An Overview on the Crystal Toxins from Bacillus thuringiensis

Strains of Bacillus thuringiensis (Bt) are known to produce crystalline proteins (δ-endotoxins) concomitantly with sporulation during their stationary phase of growth, which are demonstrated as lethal to lepidopeterous, coleopeterous and dipterous insects in addition to mites, nematodes, protozoa and flukes. Upon ingestion, the δ-nascent endotoxin is an inactive protoxin complex of (Cry alone or Cry and Cyt toxins together) high molecular mass, which is cleaved upon ingestion into the active component proteins at the high alkaline environments in the digestive tract of these agricultural pests. Conventionally, Bt-crystals are being produced employing submerged or liquid fermentation techniques in commercial media, but recently many workers have used solid-state fermentation strategy for the enhanced production of Bt-toxin at low cost. Apart from δ-endotoxin, some isolates of Bt produce another class of insecticidal small molecules called β-exotoxin (thuringiensin), which may be harmful to humans. Moreover, resistance to Bt developed in various target pest is yet another concern for Bt-industry. Following a brief introduction, this review addresses various toxins produced by various strains of Bt, Bt production media and media formulations with emphasis to solid-state fermentation, general structure of Cry toxin, its mode of action, target pests, bioassay, resistance to Bt toxins and resistance management. Briefly, this review would provide the readers an overview on the general aspects of Bt toxin, its general structure and mechanism of action.


Introduction
Biological pesticide is one of the most promising alternatives over conventional chemical pesticides, which offers less or no harm to the environments and biota.Bacillus thuringiensis (commonly known as Bt) is an insecticidal Gram-positive spore-forming bacterium producing crystalline proteins called delta-endotoxins (δ-endotoxin) during its stationary phase or senescence of its growth.Bt was originally discovered from diseased silkworm (Bombyx mori) by Shigetane Ishiwatari in 1902.But it was formally characterized by Ernst Berliner from diseased flour moth caterpillars (Ephestia kuhniella) in 1915 [1].The first record of its application to control insects was in Hungary at the end of 1920, and in Yugoslavia at the beginning of 1930s, it was applied to control the European corn borer [2].Bt, the leading biorational pesticide was initially characterized as an insect pathogen, and its insecticidal activity was ascribed largely or completely to the parasporal crystals.It is active against more than 150 species of insect pests.Bt is normally marketed (as a mixture of dried spores and toxin crystals) under various trade names worldwide for controlling many plant pests, mainly caterpillars belonging to Lepidoptera (represented by butterflies and moths), mosquito larvae and a few others including unconventional targets like mites.The share of Bt products in agrochemical (fungicide, herbicide and insecticide) market is about only 1%.The first commercial Bt product was produced in 1938 by Libec in France, but the product was used only for a very short time due to World War II, and then in the USA in the 1950s [3].The toxicity of Bt culture lies in its ability to produce the crystalline protein, this observation led to the development of bioinsecticides based on Bt for the control of certain insect species among the orders Lepidoptera, Diptera, and Coleoptera [3][4][5].Nowadays, Bt isolates are reported also active against certain nematodes, mites and protozoa [6].It is already a useful alternative or supplement to synthetic chemical pesticide for applications in commercial agriculture, forest management, and mosquito control, and also a key source of genes for transgenic expression to transfer pest resistance in plants.Due to this economic interest, numerous approaches have been developed to enhance the production of Bt bioinsecticides.The insecticidal activity of Bt is known to depend not only on the activity of the bacterial culture itself, but also on abiotic factors, such as the medium composition and cultivation strategy.

