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Bacillus thuringiensis is a bacterium that produces insecticidal products upon sporulation. These products have a very narrow host range and are easily degraded by the environment. This makes B. thuringiensis an environmental friendly solution for the control of pests of among others: maize, cotton and potatoes and for the control of mosquitoes and blackflies. To improve on some shortcomings of the insecticidal spray, transgenic Bt-crops have been developed. It is a big concern that insects may become resistant to B. thuringiensis.
Bacillus thuringiensis is a gram-positive, rod-shape, spore-forming bacterium. It is found in soil, in grain storage plants and on plant surfaces (de Maagd et al., 1999). During sporulation, the bacterium forms a parasporal inclusion body, the crystal. The crystal is made of Cry and Cyt proteins that could be insecticidal (Côté, 2007).
In the previous century sudden collapse disease was a problem in silkworms (Bombyx mori) at rearing facilities in Japan. Isiwate isolated the bacterium, a 'bacillus' responsible for the disease. A decade later, Beliner, isolated a similar bacterium responsible for the death of Mediterranean flour moth (Anagasta kuehniella) larvae. He named the bacterium Bacillus thuringiensis, because the flour in which the larvae were found was from Thuringia, Germany. In 1938 the first B. thuringiensis-based commercial formulation, Sporeine, was produced in France (Côté, 2007).
The first large scale commercial product, ThuricideTM was produced in the 1950's (de Maagd et al., 1999). From 1956 to 1958 the FDA (Food and Drug Administration) granted a temporary exemption from tolerance and the PRD issued an experimental permit for initial field work and safety studies. These included tests on human volunteers. Bacillus thuringiensis was used on vegetables and feed crops. The FDA granted full exemption from tolerance on 14 April 1960, allowing B. thuringiensis to be used commercially on food and forage crops (Heimpel, 1971).
By the late 1960's about 150 B. thuringiensis strains had been isolated, with 9 serotypes known. Through the years B. thuringiensis was isolated from mosquito larvae (1970's in Israel) and yellow mealworms (1980's), showing that B. thuringiensis doesn't just have anti-Lepidopteran activity, but kills Dipterans and Coleopterans to. By the late 1990's it was estimated that 60 000 different B. thuringiensis strains was kept in collections around the world. Some patents claim activities against Hymenoptera, Homoptera, Hemiptera and Mallophaga orders, although it has not been described in scientific literature. There have also been reports of anti-Nematoda and anti-Protozoa activities (Côté, 2007).
Two B. thuringiensis protein genes have been identified with insecticidal properties: The "crystal" cry genes and "cytolytic" cyt genes (Bobrowski, 2002). B. thuringiensis produce more than one toxin, one of which is the most important insecticidal, Î´-endotoxin (Falcon 1971). The bacterium also produces Lecithinase C, a liable exotoxin, and a thermostable exotoxin. The mode of action of the liable exotoxin is not known. It could be produced by B. thuringiensis or be a product of fermentation. The thermostable exotoxin is not a true toxin, but rather a toxic metabolite. Its role in the pathogenesis of B. thuringiensis is uncertain, but it could be a nucleotide-like compound that damages the ATP metabolism of hosts (Lysenko and KuÄera, 1971). Cyt proteins are haemolytic (Gao et al., 2008).
Figure 2: Phase contrast photomicrograph of B. thuringiensis (Norris, 1971).
When spores are ingested by a susceptible host, they germinate in the midgut. After germination the spores enter the hemocoel where they multiply rapidly and destroy certain tissues. This is called "septicemia". Upon germination the crystal is also released (Falcon 1971) and dissolves in the alkaline midgut. Inactive Cry proteins, protoxins, are released and activated by specific proteases. The activated toxin (Î´-endotoxin) binds to receptors on epithelial midgut cell membranes (de Maagd et al., 1999), causing conformational changes that allow insertion of the toxin into the membrane. The toxins then cause the formation of ion channels that leads to colloid osmotic cell lysis and the death of the larva (Côté, 2007; de Maagd et al., 1999). The crystals kill or weaken the larva so that the bacteria can produce septicemia. After death the host disintegrates and the spores are released in the environment (Falcon 1971).
