Novel Approaches in Insecticide Resistance Management (IRM)

INSECTICIDE RESISTANCE MANAGEMENT IN INSECTS- NOVEL
APPROACHES
Presenting By:
Suresh R. Jambagi
1st Ph.D. (Entomology)
PAMB0021
UAS, GKVK Bangalore
1

CONTENT
Introduction
Mechanisms of insecticide resistance
IRM Concept: Principles
IRM Strategies
Novel approaches in IRM
Challenges to manage resistance in insects
Conclusion
2
Suresh R. Jambagi Ph.D. (Entomology) UAS GKVK Bangalore

Insecticide Resistance?
A heritable change in the sensitivity of
a pest population that is reflected in the
repeated failure of a product to achieve the
expected level of control when used
according to the label recommendation for
that pest species.
(IRAC)
3

Why Does Resistance Develop?
1. Resistance is quick to develop to compounds with high effective kill,
long residual and are highly selective at a single biochemical target site.
2. Continued and frequent use of a insecticides
3. Use of application rates -below or above those recommended on the
label.
4. Poor coverage of the area being treated
5. Frequent treatment of organisms with large populations and short
generation times.
4

6. Failure to incorporate non-pesticidal control practices when
possible
7. Simultaneous treatment of larval and adult stages with single
or related compounds.
8. The toxicant is converted into a non toxicant form in the
body of insect by various enzymes.
10. Genetic mutation and inheritance
5

Why Study Insecticide Resistance?
A knowledge of insecticide resistance is an important
from the point of view of proper selection of
insecticides.
6

Resistance development in insects:
Sl.
no
Year Insect Scientific name Insecticide
1 1914 San Jose scale Quadraspidiotus
perniciosus
Lime
sulphur
2 1916 Citrus red scale Aonidiella aurantii HCN
3 1947 House fly Musca domestica DDT
4 1952 Mosquito Culex fatigans DDT
5 1952 Human louse Pediculus humanus corporis HCH
6 1953 Bed bug Cimex lectularius DDT
7 1961 Rat flea Xenopsylla cheopsis DDT
8 1963 Cattle tick Boophilus microplus Lindane
9 1963 Singhara beetle Galerucella birmanica DDT
10 1965 Tobacco caterpillar Spodoptera litura HCH
11 1971 Red flour beetle Tribolium castaneum DDT
Patel et al., 2018 7

Resistance across pest orders
(IRAC)
8

World scenario:
(IRAC)
9

Resistance in
insects
Pre adaptive mechanism
(genetic)
House fly-
Kdr-0 in chromosome 3 (DDT
resistance)
Kdr NPR in chromosome 5
(pyrethroids)
Drosophila-
Single dominant resistant
gene on chromosome 5 (DDT
&BHC)
Post adaptive
mechanism
(physiological)
Conversion of insecticides to-
Nontoxic metabolites
Excretion of toxicants
Storage of toxicants
Behavioral aspects
10

Mechanisms of insecticide resistance in insects
11

1. Altered target-site resistance:
• The site where the toxin usually binds in the insect becomes
modified to reduce the insecticide's effects.
• AchE insensitivity : Resistance to OP and Carbomates
• Sodium Channel insensitivity: Resistance to DDT and Pyrethroids
• GABA receptor (Cl-channel) insensitivity: Resistance to
cyclodiens, fipronil, and avermectins.
• nAchR insensitivity: Resistance to nicotine, neonicotinoids and
spinosad
12

2. Behavioural resistance :
 Resistant insects may detect or recognize a danger and avoid the
toxin by changing their normal activity.
 This mechanism of resistance has been reported for several classes
of insecticides, including organochlorines, organophosphates,
carbamates and pyrethroids.
 Examples: Anopheles mosquito: SS lives and bites inside home, but
RR remains outdoor and flies into house to bite because DDT was
applied to interior walls
• House flies: Avoid treated surface
• Cockroaches: Avoid treated surface and baits
• Diamondback moth: Avoid permethrin
13

3. Penetration resistance:
 It is due to modified composition and structure of
integument
 Resistant insects may absorb the toxin more slowly
than susceptible insects
 Binding protein and lipid reservoir traps insecticide in
the cuticle.
 Penetration resistance is often present along with
other forms of resistance.
 Slight resistance
14

4. Metabolic resistance:
METABOLISM: (Greek = Change)
When an insecticide is applied on any living organism
(plant/ animal), it undergoes some chemical changes resulting
in the formation of new products called as metabolites and
the process is metabolism.
 The extent and nature of the metabolites vary with the
chemical, the organism ( species, strain, age, sex etc.), time
and environmental factors.
 These metabolites may be more toxic, equitoxic or less
toxic than original compound.
Metabolism
Activation
Converts inactive
compound to
active compound
Active compound
to another active
compound
Detoxification
Compound to non
toxic compound
15