Bt Toxins
Bt produces one or more types of parasporal crystalline proteins (called δ-endotoxins) concomitantly with sporulation.Cryastal (Cry) or cytolytic (Cyt) proteins singly or in their combination constitute the δ-endotoxins [7].Cry proteins are parasporal crystalline inclusions produced by Bt that exhibit experimentally verifiable toxic effect to a target organism or have significant sequence similarity to a known Cry protein.Cyt proteins are also parasporal inclusions exhibiting hemolytic (cytolitic) activity with obvious sequence similarity to a known Cyt protein.These toxins are highly specific to their target insect, but innocuous to humans, vertebrates and plants, and are completely biodegradable [8].These crystalline proteins are mainly encoded by extra-chromosomal genes located on the plasmids.The parasporal crystalline proteins produced during the stationary (senescence) phase of its growth cycle account for 20% -30% of the dry weight of the cells of this phase [9].Expression of most Cry genes (e.g., cry1Aa, cry 2A, cry 4A, etc.) are well regulated in the sporulation phase of growth.Studies have shown that several Cry proteins-when expressed in either E. coli or B. subtilis-expressed as 130 to 140 kDa protoxin complex molecules that retain their biological activity.More than 200 types of endotoxin gene have been cloned from various strains of Bt, and sequenced so far.The plasmid profiles of most Bt strains are rather complex, with molecular weight varying from 2 to 200 kb and the number of plasmids ranging from 1 to 10 in most strains [10].The self-assembly of these 130 kDa proteins is spontaneous, mediated primarily by the C-terminus of the protein.Their cysteine-rich carboxyl terminus is highly conserved among lepidopteran-specific Cry proteins, which generates a number of disulfide bridges that allow good crystal packing and also protects the toxin from the attack of various proteases.Commercial insecticides derived from Bt have a long history of successful use in the biocontrol of insect pests [11,12].Many studies examined the composition and methods of preparation of nutrient media for entomopathogenic bacteria [13,14].Chromsomal insertion of Cry gene may enhance the production of δ-endotoxins in Bt strains [15].Erythromycin resistance may affect the sporulation processes in Bt and B. subtilis [16,17].Most Bacillus strains produce a mixture of structurally different insecticidal crystal proteins (Cry proteins), which are encoded by different Cry genes which target different insect orders (Table 1).Each of these proteins may contribute to the insecticidal spectrum of a strain that makes it selectively toxic to a wide variety of insects belonging to the Lepidoptera, Coleoptera, Diptera, Hymenoptera and Mallophaga, as well as to other invertebrates [11,[18][19][20].Bt strains are able to produce exoenzymes, such as proteases and α-amylases [21].Apart from δ-endotoxin, some isolates of Bt produce another class of insecticidal small molecules called β-exotoxin, the common name for which is thuringiensis [22].

General Structure of Cry Toxin
The major component of crystals toxic to lepidopteron larvae is a 130 kDa protein (protoxin), which upon cleavage in the insect yields the functional (insecticidal) proteins of lower molecular weight; very often the crystal formed is an assemblage of many proteins [59].A Bt isolate (Soil-47) showed distinct bands of 32.1 and 34.6 kDa.
The band corresponding to 32.1 kDa protein could arise from the type Cry1 and/or Cry 4 gene, while the other (34.6 kDa) protein is possibly encoded by the type Cyt gene [60].An unexpected finding was that a 20 kb heterologous DNA fragment was found intimately associated with the crystals from Btk HD73, The DNA is not susceptible to nuclease attack unless the protoxin is removed or proteolyzed to toxin.The active toxin is not associated with DNA; however, evidence was obtained which indicated that the DNA was involved in the generation of toxin from the crystal protein.[61].
Structure determination of Bt toxins remains one of the most important tools in understanding and improving the utility of these proteins.Crystal structure of Cry III A has been published first [62] and several others are now available.Xia et al. [63] predicted the first theoretical model of the three dimensional (3D) structure of a Cry (Cry 5Ba) toxin by homology modeling on the structure of the Cry1Aa toxin, which is specific to Lepidopteran insects.The three-domain structure of CryIIIA consisted of the following: an α-helical barrel (domain I) which shows some resemblance to membrane-active or sporeforming domains of other toxins [64]; a triangular prism of "Greek key" beta sheets (domain II); and a β-sheet jelly-roll fold (domain III) [62].Members of this 3-domain Cry family are used worldwide for insect control, and their mode of action has been characterized in some details [8].

