Chapter 15. Microbial Insecticides
-Bacillus thuringiensis -Baculovirus
-Bacillus thuringiensis
-Baculovirus
Insecticides
Major uses: Crop protection & control of disease vectors (e.g. mosquitoes)
Problems asssociated with chemical insecticides
Degrade slowly: persist in the environment (15-20 years for DDT) Pollution of surface and groundwater Bioaccumulation: concentration in animal tissues is magnified through the food chain algae, bacteria ---> daphnia ---> fish ---> eagles, humans etc. Nonselective: toxic to nontarget organisms (wildlife, beneficial insects, farm animals, humans) Development of resistance by target organisms Requires application of more insecticide and introduction of new insecticides
Degrade slowly: persist in the environment (15-20 years for DDT)
Pollution of surface and groundwater
Bioaccumulation: concentration in animal tissues is magnified through the food chain
algae, bacteria ---> daphnia ---> fish ---> eagles, humans etc.
Nonselective: toxic to nontarget organisms (wildlife, beneficial insects, farm animals, humans)
Development of resistance by target organisms
Requires application of more insecticide and introduction of new insecticides
Microbial insecticides have fewer problems
Ex. Toxins produced by bacteria (Bacillus thuringiensis) Pathogens that infect insects (Bacillus popilliae, Baculovirus)
Ex. Toxins produced by bacteria (Bacillus thuringiensis)
Pathogens that infect insects (Bacillus popilliae, Baculovirus)
Bacillus thuringiensis as an insecticide
Gram + spore former, produces a protein that kills insects
See Figs. 15.1 and 15.3
Several subspecies are toxic to different insect orders
See Table 15.1 Subsp. toxicities kurstaki ---> lepidopteran larvae (moths, butterflies) israelensis ---> dipteran larvae (mosquitoes) tenebrionis ---> coleopterans (potato beetle, boll weevil)
See Table 15.1
Subsp. toxicities
kurstaki ---> lepidopteran larvae (moths, butterflies) israelensis ---> dipteran larvae (mosquitoes) tenebrionis ---> coleopterans (potato beetle, boll weevil)
kurstaki ---> lepidopteran larvae (moths, butterflies)
israelensis ---> dipteran larvae (mosquitoes) tenebrionis ---> coleopterans (potato beetle, boll weevil)
Disadvantages
Applied by spraying plant surfaces and must be ingested by insect Boring, sucking and root eating insects not affected Kill insect only at a particular stage of insect's life cycle Timing of application is crucial Specificity Use of just one subsp. will not kill multiple crop pests Expensive Ex. kurstaki costs 1.5-3 X more than chemical insecticides Rapidly degraded by sunlight May not persist long enough for control of insect pest (+) However, this prevents development of resistance
Applied by spraying plant surfaces and must be ingested by insect
Boring, sucking and root eating insects not affected
Kill insect only at a particular stage of insect's life cycle
Timing of application is crucial
Specificity
Use of just one subsp. will not kill multiple crop pests
Expensive
Ex. kurstaki costs 1.5-3 X more than chemical insecticides
Rapidly degraded by sunlight
May not persist long enough for control of insect pest (+) However, this prevents development of resistance
May not persist long enough for control of insect pest
(+) However, this prevents development of resistance
Cloning cry Genes from Bacillus thuringiensis subsp. kurstaki
See Fig. 15.4
Cloning procedures
1. Plasmids were separated from chromosomal DNA by centrifugation through a gradient of cesium chloride 2. Small plasmids were discarded because they were not big enough to code for a protein with a molecular weight of 130,000 (130 k-Da). 3. Medium and large plasmids were separated from each other by centrifugation through a sucrose gradient. 4. The medium and large plasmids were partially digested with Sau 3AI and ligated into the Bam HI restriction site of the cloning vector pBR322. Why was it possible to ligate DNA cut with the two different restriction enzymes? 5. The DNA was introduced into E. coli by transformation to create a DNA library. 6. The library was screened for the expressed toxin using an immunoassay. The immunoassay used an antibody that bound to the toxin protein. The antibody was radioactively labeled by binding protein A containing a radioactive isotope of iodine, 125I. Protein A is produced by Staphylococcus aureus and binds to the constant domain of antibodies. Screening consisted of exposing a membrane colony lift to the radioactive antibody which was detected by autoradiography
1. Plasmids were separated from chromosomal DNA by centrifugation through a gradient of cesium chloride
2. Small plasmids were discarded because they were not big enough to code for a protein with a molecular weight of 130,000 (130 k-Da).