Figure 3: The primary structure of three Bacillus thuringiensis
Cry-proteins. The proteases in the insect's gut remove the
grey N- and C-terminal ends. This activates the toxin.
Transgenic plants produce C-terminally trimmed or full
length protoxins (de Maagd et al., 1999).
Figure 4: The structure of Cry1Aa. The blue domain inserts the
toxin into the membrane and forms the pores.
Receptor recognition and binding is directed by the
green and red domains (de Maagd et al., 1999).
The genes needed for crystal formation are carried on a plasmid (Côté, 2007).
Different B. thuringiensis Varieties
Bacillus thuringiensis has several varieties. The susceptibility of insects to the toxin varies between varieties and within varieties (Falcon 1971). The crystal formed in the spores is antigenic and more than one antigen can be distinguished, differentiating between different serotypes (Lysenko and KuÄera, 1971). B. thuringiensis serotypes that do not produce the crystal protein produce the heat-stable toxin (Bond et al., 1971).
B. thuringiensis strains produce crystals that vary in size, shape and number. There are 5 Bt crystals (Bobrowski, 2002):
Cry 4 & Cyt Proteins: Amorphous and composite
Uses for Bacillus thuringiensis pesticides
B. thuringiensis insecticides can be used to control Lepidopteran, Dipteran and Coleopterin pests (Côté, 2007). It is widely used as pesticides on crops and in forestry, and for control of mosquitoes and blackflies (Gough et al., 2005).
Different Cry proteins are lethal to different insects. Similarly, certain variants are used to control certain pests.
Toxicity of Cry proteins (Gao et al., 2008):
Cry1Aa, Cry1Ab, and Cry1Ac: Plutella xylostella and Heliothis armigera
Cry3, Cry7, and Cry8: Some Coleoptera species
Cry2: Lepidoptera and/or Diptera
Cry1Ba: Sheep Blowfly (Gough et al., 2005)
Toxicity of variants:
B. thuringiensis var. israelensis: Mosquitoes and some Diptera (Yamamoto et al., 1983).
B. thuringiensis var. kurstaki: Numerous Lepidoptera (butterflies, moths) families in agriculture and forestry (Mohammedi et al., 2006).
B. thuringiensis var. tenebrionis: Larvicide for some Coleoptera families, particularly for Colorado potato beetle (Mohammedi et al., 2006).
Bacillus thuringiensis can also be used as a feed additive to inhibit muscoid fly production in the faeces of poultry and cattle. The thermostable exotoxin is the active factor (Laird, 1971).
Honey bees have been used to transfer Bacillus thuringiensis var. kurstaki to flowers of sunflower, achieving the control of the sunflower moth, along with an increase in the pollination and seed-set (Jyoti and Brewer, 1999).
B. thuringiensis can be used in combination with other agents to increase effectiveness (Falcon 1971):
B. thuringiensis + a nuclear polyhedrosis virus = enhanced control of alfalfa caterpillar.
B. thuringiensis + a nuclear polyhedrosis virus = effective for control of Malacosoma fragile.
B. thuringiensis + Beauveria bassiana (Fungus) = more effective to control P. brassicae.
B. thuringiensis activate latent infection of granulosis virus in Hyphantria cunea.
Table 1: Certain pest successfully controlled by B. thuringiensis (Falcon, 1971).
Tent caterpillar, Fall webworm, Eye-spotted budmoth, Winter moth, Tentiform leaf miner, Apple rust mite, Red-banded leaf roller, Eastern tent caterpillar, Ermine moth, Web moth, Codling moth.
Pieris brassicae, P.rapae, Plutella maculipennis.
Leafworm, Leaf perforator, Pink ballworm.
Grape leaf borer.