Mode of Action
Mode of action of δ-endotoxin involves several events that must be completed several hours after the ingestion in order to lead to insect death.Following ingestion of the inactive protoxin, the crystals are solubilized by the alkaline conditions in the insect midgut and are subse-quently proteolytically converted into a toxic core fragment [65].This activated toxin binds to receptors located on the apical microvillus membranes of epithelial midgut cells.For Cry1A toxins, at least four different binding sites have been described in different lepidopteran insects: a cadherin-like protein (CADR), a glycosylphosphatidylinositol (GPI)-anchored aminopeptidase-N (APN), a GPIanchored alkaline phosphatase (ALP) and a 270 kDa glycoconjugate [66].Cry toxins interact with specific receptors located on the host cell surface and are activated by host proteases following receptor binding, which would result is in the formation of a pre-pore oligomeric structure that is insertion competent.In contrast, Cyt toxins directly interact with membrane lipids and insert into the membrane.Recent evidence suggests that Cyt synergizes or overcomes resistance (for instance, to mosquitocidal-Cry proteins) by functioning as a Cry-membrane bound receptor [8].
Once activated, the endotoxin binds to the gut epithelium and causes cell lysis by the formation of cationselective channels, which leads to death.The activated region of the δ-endotoxin is composed of three distinct structural domains: an N-terminal helical bundle domain involved in membrane insertion and pore formation; a beta-sheet central domain involved in receptor binding; and a C-terminal beta-sandwich domain that interacts with the N-terminal domain to form a channel.After binding, toxin adopts a conformation suitable for allowing its insertion into the cell membrane.Subsequently, oligomerization occurs, and this oligomer forms a pore or ion channel induced by an increase in cationic permeability within the functional receptors contained on the brush borders membranes.This allows the free flux of ions and liquids, causing disruption of membrane transport and cell lysis leading to insect death [65,55].The complete nature of this process is still elusive.
Differences in the extent of solubilization sometimes explain differences in the degree of toxicity among Cry proteins [67].A reduction in solubility is speculated to be one potential mechanism for insect resistance [68].Cry3A protein may be necessary for the solubilization of toxins in the midgut of insects [69].Most recently, two models were proposed for the action of crystal proteins i.e., the sequential binding model and signaling pathway model [70].

Target Pests
It is well documented that many insects are susceptible to the toxic activity of Bt; of them, lepidopterans have exceptionally been well studied, and many toxins have shown activity against them [66].The order Lepidoptera encompasses majority of susceptible species belonging to agriculturally important families such as Cossidae, Ge-lechiidae, Lymantriidae, Noctuidae, Pieridae, Pyralidae, Thaumetopoetidae, Tortricidae, and Yponomeutidae [71].A novel crystal proteins exhibiting insecticidal activity against lepidopterans has been reported from Bt strains [72].
Dipterans are also important target pests, and many of them are highly susceptible to Bt. Discovery of novel strains of Bt containing parasporal crystal proteins having pesticidal properties against whiteflies, aphids, leaf hoopers, and possibly other sucking insects of agronomic importance extended the potential applications of this bacterium.However, the novel toxic activities found in these novel strains are not limited only to insects, as some of them produce crystals with activity against nematodes, protozoans, flukes, collembolans, mites and worms, among others [66].