3. Medium and large plasmids were separated from each other by centrifugation through a sucrose gradient.
4. The medium and large plasmids were partially digested with Sau 3AI and ligated into the Bam HI restriction site of the cloning vector pBR322.
Why was it possible to ligate DNA cut with the two different restriction enzymes?
5. The DNA was introduced into E. coli by transformation to create a DNA library.
6. The library was screened for the expressed toxin using an immunoassay.
The immunoassay used an antibody that bound to the toxin protein. The antibody was radioactively labeled by binding protein A containing a radioactive isotope of iodine, 125I. Protein A is produced by Staphylococcus aureus and binds to the constant domain of antibodies. Screening consisted of exposing a membrane colony lift to the radioactive antibody which was detected by autoradiography
The immunoassay used an antibody that bound to the toxin protein.
The antibody was radioactively labeled by binding protein A containing a radioactive isotope of iodine, 125I.
Protein A is produced by Staphylococcus aureus and binds to the constant domain of antibodies.
Screening consisted of exposing a membrane colony lift to the radioactive antibody which was detected by autoradiography
Genetic Engineering B. thuringiensis and cry Genes
1. Express toxin gene continuously during vegetative growth
See Fig. 15.5 Most cry genes expressed only during sporulation phase of growth i.e. at the end of the growth cycle Sporulation specific promoters control expression Place cry gene under control of promoter of a gene that is constitutively expressed during vegetative growth Ex. Tetr gene or cryIIIA gene Higher yields of toxin would be produced in less time
See Fig. 15.5
Most cry genes expressed only during sporulation phase of growth
i.e. at the end of the growth cycle Sporulation specific promoters control expression
i.e. at the end of the growth cycle
Sporulation specific promoters control expression
Place cry gene under control of promoter of a gene that is constitutively expressed during vegetative growth
Ex. Tetr gene or cryIIIA gene
Higher yields of toxin would be produced in less time
2. Broaden spectrum of toxicity against more insect species
i) Transform B. thuringiensis strains with several cloned cry genes that encode proteins that kill different insect species See Table 15.3
i) Transform B. thuringiensis strains with several cloned cry genes that encode proteins that kill different insect species
See Table 15.3
3. Increase availability of toxin to insects by introducing cry genes into other species of bacteria
i) Mosquito control Heavy parasporal crystals sink below feeding zone of mosquitoe larvae Transfer cry genes to aquatic microorganisms that inhabit feeding zone and are food source for mosquito larvae Cyanobacteria (photosyntheitc), Caulobacter crescentus, Asticcacaulis excentricus Ex. Asticcacaulis excentricus Dwells near surface and doesn't sink Grows on a cheap medium Low protease activity protects protoxin from inactivation Adapted to high levels of UV light present near water surface ii) Control of insects that feed on plant roots Spraying crops w/ B.t. doesn't kill insects that feed on roots Pseudomonas fluorescens inhabits rhizosphere Protect roots by transferring cry genes to this organism Inoculate seeds w/ recombinant organism before planting iii) Control of sucking and boring insects Ingest very little protoxin during feedong Clavibacter xyli inhabits xylem of grasses Transfer of cry gene to C. xyli protects corn from dmage by European corn borer
i) Mosquito control
Heavy parasporal crystals sink below feeding zone of mosquitoe larvae Transfer cry genes to aquatic microorganisms that inhabit feeding zone and are food source for mosquito larvae Cyanobacteria (photosyntheitc), Caulobacter crescentus, Asticcacaulis excentricus Ex. Asticcacaulis excentricus Dwells near surface and doesn't sink Grows on a cheap medium Low protease activity protects protoxin from inactivation Adapted to high levels of UV light present near water surface