Phytophagous mite, Peach twig borer.
Western yellow-striped armyworm.
On defined media containing glucose and salts Bacillus thuringiensis grows slowly with poor sporulation. With additional amino acids or casein hydrolysate with balanced nitrogen:carbon content this can be improved. The optimum growth temperature for sporulation is 30Â°C (Norris, 1971).
B. thuringiensis must first be produced by fermentation where it multiplies as vegetative cells and enters sporulation. During sporulation the parasporal crystals are produced and they are released after cell lysis. To transform the bacterium for commercial exploitation, the biomass is harvested (dried or not) which is the technical grade unformulated powder or paste that serves as the active ingredient for insecticidal products. The formulated products can contain microbial growth inhibitors, surfactants, spreader-stickers, UV protectants, etc. The formulation increases persistence, improves the shelf-life of the product, enhance deposition and affiance and makes it easier to apply. The final products can be dried wettable powders, dusts, pellets, etc. or liquid aqueous or emulsifiable oils (Côté, 2007).
The formulations are diluted for agricultural use. They are designed for long-term storage, easy usage and to be compatible with spraying equipment. For use on forest pests the formulation is highly concentrated (Côté, 2007).
Problems with insecticidal sprays
B. thuringiensis is unstable in the environment (de Maagd et al., 1999). 24 hours after spraying the insecticidal activity is reduced by 50%. This is due to UV degradation of the crystals, rainfall, etc. (Côté, 2007)
When Bacillus thuringiensis spores were placed on a membrane filter or glass slide and exposed to sunlight, it was found that natural sunlight exposure for 30 minutes inactivated 50% of spores and an exposure time of 60 minutes inactivated 80% of the spores (Falcon 1971).
Due to the degradation by UV light it is necessary for several applications of the pesticide during the growing season. This increases the costs to control the pests. Improvements have been made, but it stays the biggest problem (de Maagd et al., 1999).
To protect the chemical pesticide from environmental degradation or to provide controlled release of the active ingredient, it is coated with synthetic polymers. For B. thuringiensis, it is covered in UV-absorbing compounds, clay, chemical polymer matrices and various starches and biopolymer matrices (Côté, 2007).
B. thuringiensis pesticides can't penetrate tissues, and consequently can't reach insects in all parts of the plant (de Maagd et al., 1999). The effect that Bacillus thuringiensis will have as control agent depends on the behaviour of the larvae. If the larvae move deep into the whorl of the plant and avoid exposure to the spores, the pesticide will not be successful (Falcon 1971). The sprays only kill insects that come within direct contact of the crystals. Therefore it is not effective for sap-sucking insects, insects that bore into the plant tissues, insects that occur on the roots or piercing insects (de Maagd et al., 1999).
Ignoffo determined the optimal, minimum and maximum temperatures for larval death in pink bollworm. At 40.1Â°C the best results were observed and no septicemia was detected at 51.2Â°C and 8.6Â°C (Falcon 1971).
Often, crops are inhabited by numerous different pests. B. thuringiensis pesticides have very narrow specificity and a single B. thuringiensis product may not be able to control all of them (de Maagd et al., 1999).
To continue the use of Bacillus thuringiensis as a biocontrol agent, it is crucial to prevent the evolution of resistance in the target pest populations. Bacillus thuringiensis has been used for over 30 years and since then only the diamondblack moth (Plutella xylostella) has been reported to be resistant outside of the laboratory. In 2000 a survey showed that Trichoplusia ni populations in greenhouses in British Columbia evolved resistance against B. thuringiensis. The greenhouse environment could have contributed to the resistance because it protects the pest and pathogen from the elements (Janmaat 2007).
The Bacillus thuringiensis endotoxin is not toxic to mammals. The toxin is degraded to an atoxic state by the stomach pepsin which has a pH optimum of 2 (Cooksey 1971).