Production Media and Media Formulations
Indeed, large quantities of spores with high insecticidal activity are required for practical applications.This means that while handling Bt as bioinsecticide, a high spore count is not sufficient to ensure toxicity, but it is necessary to reach high δ-endotoxin titers.One of the most underreported aspects of Bt is that of the production and formulation, although there are certain work existed in connection with Bt growth on several synthetic or complex media [73].There are several formulations of media proposed by different authors.Our group explored the efficacy of various raw agricultural products as supplement to LB for enhancing the toxin production, and found potato flour as an efficient supplement to commercial Luria-Bertani (LB) medium [7].To develop a costeffective process for the production of Bt-based insecticide, it is imperative to cultivate the bacterial strain in a nutrient rich medium to obtain the highest yields of spore-crystal complexes.Conventionally, Bt-crystals are being produced employing submerged or liquid fermentation (SmF) techniques, but recently many workers use nutrient-rich waste water or sludge from various treatment plants as the medium for the production of Bt-toxin [74].Solid-state fermentation: Solid-state fermentation (SSF) has been developed in eastern countries over many centuries, and has enjoyed broad application in these regions to date [75].The term SSF denotes cultivation of microorganisms on solid, moist substrates in the absence of a free aqueous phase (water).There are several advantages for SSF; for example, high productivities, extended stability of products and low production costs, which say much about such an intensive biotechnological application.With increasing progress and application of rational methods in engineering, SSF will reach higher levels regarding standardization and reproducibility in future.This can make SSF as the preferred technique in the special fields of application such as the productions of enzymes and secondary metabolites, especially foods and pharmaceuticals [76].
Different production media and media compositions can change either the relative toxicity against several target insects or the insecticidal potency of products obtained from the same Bt strains [77].According to Farrera et al., [78], media with different composition showed changes in crystal production, i.e. different amounts of Cry proteins produced per spore would vary.The ingredients in the media affect the rate and synthesis of the different δ-endotoxins and also the size of the crystals produced.Using barley as the carbon source, Amin [79] developed a cost-effectively protocol for the mass production of Bt.
Several media based on complex substrates such as corn steep liquor [80], peptones [81] blackstrap molasses and Great Northern White Bean concentrate [82], or LB supplemented with agricultural products [7] have been found efficient for Bt bioinsecticide production.Various investigators modified such commercial media by supplementing it with mineral nutrients or various salts, i.e., enriched medium.Zouari et al. [73] showed that Bt subspecies kurstaki produced 1 g/L of δ-endotoxin in 4.5 g/L total dry biomass in a complex liquid medium, in which the sugar was replaced by gruel hydrolysate.A mixture of extracts from potato and Bengal gram or bird feather and de-oiled rice bran or wheat bran, chickpea husk and corncob was used to cultivate Bt israelensis and found that the mosquitocidal activity of the crude toxin was higher than that produced in the conventional medium [83].Valicente et al. [84] used LB medium supplemented with various salts, and agricultural by-products like soybean flour (0.5%) and liquid swine manure (4%) to increase Bt biopesticide production by SmF, which resulted in 1.18 g/L dry cell mass.Zhuang et al. [85] also claimed that they have purified δ-endotoxin (up to 7.14 mg/g medium) by one step centrifugation from wastewater sludge-based medium, however they did not provide any physical evidence for the purified crystals.From these reports, it seems that maximum yield of Bt toxin could be attained is 3.6 g/L [84] in SmF or 7.14 g/Kg medium in SSF [85], where they did not provide the actual cost effect.

