Advances in Microbiology, 2013, 3, 462-472
http://dx.doi.org/10.4236/aim.2013.35062 Published Online September 2013 (http://www.scirp.org/journal/aim)
An Overview on the Crystal Toxins from
Bacillus thuringiensis
Veloorvalappil N. Jisha, Robinson B. Smitha, Sailas Benjamin*
Enzyme Technology Laboratory, Biotechnology Division, Department of Botany, University of Calicut, Kerala, India
Received July 17, 2013; revised August 17, 2013; accepted August 30, 2013
Copyright © 2013 Veloorvalappil N. Jisha et al. This is an open access article distributed under the Creative Commons Attribution
License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
ABSTRACT
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 com-
mercial 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 fermenta-
tion, 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.
Keywords: Bacillus thuringiensis; δ-Endotoxin; Resistance
1. Introduction
Biological pesticide is one of the most promising alterna-
tives 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 crys-
talline proteins called delta-endotoxins (δ-endotoxin)
during its stationary phase or senescence of its growth. Bt
was originally discovered from diseased silkworm (Bom-
byx 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 Euro-
pean 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 oth-
ers including unconventional targets like mites. The
share of Bt products in agrochemical (fungicide, herbi-
cide 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 con-
trol of certain insect species among the orders Lepidop-
tera, Diptera, and Coleoptera [3-5]. Nowadays, Bt iso-
lates are reported also active against certain nematodes,
mites and protozoa [6]. It is already a useful alternative
or supplement to synthetic chemical pesticide for appli-
cations in commercial agriculture, forest management,
and mosquito control, and also a key source of genes for
*Corresponding author.
C
opyright © 2013 SciRes. AiM

V. N. JISHA ET AL. 463
transgenic expression to transfer pest resistance in plants.
Due to this economic interest, numerous approaches have
been developed to enhance the production of Bt bioinsec-
ticides. 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.
2. Bt Toxins
Bt produces one or more types of parasporal crystalline
proteins (called δ-endotoxins) concomitantly with sporu-
lation. 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 pro-
duced 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 com-
plex 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 mo-
lecular 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 con-
served among lepidopteran-specific Cry proteins, which
generates a number of disulfide bridges that allow good
crystal packing and also protects the toxin from the at-
tack of various proteases. Commercial insecticides de-
rived from Bt have a long history of successful use in the
biocontrol of insect pests [11,12]. Many studies exam-
ined the composition and methods of preparation of nu-
trient media for entomopathogenic bacteria [13,14]. Chro-
msomal insertion of Cry gene may enhance the produc-
tion 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 mix-
ture 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, Dip-
tera, Hymenoptera and Mallophaga, as well as to other
invertebrates [11,18-20]. 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-exo-
toxin, the common name for which is thuringiensis [22].
Table 1. Endotoxin produc ing Bac illi and tar get or ganisms.
Bacillus spp. Target pest Reference
B. laterosporus &
Brevibacillus laterosporus
Musca domestica and
Aedes aegypti [25]
B. sphaericus
& Bt israelensis Culex quinquefasciatus, C. pipiens, C. tarsalis [26-28]
Bt
Acyrthosiphon pisum,
Aedes aegypti,
Autographa californica ,
Cacyreus marshalli,
Epinotia aporema,
Lobesia botrana,
Manduca sexta,
Manduca sexta,
Pectinopho gossypiella,
Pieris brassicae,
Plutella xylostella,
Rhizoglypus robini
Spodoptera exigua,
Spodoptera frugiperda
[29-43]
Bt berliner Lygus hesperus [44]
Bt BRL 43
First instar larvae of cotton leaf
worm, cotton boll worm and
black cut worm
[45]
Bt finitimus B-1166 VKPM- [46]
Bt galleriae Galleria mellonella L. [47]
Bt H14 Leishmania. major [48]
Bt IPS 78/11 Manduca sexta [49]
Bt israelensis IPS78/11 Lucilia cuprina, L. sericata,
and Calliphora stygia [50]
Bt sotto Cabbage butterfly [51]
Bt kurstaki
Helicoverpa zea; Scrobi
p
alpula
absoluta: Malacosoma neustria
and Lymantria dispar larvae
[52,53]
Btk (serotype H3a,
3b, 3c) strain BNS3 Prays oleae [54]
Btk HD1 &
Btk HD73
Manduca sexta Heliothis
irescens [55]
Bt tolworthi
Spodoptera frugiperda,
Ostrinia nubilalis and
Plutella xylostella, S.exigua
[56,57]
Bt. tenebrionis synanthropic mites [58]