Zhou et al. (2008) found that small amounts of B. thuringiensis used as biopesticides occurred in food and beverages. Although it was showed that B. thuringiensis is safe for humans, mammals, birds and non-target insects, further investigation is still done to study the impact of B. thuringiensis on vertebrates.
The question is whether there could develop Bacillus thuringiensis strains which are virulent to vertebrates. Pathogens that occur naturally in an environment are relatively stable. To become virulent to vertebrates several genetic changes need to occur for that pathogen to become successful in the new environment. Due to the low rate at which genetic change occur, the probability of this switch is very low and highly unlikely (Heimpel, 1971).
To improve on the problems experienced with chemical pesticides, transgenic plants were developed. Transgenic plants were created that express the crystal proteins continuously. The toxin is therefore protected against degradation and will kill boring larvae (de Maagd et al., 1999).
In the first attempts to produce B. thuringiensis transgenic plants, the cry genes were transformed into tobacco and tomato plants using Agrobacterium tumefaciens. After tests, the B. thuringiensis genes were modified for optimal expression in plants (de Maagd et al., 1999).
Potatoes were the first plants that were engineered to express the Cry3A protein. This was done in 1995 to protect the plants from Colorado potato beetles. The Cry1Ac protein was expressed in Bt-cotton that was developed in 1996. This protein protects the crops against cotton and pink bollworm, but most especially against tobacco budworm. This transgenic plant increased yield with 14% and reduced insecticide use with 1.1 million litres. Since 1996 several companies produced Bt-maize that express the Cry1Ab protein (de Maagd et al., 1999).
Figure 5: Producing transgenic B. thuringiensis crops.
Potential Problems with Transgenic Plants
There is a concern that the predators and parasites that feed on the pests could be killed to. But this is unlikely due to the high specificity of B. thuringiensis (de Maagd et al., 1999).
The other concern is that resistance to the toxins may arise due to the continuous exposure to the toxin. Resistance may be due to the loss of receptors for binding in the midgut or due to increased protease activity. No resistance to Bt plants have been showed yet (de Maagd et al., 1999).
Approaches to prevent resistance (de Maagd et al., 1999)
Transform more than one gene into plants. The proteins expressed should have different modes of action.
Engineer plants that only produce the toxins from parts of the crop that is not economically important (tissue specific promoters).
Engineer plants that only express the protein after a certain amount of damage is caused by the pest (inducible promoters).
Crop rotation with non-transgenic plants will slow down the development of resistance.
Much molecular knowledge and further research needs to be done to prevent resistance. This should be done as quickly as possible. If resistance is developed for transgenic Bt-plants a very valuable defence mechanism will be lost. Farmers may not see the necessity to rotate their crops. It might lead to a decrease in their yield, but they should be informed on the consequences if crops are not rotated.
The role of B. thuringiensis in the environment is still unclear. It has been proposed that it protects plants against insect pests or that it is a normal soil bacterium that lives in insects or nematodes. Epizoonotics caused by B. thuringiensis is rare in the environment, and is mostly reported in rearing facilities. Further, B. thuringiensis is often found in environments where insects do not occur (Côté, 2007).
The role of the crystal is also still unclear. It might give the bacterium the advantage to replicate in susceptible insects, but some crystal proteins are specific cytocidal against human cancer cells. It has been proposed that the crystal might harbour nutrients to favour germination of the spore (Côté, 2007).
Bacillus thuringiensis and Cancer
The Cry31Aa1 protein shows cytocidal activity against some human cancer cells. St-Jean-sur-Richelieu is investigating further with a patent application filed (Côté et al., 2005).
Bacillus thuringiensis is a save and environmental friendly solution for the control of insects. It is a brilliant example of how microorganisms can be used in everyday life. The most important concern in the use of Bt products in preventing insects to become resistant to it. With careful and considerate use it is preventable. There are still possibilities for improving the Bt-crops by transforming more than one gene from different strains into the plants to produce a plant resistant to more insects. With more studies on the exact role of B. thuringiensis in the environment many more strains may be found with insecticidal activities against other insects.