Bacillus thuringiensis (Bt) is a naturally occurring soil bacterium that produces crystal proteins during sporulation that are toxic to certain insect orders. Various Bt subspecies produce different toxins targeting Lepidoptera, Coleoptera, or Diptera. The toxins work by forming ion channels in the gut of susceptible insects, which leads to cell damage and death. Bt has been used widely as a biological insecticide by spraying spores to control pests like cabbage worms, gypsy moths, and spruce budworm. It is effective when eaten and breaks down quickly in the environment to avoid insect resistance.

1. Bio-control agents:
Insecticidal toxins of Bacillus
thuringiensis
Presented by:
Manisha.G
M.Sc. Biochemistry
Final year
Dept. of biochemistry &bioinformatics
GIS, GITAM university

2. Contents
 History
 Definition of biological control
 Pros and cons of bio control agents
 Introduction to Bacillus thuringiensis
 Properties
 Examples
 Its mode of action
 Administration

3. History
 Insects have largest number of described
species- >7,50,000.
 Cause massive crop damage and act as vectors
for both human and animal diseases.
 During 1940s, a number of chemical insecticides
were developed as a means of controlling the
proliferation of insect populations.
 DDT- chlorinated hydrocarbon, nervous system
and muscle tissue

4. • Dieldrin, aldrin, chlordane, lindane, toxophene were applied against
crop pests and insects that carry infectious agents.
• Malathion, parathion, diazinon- organophosphates, inhibit enzyme
acetylcholinesterase in insects, which hydrolyses the nerve transmitter
acetylcholine.
• These were found to persist in the environment for >20years.
• During 1950s higher concentrations were applied to control pests.
• They lack specificity and beneficial insects were killed along with pests.
• Given all the drawbacks associated with the use of chemical insecticides,
alternative means of controlling harmful insects have been sought.

5. Definition of biological control:
 In entomology, it has been defined as the use of live predatory insects,
entomopathogenic nematodes, or microbial pathogens to suppress populations
of different pest insects.
 In plant pathology, the term applies to the use of microbial antagonists to
suppress diseases as well as the use of host specific pathogens to control weed
populations.
 In Toto, the organism that suppresses the pest or pathogen is referred to as the
Biological Control Agent (BCA).

6.  The most commonly used biopesticides are living organisms, which are pathogenic for the pest
of interest.
 These include:
 biofungicides (Trichoderma),
 bioherbicides (Phytopthora) and
 bioinsecticides (Bacillus thuringiensis).
 One bacterial species like Bacillus thuringiensis may be effective on Aedes aegypti while another
B. sphaericus strain can be effective on a different types of mosquito like Culex quinquefasciatus.

7. Pros and cons of bio control agents
On the positive side,
1) These compounds are highly specific for a target insect species
2) Biodegradable
3) Slow to select for resistance
On the negative side,
1) Their low potency
2) High cost of production limit their use for a variety of applications.

8. Introduction
 Bt is a naturally occurring soil bacterium.
 It was first detected in 1902 in the dying larvae of
Bombyx mori by Ishiwata, who reported his finding in
the book:"Pathology of the Silkworm".
• It was first isolated from the larvae of Ephestia
kuehniella by Berliner in 1911 after he noted that it
had the capacity to kill certain insects in their larva
stage.
• Natural Bt is highly specific, with toxicity limited to only some species of one
of the major groups of insects—typically Lepidoptera (butterflies/moths),
Coleoptera (beetles), or
Diptera (flies/mosquitos).

9.  Bacillus thuringiensis is a gram-positive, spore-forming
bacterium which, during sporulation, produces protein
crystals (CRY). It is characterized as a widespread
insect pathogen, and its insecticidal activity is
attributed to the parasporal crystals.
 A variety of strains have been isolated from different
habitats and, to date, more than 100 crystal protein
genes have been sequenced.
 There are more than 150 different subspecies of
B.thuringiensis.
 Each of the subspecies produces a different toxin that
can kill specific insect.

10.  The toxicity of these crystal proteins
against certain insects and their high
specificity led to the development of
bio-insecticides for the control of pest
insect species among the orders
Lepidoptera, diptera, and coleoptera.