Heavy parasporal crystals sink below feeding zone of mosquitoe larvae
Transfer cry genes to aquatic microorganisms that inhabit feeding zone and are food source for mosquito larvae
Cyanobacteria (photosyntheitc), Caulobacter crescentus, Asticcacaulis excentricus
Ex. Asticcacaulis excentricus
Dwells near surface and doesn't sink Grows on a cheap medium Low protease activity protects protoxin from inactivation Adapted to high levels of UV light present near water surface
Dwells near surface and doesn't sink
Grows on a cheap medium
Low protease activity protects protoxin from inactivation
Adapted to high levels of UV light present near water surface
ii) Control of insects that feed on plant roots
Spraying crops w/ B.t. doesn't kill insects that feed on roots Pseudomonas fluorescens inhabits rhizosphere Protect roots by transferring cry genes to this organism Inoculate seeds w/ recombinant organism before planting
Spraying crops w/ B.t. doesn't kill insects that feed on roots
Pseudomonas fluorescens inhabits rhizosphere
Protect roots by transferring cry genes to this organism Inoculate seeds w/ recombinant organism before planting
Protect roots by transferring cry genes to this organism
Inoculate seeds w/ recombinant organism before planting
iii) Control of sucking and boring insects
Ingest very little protoxin during feedong Clavibacter xyli inhabits xylem of grasses Transfer of cry gene to C. xyli protects corn from dmage by European corn borer
Ingest very little protoxin during feedong
Clavibacter xyli inhabits xylem of grasses
Transfer of cry gene to C. xyli protects corn from dmage by European corn borer
Baculoviruses
See Table 15.5 Ex. pine saw fly, cotton bollworm, gypsy moth, cabbage looper
See Table 15.5
Ex. pine saw fly, cotton bollworm, gypsy moth, cabbage looper
Advantages/disadvantages
+/ - Limited host ranges Want to controll multiple pests but not to affect beneficial insects + Resistance of insect to virus rarely develops - Slow to kill May still cause crop damage before dying - High production costs Viruses must be produced in cultured insect cells (or whole insects)
+/ - Limited host ranges
Want to controll multiple pests but not to affect beneficial insects
+ Resistance of insect to virus rarely develops
- Slow to kill
May still cause crop damage before dying
- High production costs
Viruses must be produced in cultured insect cells (or whole insects)
Genetic Engineering of Baculoviruses
1. Introduce genes that disrupt levels of hormones controlling stages of insect life cycle.
Ex. some insect hormones are esters: introduce esterase gene. Esterase R-C=O(O-CH3) + H2O ------------------> R-COOH + CH3OH (juvenile hormone) ...............................................INACTIVE Treated insect larvae stopped growing and feeding on plant -Disadvantage: hormone's influence occurs only at specific stage of life cycle, making the timing of application to crops critical for effectiveness
Ex. some insect hormones are esters: introduce esterase gene.
Esterase
R-C=O(O-CH3) + H2O ------------------> R-COOH + CH3OH (juvenile hormone) ...............................................INACTIVE
Treated insect larvae stopped growing and feeding on plant
-Disadvantage: hormone's influence occurs only at specific stage of life cycle, making the timing of application to crops critical for effectiveness
2. Introduce genes that increase viral pathogenicity.
Ex. Scorpion gene that encodes an insect-specific neurotoxin Insects infected with the engineered baculovirus caused less plant damage than those infected with the wild-type virus
Ex. Scorpion gene that encodes an insect-specific neurotoxin
Insects infected with the engineered baculovirus caused less plant damage than those infected with the wild-type virus
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