V. N. JISHA ET AL.
464
Beta-exotoxin and the other Bacillus toxins (δ-endo-
toxins) may contribute to the general insecticidal toxicity
of the bacterium to lepidopteran, dipteran, and coleop-
teran insects. Beta-exotoxin is known to be toxic to hu-
mans and almost all other forms of life and, in fact, its
presence is prohibited in Bt products [23]. Engineering of
plants to contain and express only the genes for δ-endo-
toxins avoids the problem of assessing the risks posed by
these other toxins that may be produced in microbial pre-
parations [24].
3. General Structure of Cry Toxin
The major component of crystals toxic to lepidopteron
larvae is a 130 kDa protein (protoxin), which upon cleav-
age in the insect yields the functional (insecticidal) pro-
teins of lower molecular weight; very often the crystal
formed is an assemblage of many proteins [59]. A Bt iso-
late (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 het-
erologous DNA fragment was found intimately associ-
ated with the crystals from Btk HD73, The DNA is not
susceptible to nuclease attack unless the protoxin is re-
moved 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 gen-
eration 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 spore-
forming 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-do-
main Cry family are used worldwide for insect control,
and their mode of action has been characterized in some
details [8].
4. 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 frag-
ment [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 glycosylphosphatidyl-
inositol (GPI)-anchored aminopeptidase-N (APN), a GPI-
anchored alkaline phosphatase (ALP) and a 270 kDa gly-
coconjugate [66]. Cry toxins interact with specific re-
ceptors 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 mos-
quitocidal-Cry proteins) by functioning as a Cry-mem-
brane bound receptor [8].
Once activated, the endotoxin binds to the gut epithe-
lium and causes cell lysis by the formation of cation-
selective 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 allow-
ing its insertion into the cell membrane. Subsequently,
oligomerization occurs, and this oligomer forms a pore or
ion channel induced by an increase in cationic perme-
ability within the functional receptors contained on the
brush borders membranes. This allows the free flux of
ions and liquids, causing disruption of membrane trans-
port 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].
5. Target Pests
It is well documented that many insects are susceptible to
the toxic activity of Bt; of them, lepidopterans have ex-
ceptionally 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-

V. N. JISHA ET AL. 465
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 hoop-
ers, and possibly other sucking insects of agronomic im-
portance extended the potential applications of this bac-
terium. 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].
6. 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 sup-
plement to LB for enhancing the toxin production, and
found potato flour as an efficient supplement to commer-
cial Luria-Bertani (LB) medium [7]. To develop a cost-
effective process for the production of Bt-based insecti-
cide, 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 fermen-
tation (SmF) techniques, but recently many workers use
nutrient-rich waste water or sludge from various treat-
ment 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 cen-
turies, and has enjoyed broad application in these regions
to date [75]. The term SSF denotes cultivation of micro-
organisms on solid, moist substrates in the absence of a
free aqueous phase (water). There are several advantages
for SSF; for example, high productivities, extended sta-
bility of products and low production costs, which say
much about such an intensive biotechnological applica-
tion. 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 spe-
cial fields of application such as the productions of en-
zymes 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 ob-
tained from the same Bt strains [77]. According to Far-
rera 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 ingre-
dients 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 pro-
duction 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 sup-
plementing it with mineral nutrients or various salts, i.e.,
enriched medium. Zouari et al. [73] showed that Bt sub-
species 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 supple-
mented 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 wastewa-
ter 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 ac-
tual cost effect.
7. Bioassay
Well-designed studies under confined conditions re-
quired 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