11. PROPERTIES OF INSECTICIDAL TOXINS:

12. Examples:
 B.thuringiensis subsp kurstaki -lepidopteran larvae (moths, butterflies, skippers,
cabbage worms and spruce bud worms).
 B.thuringiensis subsp tenebrionis – coleopteran (beetles) such as potato beetle
and boll weevil.
 Some species of B.thuringiensis produce insecticidal toxins that are directed
against hymenoptera (sawflies, wasps, bees and ants), orthoptera
(grasshoppers, crickets) and mallophaga (lice).

13. Different domains involved in the toxicity of B.thuringiensis toxin in the mid-gut of targeted
insect. Source:Sharma et al., 2000. Bt bacteria are used by farmers, foresters and gardeners
to destroy butterfly larvae, mosquito larvae and beetles. The Danish field study, which was
undertaken in 1993 and 1994, is one of the first in the world where plants have been
systematically sprayed with Bacillus thuringiensis bacteria, and where the research has been
ecologically oriented.

14. Mode of action
1. Bacillus thuringiensis is only effective when eaten by specific family of insects
with a specific (usually alkaline) gut pH and the specific gut membrane
structures required to bind the toxin. (typically butterflies, moths, beetles, flies
and mosquitoes).
2. Not only must the insect have the correct and be at a susceptible stage of
development, but the bacterium must be eaten in sufficient quantity.
3. When ingested by a susceptible insect, the spores feed on natural intestinal flora
then it burst releasing the protein toxin (Crystalline protein) damaging the gut
lining (the intestinal walls), leading to a kind of leaky gut condition.
4. Affected insects stop feeding and die from the combined effects of starvation,
tissue damage and gastrointestinal infections by other pathogens like bacteria
and fungus.
5. The natural Bt spores do not usually spread to other insects or cause disease
outbreaks on their own as occurs with many pathogens.

15. BT crystalline Toxin 200px.
Normal gut bacteria
BT SPORES

17. Formation of ion channel:
• In its active form, the toxic protein inserts itself into the membranes of the gut epithelial cells of the
insect.
• Creates an ion channel.
• Leads to an excessive loss of cellular ATP.

18. DEATH
Summary:

20. • In 1979, approximately 1% of the forest area in Canada that was treated with an
insecticide to combat the spruce budworm (about 2 million hectares, or 8,000
square miles) was sprayed with B. thuringiensis subsp. kurstaki. The remainder
of the treated forests were sprayed with chemical insecticides.
• By 1986, the use of B. thuringiensis subsp. kurstaki had increased dramatically.
It was used to treat approximately 74% of the forests sprayed in that year for
spruce budworm.
• In other countries, B. thuringiensis subsp. kurstaki has been used against tent
caterpillars, gypsy moths, cabbage worms, cabbage loopers, and tobacco
hornworms.
Usage:

21. • For the biological control (bio control) of insect pests, B. thuringiensis subsp.
kurstaki is typically applied by spraying approximately 1.3 × 108 to 2.6 × 108
spores per square foot (1 square foot is equivalent to 0.093 m2) of the target
area.
• Administration of the spores is timed to coincide with the peak of the larval
population of the target organism, because the parasporal crystals, being
sensitive to sunlight, are short-lived in the environment.
• Under simulated conditions, sunlight degrades over 60% of the tryptophan
residues of the parasporal crystal within a 24-hour period, thereby rendering
the protein inactive.
• Depending on the amount of sunlight present, parasporal crystals may persist
in the environment for as little as a day or as long as a month. The lack of
persistence of the insecticidal protoxin in the natural environment means that
natural selection of resistant insects is highly unlikely.
Administration:

22. Reference:
 Aronson, A. (1993) In Bacillus subtilis and Other Grampostive Bacteria.
Sonenshein, A., Hoch, J.A., and Losick, R. (eds). Washington D.C.: Am Soc
Microbiol, pp. 953- 963.
 Bacillus thuringiensis: an environmental bio pesticide. Theory and practical.
 Donovan, W.P., Rupar, M.J., Slaney, A.C., Malvar, T., Gawron-Burke, M.C., and
Johnson, T. (1992) Appl Env Microbio158: 3921-3927.
 Li., J., Carroll, J., and Ellar, D.J. (1991) Nature353: 815-821.
 Molecular biotechnology : principles and applications of recombinant DNA (4th
edition) by Bernard R. Glick, Jack J Pasternak, Cheryl L Patten.