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Molecular basis of insecticide resistance with special reference to
Bacillus thuringiensis
Presented by-Deba Jit DasPhD 3rd year, 1st semester
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What does insecticide resistance mean?
Mechanism of development of resistance
Resistance against Bacillus thuringiensis
Strategies to delay resistance development against Bt
Genetic regulation of relative fitness in Cry1Ac resistant Helicoverpa
armigera larvae
Points to be discussed..
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The decreased susceptibility of a pest population to
a pesticide that was previously effective at controlling
the pest
Or
The genetically acquired ability of an organism to
survive a pesticide application at doses that once
killed most individuals of the same species
What is insecticide resistance??
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Phase I- Due to selection of variants in the population
according to genetic principle, the resistance which is
initially present in the population is expressed.
Phase II- Acceleration of resistance takes place by
induction of pre-existing detoxifying enzymes towards
enhanced activity, resulting in faster breakdown of the
chemicals.
Two phases of resistance
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How does resistance occur in pest population??
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I. Behavioral Resistance
Mechanisms of insecticide resistance…
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Development of ability to avoid a dose that would prove lethal.
Stimulus dependent and matter of hypersensitivity or hyperirritability
Avoid lethal dose or treated surface
Behavioural resistance??
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Physiological resistance
Reduced penetration
Target site insensitivity
Enhanced detoxification
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Cuticle contains more protein and Lipid.
Increased sclerotization.
Binding protein and lipid reservoir traps
insecticide in the cuticle.
Slight resistance.
What is reduced penetrance??
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Target site insensitivity
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What are the different targets of different group of insecticides??
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InsecticideDetoxification of insecticides by enzymes
Target site
Enhanced detoxification
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Detoxifying Enzymes
a. Hydrolases –Carboxyl Esterase (CarE)
i. Esterase gene amplification
i i. Esterase mutation
b. Mixed fuction oxidases (MFO’s)
Cytochrome P450 monoxygenases (P450)
c. Glutathion-S-transferases (GST)
What are the different detoxifying enzymes??
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General defense mechanism in insects against insecticides…
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Resistance development against Bacillus
thuringiensis
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Bacillus thuringiensis
Bacillus thuringiensis (Bt ) is a common gram positive,
spore-forming, soil bacterium.
In stress condition Bt undergo sporulation and synthesize
insecticidal crystal proteins (ICPs) or Cry proteins.
Benefits of Bt proteins
High specificity and potency,
Reduction in chemical pesticide applications
Increased crop yield.
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How does Cry protein act??
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Mechanisms of resistance against Cry protein..
Four major mechanisms of resistance of insects against
Bt toxins are
Alteration in protease activity
Sequestration of the toxin by glycolipid moieties
Alteration in cry toxin receptor
Alteration in ABCC2 transporter
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Schematic representation of different mechanism of resistance against Cry toxin
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How does mutated ABCC2 transporter induce resistance in insects??
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How can the toxicity of cry protein be enhanced??
Chitinase improves accessibility of the toxin to the epithelial
membrane.
Serine protease inhibitors reduce degradation of Cry or of
toxin-receptors.
Introduction of intramolecular cleavage sites
Expression of hybrid toxin
Deletion of helix -1 that induces toxin-oligomerization and α
skip cadherin interaction.
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Sites for modification of Cry toxins…
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Strategies to delay resistance development against Bt
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Genetic regulation of relative
fitness in Cry1Ac resistant
Helicoverpa armigera larvae
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Up-regulated death associated LIM-only protein contributes to fitness costs of Bacillus thuringiensis
Cry1Ac resistance in Helicoverpa armigeraHuiming Guo , Guoqing Lu , Xiaofeng Su , Gemei Liang , Chenxi Liu ,
Hongmei Cheng (2014)
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Enhanced detoxificationBrief overview of the study….
cDNA-AFLP analysis was used to identify differentially
related gene both in susceptible and resistant insects
Ha-DALP gene was differentially expressed in resistant
Helicoverpa larvae
Ha-DALP gene is associated with transcriptioanal
regulation, cell signalling and cell fate determination
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Enhanced detoxificationObjective of the study….
To analyze the expression pattern of Ha-DALP
gene in both Cry1Ac resistant (BtR) and
susceptible (96S) insects
To compare the fitness-trade off between two
groups of insects
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Enhanced detoxificationExperimental procedures and findings
Two strains of H.armigera were used for the study
purpose
Cry1Ac resistant Bt strain (BtR)
Cry1Ac susceptible strain (96S)
Resistant insects were selected from the susceptible
strain reared for 75 generations in the presence of
Cry1Ac protoxin
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Enhanced detoxificationRNA extraction and cDNA cloning
RNA was extracted from different stages (2 n d , 3 r d ,4 t h , and
5 t h instar) and tissues (epidermis, midgut and whole body)
RACE was performed to obtain the full lenth sequence of
HaDALP gene
Sequence comparison with Menduca sexta HaDALP gene
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Multiple sequence alignment result
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Ha-DALP gene expression at
different stage of larval
growth and in different
tissues was checked by qRT-
PCR.
96S and Bt-R Ha-DALP expression analysis
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FISH was carried out to determine specific expression of Ha-DALP gene in different stages and tissues
of 96s and Bt-R insects.
Analysis of Ha-DALP expression by FISH
FISH analysis of HaDALP transcription levels in the 96S 2nd instar larvae
FISH analysis of HaDALP transcription levels in the Bt-R 2nd instar larvae
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To express Ha-DALP specific dsRNA a recombinant
plasmid (RNAi-DALP) was constructed
Primers were designed to to amplify a 423 bp fragment
from Ha-DALP gene
RNAi-DALP plasmid was transformed into competent
HT115 cells
Bacteria containing Ha-DALP specific dsRNA were
harvested and used for H. armigera feeding bioassay
RNAi analysis in 96S larvae
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qRT-PCR and RT-PCR analysis of Ha-DALP silencing
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Enhanced detoxificationMeasurement of larval weight loss
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Purified Ha-DALP protein feeding bioassay
Recombinant expression vector pET32a-HaDALP was
constructed using primers designed from Ha-DALP gene
sequence
HaDALP protein was isolated and purified by affinity
purification
Purified HaDALP protein used for feeding the
experimentel groups
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Effect of feeding purified Ha-DALP protein to 96S larvae
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Conclusion of the discussion
To overcome the selection pressure a large group insects has
developed resistance against insecticides as well as Bt
But there is enough score for us to enhance the efficacy of Cry toxin
Genetic change in insects resulting in Bt resistance can also affect
their fitness
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