In recent years, there has been increased use of biopesticide throughout the world. Biopesticides are mainly pesticides derived from the living organism like bacteria, animals, etc. The interest in GM crops and biopesticides has increased due to the increased laws and regulation on the use of chemical pesticides like organophosphates. Due to the ill effects of chemicals pesticides and fertilizers on human health, other organisms and soil the various governments have made policies which restrict the use of chemical pesticides and enhance use of biopesticides[3,4]. So the use of broad spectrum pesticides is reduced and use of narrowly targeted pesticides is favoured. The advancement of recombinant DNA technology has made possible to use various toxins from organisms to be expressed and cloned in plants which acts as resistance against various pests and diseases. One of the major biopesticide is derived from soil bacterium Bacillus thuringiensis. 90% biopesticides used today are derived from Bacillus thuringiensis .
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This bacterium was first discovered in Japan[6,8].Its was first experimented of crops like tobacco and tomato.It is a gram positive bacterium. It's isolated throughout the world from soil, animals, insect, many habitats, etc. Bacillus thuringiensis is a spore forming bacteria which produces crystal proteins which have insecticidal properties during sporulation[1,6,7]. These crystalline proteins are made up of protoxin subunits called δ endotoxins. The Bt toxin gene inserted into genetically modified crops shows resistance against lepidopterans, dipterans and coleopterans[1,6].
Various genes encoding protoxin form have been discovered of Bacillus thuringiensis. They are mainly classified as cry proteins. The cry proteins are of various groups cry I, cry II, cry III. The each cry protein has various other forms like cry IA, cry IIA, etc[6,4]. The various forms of cry proteins have structural similarity in them and hence functional similarity. The classification of these genes is also related to biological activity of their product. The classification is also based on various host range the toxins act on like cry I acts against Lepidopterans ,cry II act against lepidoterans and Dipterans, cry III acts against Coleopterans and cry IV act against Dipterans. The toxins are modified so that they can be soluble and more active in the plant cell[1,6].
Bacillus thuringiensis spores and crystals  File:Bacillus thuringiensis.JPG
Mode of action:
The transfer of Bt toxin into the plant like maize, cotton, rice, tomato ,etc and its expression is necessary for the protection of plants against various pests. As the toxins remain into the plant tissue they are environmentally safe for the use. The plants to be modified using Bt toxin are modified using regulatory sequences like introns, promoters and enhancers. The toxicity occurs in the midgut region of the insect.[4,6]
The digestive system of the insect is compartmentalized and so are the digestive enzymes. There is high alkaline Ph present in the digestive system of the insect so that a process of digestion takes place without disruption. This high alkaline Ph prevents any toxin from inactivating digestive process. When the food is taken by insect it first enters the foregut and is broken down into small pieces which can pass through peritrophic membrane. This membrane prevents entry of large food molecules and large toxin molecules from the plants and hence protects the midgut region and also acts as membrane through which nutrients are absorbed. This membrane is made up of proteins and mucopolysaccharides. The digested food is then absorbed by the epithelium present in the midgut region. The goblet cells in lumen helps in maintain high Ph and K+ concentration which helps in absorption of nutrients.
After ingestion of Bt toxin it takes various steps like solubilisation of crystal protein, activation of toxin and insertion of toxin into midgut epithelium to kill the insect. The Bt should be in small spore form to effectively enter the midgut region [1,4,8]. The spores are protected by crystalline proteins from the alkaline Ph. The δ endotoxins play an important role in Bt mediated toxicity. The cry proteins when solubilised are in inactive form i.e in protoxin form. The cry proteins need to be proteolytically processed which is done with the help of help high alkaline Ph and digestive enzymes present in the gut region.
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The activation of toxin is done by gut proteases which cut down proteins in to smaller poly peptides and by removal of both carboxyl terminal and amino acid terminal ends of the proteins. After activation cry toxins bind to its receptors, amino peptidase acts as receptors for the toxins. Then the toxin binds to various sites on cell surface which prevents resistance against all the cry proteins[1,4,7].
The cry proteins consist of three domains which help in insecticidal activity of toxin. Domain I consist of 3 α helical domains arranged around the central helix. This domain helps in membrane insertion. Domain II consist of 3 β folded sheets which are symmetrical and helps in recognition and binding of toxin. Domain III consist of 2 symmetrical β folded sheets which helps in recognition, binding and pore formation and channel specificity. Domain II and III both have different conformation [1,4,7,8].
Domain structure of Cry protein
The toxin attaches in two steps the first one is reversible which deals with attachment factors onto membrane and second step is irreversible which deals with insertion of toxin in membrane. After attachment of toxin of toxin to membrane domain I rearranges itself and inserts all the 3 helices in to membrane. Domain 3 attaches itself to the inserted helices and the insertion of toxin becomes irreversible. This causes formation of pores and this pores form the K+ selective ion channel. The formation of K+ causes the sudden physiological changes in the gut region. The K+ gradient in epithelial cells is disrupts and Ph balance of midgut lumen is also disrupted[1,4,8]. It increases the hemolymph K+ concentration which causes cell lysis of the toxin attached cells due to decrease in Ph and osmotic lysis. This then helps in germination of spores due to presence of neutral environment and nutrients from dead cells. This causes paralysis of gut region. Most of the insects die due to the starvation due to paralysis of digestive system and feeding inhibition rather than toxin itself.
Schematic representation of Mode of action of Bt toxin 
Resistance against Bt toxins in lepidopterans, dipterans and coleopterans :
The resistance against Bt toxin is mainly achieved by 3 methods :
Altered binding of cry protein to binding site, this is done by decreased affinity to binding site or reduction in number of binding sites.
Alteration in proteolytic processing which causes decreased rate of activation of toxin and increased rate of toxin degradation.
Instant and rapid regeneration of cells affected by toxins which prevents digestive paralysis and feeding inhibition.
Effect on environment and health:
The safety assessment of Bt crops have been attracted various researchers, farmers and consumers. There were many issues raised against the safety of Bt crops. So wide range of research have been conducted on the Bt crops. Several researches were conducted on hazards of genetically modified crops and it was concluded that Bt crops had no environmental hazard. It has been found that Bt toxin released in soil had no effect on organism like earthworms, nematodes or other plants present in soil as plants cannot uptake Bt toxin from soil[2,5]. The effect of Bt products and spores on mammals were studied in laboratory and found that there was no possible infectivity or toxicity seen. There has been no adverse effect seen on human health on consumption or coming in contact with Bt products. It has been found that Bt toxin does not have any adverse effect on human or marine organisms even if present in water consumed by humans or other water bodies. Bt products are being widely used in United states America and Canada every year from last several decades.
There should be more research done on the Bt toxins for improvement of current Bt products as it is limited to very few class of pest as compared to other broad spectrum pesticides and because of its short residual activity. There should be more study conducted on development various resistance mechanism in case pests get resistance against the Bt toxin over a period of time. The advantage of Bt toxin also include low mammalian and non targeted impact. Overall Bt toxin is cost effective and environment friendly method to solve the global pest problems. Discovery of new method to target specific insect which is does not have adverse effect on human health and environment using recombinant microorganism and proteomic technology should be developed.
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