Hi there, This was topic on which I presented a talk in our department. As we know Insecticide such as pyrethroids are being most widely used for controlling insect pest in agriculture because of their safe, cheap, effective and long-lasting nature (Bulter et al. 2011). However, the widespread development of insecticide resistance, especially resistance to pyrethroid and the fact that resistance to an insecticide generally confers cross-resistance to other insecticides has become a serious problem challenging the control of agriculturally, economically, and medically important insect pests and resulting in increase of insect vector-borne diseases in many parts of the world (Zaim 2002; Bulter 2011). Three major mechanisms are involved in insecticide resistance: (1) increased metabolic detoxification of insecticides; (2) decreased sensitivity of the target proteins on which an insecticide acts, known as target site insensitivity; and (3) decreased cuticular penetration/or increased sequestration/storage. Bacillus thuringiensis(Bt)Cry protein is one of the most effective biopesticides that can act against a large group of insect orders like Lepidoptera, Coleoptera, Diptera etc. But through the course of evolution insects have developed resistance so that they can combat against Bt. Mutation(s) in the target site; reduced protease activity; sequestration of toxin molecules; mutations in the ABCC2 transporter protein are few mechanisms which govern resistance in insects against Cry protein. Therefore, it has become prime importance to understand the molecular basis of insect resistance and what could be the strategies by which the efficacy of the Cry protein can be enhanced. The strategies for improving the efficacy of Cry toxin are (i) Expression of chitinase gene along with Cry toxins; (ii) Expression of hybrid toxin; (iii) Introduction of intramolecular cleavage sites. The insects have developed resistance against different groups of Cry proteins such as Cry2Aa, Cry1Ac, Cry2Ab etc. But the relative fitness (such as larval and pupal weight, pupal duration, and survival rate, etc.) in resistant larvae is much lower than the susceptible larvae because of genetic changes in insects. One of such genes that affects the relative fitness in Helicoverpa armigera is death associated LIM only protein (Ha-DALP) which is basically involved in cell signalling, cell fate determination, transcriptional regulation of gene expression etc.

1. Molecular basis of insecticide
resistance with special reference to
Bacillus thuringiensis
Presented by-Deba Jit Das
PhD 3rd
year, 1st
semester

2.  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..

3.  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??

4.  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

5. How does resistance occur in pest population??

6. I. Behavioral Resistance
Mechanisms of insecticide resistance…

7.  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??

8. Physiological resistance
 Reduced penetration
 Target site insensitivity
 Enhanced detoxification

9.  Cuticle contains more protein and Lipid.
 Increased sclerotization.
 Binding protein and lipid reservoir traps
insecticide in the cuticle.
 Slight resistance.
What is reduced penetrance??

10. Target site insensitivity

11. What are the different targets of different group of
insecticides??

12. Insecticide
Detoxification of insecticides by
enzymes
Target site
Enhanced detoxification

13. Detoxifying Enzymes
a. Hydrolases –Carboxyl Esterase (CarE)
i. Esterase gene amplification
ii. Esterase mutation
b. Mixed fuction oxidases (MFO’s)
Cytochrome P450 monoxygenases (P450)
c. Glutathion-S-transferases (GST)
What are the different detoxifying enzymes??

14. General defense mechanism in insects against
insecticides…

15. Resistance development
against Bacillus
thuringiensis

16. 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.

17. How does Cry protein act??

18. 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

19. Schematic representation of different mechanism of
resistance against Cry toxin

20. How does mutated ABCC2 transporter induce
resistance in insects??

21. 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 andα
skip cadherin interaction.

22. Sites for modification of Cry toxins…

23. Strategies to delay resistance development against Bt

24. Genetic regulation of relative
fitness in Cry1Ac resistant
Helicoverpa armigera larvae

25. Up-regulated death associated LIM-only protein
contributes to fitness costs of Bacillus thuringiensis
Cry1Ac resistance in Helicoverpa armigera
Huiming Guo , Guoqing Lu , Xiaofeng Su , Gemei Liang , Chenxi Liu ,
Hongmei Cheng (2014)

26. 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

27. 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

28. 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

29. Enhanced detoxificationRNA extraction and cDNA cloning
 RNA was extracted from different stages (2nd
, 3rd
,4th,
and
5th
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

30. Multiple sequence alignment result

31.  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

32.  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

33.  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

34. qRT-PCR and RT-PCR analysis of Ha-DALP
silencing

35. Enhanced detoxificationMeasurement of larval weight loss

36. 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

37. Effect of feeding purified Ha-DALP protein to 96S
larvae

38. 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