insect resistance

Multiple insects have developed a resistance to B. thuringiensis. In November 2009, Monsanto scientists found the pink bollworm had become resistant to the first-generation Bt cotton in parts of Gujarat, India - that generation expresses one Bt gene, Cry1Ac. This was the first instance of Bt resistance confirmed by Monsanto anywhere in the world.[78][79] Monsanto responded by introducing a second-generation cotton with multiple Bt proteins, which was rapidly adopted.[78] Bollworm resistance to first-generation Bt cotton was also identified in Australia, China, Spain, and the United States.[80] Additionally, the Indian mealmoth, a common grain pest, is also developing a resistance since B. thuringiensis has been extensively used as a biological control agent against the moth.[2] Studies in the cabbage looper have suggested that a mutation in the membrane transporter ABCC2 can confer resistance to B. thuringiensis.[81]

Secondary pests

Several studies have documented surges in "sucking pests" (which are not affected by Bt toxins) within a few years of adoption of Bt cotton. In China, the main problem has been with mirids,[82][83] which have in some cases "completely eroded all benefits from Bt cotton cultivation".[84] The increase in sucking pests depended on local temperature and rainfall conditions and increased in half the villages studied. The increase in insecticide use for the control of these secondary insects was far smaller than the reduction in total insecticide use due to Bt cotton adoption.[85] Another study in five provinces in China found the reduction in pesticide use in Bt cotton cultivars is significantly lower than that reported in research elsewhere, consistent with the hypothesis suggested by recent studies that more pesticide sprayings are needed over time to control emerging secondary pests, such as aphids, spider mites, and lygus bugs.[86]

Similar problems have been reported in India, with both mealy bugs[87][88] and aphids[89] although a survey of small Indian farms between 2002 and 2008 concluded Bt cotton adoption has led to higher yields and lower pesticide use, decreasing over time.[90]

Controversies
Main article: Genetically modified food controversies

There are controversies around GMOs on several levels, including whether making them is ethical, whether food produced with them is safe, whether such food should be labeled and if so how, whether agricultural biotech is needed to address world hunger now or in the future, and more specifically to GM crops—intellectual property and market dynamics; environmental effects of GM crops; and GM crops' role in industrial agricultural more generally.[91] There are also issues specific to Bt transgenic crops.

Lepidopteran toxicity

The most publicised problem associated with Bt crops is the claim that pollen from Bt maize could kill the monarch butterfly.[92] The paper produced a public uproar and demonstrations against Bt maize; however by 2001 several follow-up studies coordinated by the USDA had asserted that "the most common types of Bt maize pollen are not toxic to monarch larvae in concentrations the insects would encounter in the fields."[93][94][95][96] Similarly, B. thuringiensis has been widely used for controlling Spodoptera littoralis larvae growth due to their detrimental pest activities in Africa and Southern Europe. However, S. littoralis showed resistance to many strains of B. thuriginesis and were only effectively controlled by few strains.[97]

Wild maize genetic mixing

A study published in Nature in 2001 reported Bt-containing maize genes were found in maize in its center of origin, Oaxaca, Mexico.[98] In 2002, paper concluded, "the evidence available is not sufficient to justify the publication of the original paper."[99] A significant controversy happened over the paper and Nature's unprecedented notice.[100]

A subsequent large-scale study in 2005 failed to find any evidence of genetic mixing in Oaxaca.[101] A 2007 study found the "transgenic proteins expressed in maize were found in two (0.96%) of 208 samples from farmers' fields, located in two (8%) of 25 sampled communities." Mexico imports a substantial amount of maize from the U.S., and due to formal and informal seed networks among rural farmers, many potential routes are available for transgenic maize to enter into food and feed webs.[102] One study found small-scale (about 1%) introduction of transgenic sequences in sampled fields in Mexico; it did not find evidence for or against this introduced genetic material being inherited by the next generation of plants.[103][104] That study was immediately criticized, with the reviewer writing, "Genetically, any given plant should be either non-transgenic or transgenic, therefore for leaf tissue of a single transgenic plant, a GMO level close to 100% is expected. In their study, the authors chose to classify leaf samples as transgenic despite GMO levels of about 0.1%. We contend that results such as these are incorrectly interpreted as positive and are more likely to be indicative of contamination in the laboratory."[105]

Colony collapse disorder

As of 2007, a new phenomenon called colony collapse disorder (CCD) began affecting bee hives all over North America. Initial speculation on possible causes included new parasites, pesticide use,[106] and the use of Bt transgenic crops.[107] The Mid-Atlantic Apiculture Research and Extension Consortium found no evidence that pollen from Bt crops is adversely affecting bees.[93][108] According to the USDA, "Genetically modified (GM) crops, most commonly Bt corn, have been offered up as the cause of CCD. But there is no correlation between where GM crops are planted and the pattern of CCD incidents. Also, GM crops have been widely planted since the late 1990s, but CCD did not appear until 2006. In addition, CCD has been reported in countries that do not allow GM crops to be planted, such as Switzerland. German researchers have noted in one study a possible correlation between exposure to Bt pollen and compromised immunity to Nosema."[109] The actual cause of CCD was unknown in 2007, and scientists believe it may have multiple exacerbating causes.[110]

Beta-exotoxins

Some isolates of B. thuringiensis produce a class of insecticidal small molecules called beta-exotoxin, the common name for which is thuringiensin.[111] A consensus document produced by the OECD says: "Beta-exotoxins are known to be toxic to humans and almost all other forms of life and its presence is prohibited in B. thuringiensis microbial products".[112] Thuringiensins are nucleoside analogues. They inhibit RNA polymerase activity, a process common to all forms of life, in rats and bacteria alike.[113]

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See also
An ovitrap collects eggs from mosquitoes. The brown granules in the water are a B. t. israelensis preparation that kills hatched larvae.

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