Bioassay
Well-designed studies under confined conditions required to understand the effect of Bt toxins on different organisms.It is considered that Bt toxins also to be toxic to lepidopeterous, coleopeterous and dipterous insects in addition to mites, nematodes, protozoa and flukes [18,19,40].These proteins are usually thought to act only on the actively feeding larvae of susceptible species by a mechanism involving consumption and proteolytic processing of the protein followed by binding to, and the lysis of midgut epithelial cells.It was found that proteolytically activated insecticidal crystal proteins significantly reduced the lifespan of adult Heliothis virescens and Spodoptera exigua at concentrations of 500 μg/ml, but not 167 or 25 μg/ml at their assay conditions [86].Bt crystal proteins showed in vitro cytotoxicity against human cancer cells and leukemic T cells [87].Interestingly, Xu et al. [88] demonstrated that the Bt crystal proteins can protect plasmodium-infected mice from malaria.Moreover, non-conventional targets such as Caenorhabditis elegans (nematode) has been demonstrated for the first time [89].
Toxins of Btk strain HD1 have widely been used to control the forest pests such as gypsy moth, spruce bud worm, the pine procesionary moth, the European pine shoot moth and the nun moth [90].Direct feeding of crude pellet containing Bt-toxin [91], pollen diet formulation [92] are the normal mode of applications being practiced in entomotoxicity assays.A different feeding strategy was successfully used for the bioassay of A. guerreronis, in which the dried solid fermented powder directly brushed on the infested coconut buttons [7,21].Many authors used surfactants like BIT (1,2-benzisothiazolin-3-one), one of the inertingredients in Foray 48 B(a Btk formulation); the siloxane (organosilicone) Triton-X-100, Tween 20 and Latron CS-7 are some surfactants for Btk formulations [93].
The mortality rate of Thaumetopoea solitaria on the application of Btk toxin has been demonstrated by Er et al. [94].Purified Btk toxin inhibited the growth of monarch larvae, but did not cause mortality [95].The LC 50 value of Btk was found to be 398.1 μg/ml against caterpillars of Arctornis submarginata [96].Toxicity of several formulations of Btk to beet armyworm (Spodoptera exigua) was determined using neonate larvae in a diet incorporation bioassay.
Probit analysis (LC 50 ) has been used by many authors for ascertaining the efficacy of various Bt formulations.For instance, Yashodha and Kuppusamy [97] successfully used dipping method for testing the efficacy of Btk formulation in Tween 20 on Brinjal.Gobatto et al. [98] used various concentrations of spore suspension of Bt for estimating the probit value on mosquito and a moth.Payne et al. [40] employed artificial feeding assay for Two-spotted spider mite (T.urticae), a related mite to E. orientalis with different feeding regime.They fed the mite with 5 mg spray-dried powder of Bt broth (a mixture of pores, crystals, cellular debris) in 1 ml sucrose (10%) containing preservatives and surfactant.
Possible use of Bt preparation (Dipel 2X) as a substitute for chemical insecticides (Lannate and Hostathion) was evaluated against two major pests of potato crop, Agrotis sp. and Spodoptera exigua.The toxicity studies of Bt to four instars larvae of diamondback moth, Plutella xylostella (L.) suggested that Bt could be an important agent for the control of larval instars of Plutella xylostella.[42].The Bt diet suppressed the growth of the four mite species such as Acarus siro L., Tyrophagus putrescentiae (Schrank), Dermatophagoides farinae Hughes, and Lepidoglyphus destructor (Schrank) via feeding tests [58].