V. N. JISHA ET AL.
466
mechanism involving consumption and proteolytic proc-
essing of the protein followed by binding to, and the lysis
of midgut epithelial cells. It was found that proteolyti-
cally 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 hu-
man 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 Caenorhab-
ditis 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 formu-
lation [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-benzisothi-
azolin-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
LC50 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 (Spodop-
tera exigua) was determined using neonate larvae in a
diet incorporation bioassay.
Probit analysis (LC50) has been used by many authors
for ascertaining the efficacy of various Bt formulations.
For instance, Yashodha and Kuppusamy [97] success-
fully 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 mix-
ture of pores, crystals, cellular debris) in 1 ml sucrose
(10%) containing preservatives and surfactant.
Possible use of Bt preparation (Dipel 2X) as a substi-
tute 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, Plu-
tella xylostella (L.) suggested that Bt could be an impor-
tant agent for the control of larval instars of Plutella xy-
lostella. [42]. The Bt diet suppressed the growth of the
four mite species such as Acarus siro L., Tyrophagus pu-
trescentiae (Schrank), Dermatophagoides farinae Hughes,
and Lepidoglyphus destructor (Schrank) via feeding tests
[58].
8. 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 sev-
eral 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]. Exam-
ples of laboratory-selected insects resistant to individual
Cry toxins include the Indian mealmoth (Plodia inter-
punctella), the almond moth (Cadra cautella), the Colo-
rado potato beetle (Leptinotarsa decemlineata), the cot-
ton leafworm (Spodoptera littoralis), the beet armyworm
(S. exigua), etc. [19]. Given the multiple steps in proc-
essing 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 differ-
ent from selection that occurs in the field. Insect popula-
tions 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 popu-
lations was too narrow and thus did not include resis-
tance 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 fac-
tors affecting the viability or fecundity of resistant in-
sects, i.e., factors excluded from the controlled environ-
ment of the laboratory. Resistance mechanisms can be
associated with certain fitness costs that can be deleteri-
ous under natural conditions [101]. Natural enemies,
such as predators and parasites can influence the devel-
opment of resistance to Bt by preferring either the in-
toxicated, susceptible or the healthy resistant insects. In
the former case, one would expect an increase in resis-
tance development, while in the latter, natural enemies
can help to retard resistance development to Bt. Never-

V. N. JISHA ET AL. 467
theless, selection experiments in the laboratory are valu-
able 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 popu-
lations of diamondback moth showed different levels of
susceptibility to a formulated Bt product (Dipel). Popula-
tions from heavily treated areas proved more resistant
than those populations treated at lower levels, with the
highest level of resistance at 30-fold [100].
The resistance trait is conferred largely by a single
autosomal recessive locus [102]. This “Hawaii” resis-
tance allele simultaneously confers cross-resistance to
Cry1Aa, Cry1Ab, Cry1Ac, Cry1Fa, and Cry1Ja but not
to Cry1Ba, Cry1Bb, Cry1Ca, Cry1Da, Cry1Ia, or
Cry2Aa (369). At least one Cry1A-resistant diamond-
back moth strain has been shown to be very susceptible
to Cry9C [31].
Resistance to Btk products and resulting failure in dia-
mondback moth control has resulted in the extensive use
of Bt subsp. aizawai-based insecticides in certain loca-
tions [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 mem-
brane vesicles receptors was partially responsible for
resistance to both subspecies. Genetic determinants re-
sponsible for subspecies kurstak i-specific and subspecies
aizawai-specific resistance segregated separately from
each other and from the Cry1Ab resistance allele in ge-
netic experiments [103].
After less than 2 decades of intensive use of Btk in cru-
cifer agriculture, resistant insects have evolved in nu-
merous geographically isolated regions of the world, and
subspecies aizawai resistance is beginning to appear even
more rapidly.
Defying the expectations of scientists monitoring trans-
genic crops such as corn and cotton that produce insecti-
cidal proteins derived from Bt, target insect pests have
developed little or no resistance to Bt crops thus far, ac-
cording to US Department of Agriculture-funded scien-
tists. 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 effec-
tiveness 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 resis-
tance 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 strate-
gies to sustain the effectiveness of Bt crops [105].
9. Resistance Management
Resistance management strategies try to prevent or di-
minish the selection of the rare individuals carrying re-
sistance genes and hence to keep the frequency of resis-
tance 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 ar-
eas). Retrospective analysis of resistance development
does support the use of refugia [99]. Experience with
transgenic crops expressing cry genes grown under dif-
ferent 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 tis-
sues 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 ex-
pression 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 at-
tractive resistance management tactics is the combination
of a high-dose strategy with the use of refugia [19].
10. Conclusion
Development of resistance to Bt toxin is one of the con-
cerns of Bt-based agroindustry. It was expected that re-
sistance 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 estab-
lished to mitigate resistance before the crops were intro-
duced 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 chal-
lenges, 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.

V. N. JISHA ET AL.
468
11. Acknowledgements
JVN is grateful to the University Grants Commission,
Government of India for granting Rajiv Gandhi National
Research Fellowship, SRB is grateful to the University of
Calicut for granting the University Research Fellowship.
There exists no conflict of interest.
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