Resistance to Bt Toxins
Laboratory-selected strains: In the past, it was believed that insects would not develop resistance to Bt toxins, since Bt and insects have coevolved.Starting in the mid-1980s, however, a number of insect populations of several different species with different levels of resistance to Bt crystal proteins were obtained by laboratory selection experiments, using either laboratory-adapted insects or insects collected from wild populations [99,100].Examples of laboratory-selected insects resistant to individual Cry toxins include the Indian mealmoth (Plodia interpunctella), the almond moth (Cadra cautella), the Colorado potato beetle (Leptinotarsa decemlineata), the cotton leafworm (Spodoptera littoralis), the beet armyworm (S. exigua), etc. [19].Given the multiple steps in processing the crystal to an active toxin, it is not surprising that insect populations might develop various means of resisting intoxication.It is important, however, to keep in mind that selection in the laboratory may be very different from selection that occurs in the field.Insect populations maintained in the laboratory presumably have a considerably lower level of genetic diversity than field populations.Several laboratory experiments to select for Bt resistance in diamondback moths failed, although the diamondback moth is the only known insect reported so far to have developed resistance to Bt in the field [19].It is possible that the genetic diversity of the starting populations was too narrow and thus did not include resistance alleles.In the laboratory, insect populations are genetically isolated; dilution of resistance by mating with susceptible insects-as observed in field populations-is excluded [19].
In addition, the natural environment may contain factors affecting the viability or fecundity of resistant insects, i.e., factors excluded from the controlled environment of the laboratory.Resistance mechanisms can be associated with certain fitness costs that can be deleterious under natural conditions [101].Natural enemies, such as predators and parasites can influence the development of resistance to Bt by preferring either the intoxicated, susceptible or the healthy resistant insects.In the former case, one would expect an increase in resistance development, while in the latter, natural enemies can help to retard resistance development to Bt. Never-theless, selection experiments in the laboratory are valuable because they reveal possible resistance mechanisms and make genetic studies of resistance possible.
Field-selected strains: The first case of field-selected resistance to Bt was reported from Hawaii, where populations of diamondback moth showed different levels of susceptibility to a formulated Bt product (Dipel).Populations from heavily treated areas proved more resistant than those populations treated at lower levels, with the highest level of resistance at 30-fold [100].
Resistance to Btk products and resulting failure in diamondback moth control has resulted in the extensive use of Bt subsp.aizawai-based insecticides in certain locations [19].Insects in two colonies from Hawaii showed up to a 20-fold resistance to Cry1Ca, compared to several other colonies, including one obtained earlier from the same location, as well as moderately high resistance to Cry1Ab and Btk-based formulations [19].
A Malaysian strain simultaneously highly resistant to the kurstaki and the aizawai subspecies was apparently mutated in several loci [31].A Cry1Ab resistance allele associated with reduced binding to brush border membrane vesicles receptors was partially responsible for resistance to both subspecies.Genetic determinants responsible for subspecies kurstaki-specific and subspecies aizawai-specific resistance segregated separately from each other and from the Cry1Ab resistance allele in genetic experiments [103].
After less than 2 decades of intensive use of Btk in crucifer agriculture, resistant insects have evolved in numerous geographically isolated regions of the world, and subspecies aizawai resistance is beginning to appear even more rapidly.
Defying the expectations of scientists monitoring transgenic crops such as corn and cotton that produce insecticidal proteins derived from Bt, target insect pests have developed little or no resistance to Bt crops thus far, according to US Department of Agriculture-funded scientists.These findings suggest that transgenic Bt crops could enjoy more extended, more profitable commercial life cycles and that the measures established to mitigate resistance before the crops were introduced are paying off [104].
Evolution of resistance in pests can reduce the effectiveness of insecticidal proteins from Bt produced by transgenic crops.Field outcomes support theoretical pre-dictions that factors delaying resistance include recessive inheritance of resistance, low initial frequency of resistance alleles, abundant refuges of non-Bt host plants and two-toxin Bt crops deployed separately from one-toxin Bt crops.The results imply that proactive evaluation of the inheritance and initial frequency of resistance are useful for predicting the risk of resistance and improving strategies to sustain the effectiveness of Bt crops [105].

Resistance Management
Resistance management strategies try to prevent or diminish the selection of the rare individuals carrying resistance genes and hence to keep the frequency of resistance genes sufficiently low for insect control [106,107].
Proposed strategies include: the use of multiple toxins (stacking or pyramiding), crop rotation, high or ultrahigh dosages, and spatial or temporal refugia (toxin-free areas).Retrospective analysis of resistance development does support the use of refugia [99].Experience with transgenic crops expressing cry genes grown under different agronomic conditions is essential to define the requirements of resistance management.In transgenic plants, selection pressure could be reduced by restricting the expression of the crystal protein genes to certain tissues of the crop (those most susceptible to pest damage) so that only certain parts of the plant are fully protected, the remainder providing a form of spatial refuge.It has been proposed that cotton lines in which Cry gene expression is limited to the young bolls may not suffer dramatic yield loss from Heliothis larvae feeding on other plant structures, since cotton plants can compensate for a high degree of pest damage [108].
Another management option is the rotation of plants or sprays of a particular Bt toxin with those having another toxin type that binds to a different receptor.A very attractive resistance management tactics is the combination of a high-dose strategy with the use of refugia [19].

Conclusion
Development of resistance to Bt toxin is one of the concerns of Bt-based agroindustry.It was expected that resistance would be developed in transgenic crops such as corn and, interestingly target insect pests have developed little or no resistance to these Bt crops.It suggests that transgenic Bt crops could enjoy more extended, more profitable commercial life cycles and the measures established to mitigate resistance before the crops were introduced are paying off.Nevertheless, making Bt toxin at low cost for the farmers, especially in the developing and underdeveloped countries remains one of the major challenges, wherein SSF offers great potentials.In fact, the ill effects of the exotoxin, thuringiensin from Bt on humans (and other animals too) are a growing concern.