• The normal enzymatic metabolism of insect is
modified to increase insecticide detoxification or
prevent activation of insecticides.
• Resistant insects may detoxify or destroy the toxin
faster than susceptible insects or quickly rid their
bodies of the toxic molecules.
• Resistant strains may possess higher levels or more
efficient forms of these enzymes.
• Insects use their internal enzyme systems to break
down insecticides.
16
Patel et al., 2018

 Esterase
Resistance to OP, Carbamates and Pyrethroids
 Glutathione-S-Transferases
Resistance to DDT, OP and Pyrethroids
 Cytochrome P450 Mono-oxygeneses
Resistance to all classes of insecticides
Enzymes involved in the metabolic resistance mechanism
17

Goal
Delay evolution of resistance in pests rather than its
control/ management
An effective insecticide resistance management program could be
based on general resistance management principles endorsed by IRAC
(Insecticide Resistance Action Committee).
18

 Historically, growers respond to resistance by –
1. Increasing dosage and frequency of application which only
accelerate the development of resistance.
2. Switch to another insecticide – so begins the pesticide
treadmill.
 In theory, resistance can be minimized by –
1. Increase survivorship of Susceptible (SS) individuals.
2. Decrease survivorship of Resistant (R) individuals.
19

Figure: Graphic representation of the pesticide treadmill
The pesticide treadmill is a term indicating a situation in which it becomes
necessary for a farmer to continue using pesticides regularly because they have
become an indispensable part of an agricultural cycle.
20

Principles of IRM
1. Management by Moderation
2. Management by Saturation
3. Management by Multiple attack
(Curtis et al., 1993)
Moderation Saturation Multiple attack 21

1. Management by Moderation:
It attempts to preserve susceptibility genes are a valuable resource by
limiting the chemical selection pressure that is applied.
 Infrequent applications
 Non-persistent chemicals
 Preservation of refugia
 Insect-resistant varieties
 Improved timing of planting and harvesting
 Encouragement of biological controls.
22

 The term "saturation" does not imply
saturation of the environment with pesticides.
It is intended to indicate saturation of the
insect's defenses by means of on-target
dosages that are high enough to overcome
resistance.
This approach has more merit during the early
stages of selection when resistance genes
are rare, existing mainly in the heterozygous
state.
2. Management by Saturation:
23

 Formulations that could deliver high
dosages on target include
a. Microencapsulation
b. Attractants
c. Baited targets
 These will cause insecticide uptake at
rates that are lethal to heterozygotes.
24

 The multiple attack strategy is based on
the premise that control can be achieved
through the action of several
independently acting stresses, including
insecticides, each exerting selection
pressure that is below the level which
could lead to resistance.
3. Management by Multiple attack:
25

Achieved through:
i. Alterations, sequences or rotations of compounds from group
with different mode of action.
ii. Applications are often arranged into MOA spray windows or
blocks that are defined by stage of crop development and
biology of the pest concerned.
iii. Insecticide mixtures.
iv. Development of newer molecules with unique mode of action.
26

Concept Approach Means
1. Moderation
- Susceptible genes are a valuable
resource that must be conserved
while achieving economic control
Low selection pressure
• Low doses producing < 100%
mortality of SS
• Higher pest population thresholds
for treatment
• Less frequent application
• Localized applications
• Preservation of refugia
• Some generations untreated
• Chemicals of short environmental
persistence
2. Saturation
- Removal of selective advantage of
resistant phenotypes by saturation of
defense mechanisms
Rendering R genes functionally
recessive
• Higher doses on target can render
R genes functionally recessive,
thus RS= SS
Suppressing detoxification enzymes
• Appropriate synergist can cancel
out specific detoxification
enzymes and remove selective
advantage of RS and RR
3. Multiple attack
- Multi-directional, multi-site
selection reduces degree of pressure
by a single factor
Maintaining degree of selection by
each component factor below levels
leading to resistance
• Mixtures of chemicals
• Rotation of chemicals
• Chemicals with multisite action
27

Insecticide Resistance Management Strategies
Strategies to manage resistance are aimed at reducing the selection pressure
from pesticide to a minimum while still achieving control.
IRM Strategies
General IRM
Strategies
Bt crops
Household pests
and vectors
28

1. Rotation of the insecticides
2. Use of synergists
3. Mixtures and alternations
4. Negatively correlated insecticides
5. Development of newer insecticides
6. Use of insect pheromones
7. Use of insect hormones
8. Use of Integrated Approach (IPM)
9. Spot spraying
I. General IRM strategies:
29

1. Rotation of the insecticides:
The same insecticide should not used
for a long time particularly when
resistance has been detected.
Insects resistant to one insecticide
may not be resistant to another.
After a time lag the lost susceptibility
to the first insecticide returns
30

31

Strategy YEAR 1st Generation 2nd Generation
Strategy-I
1st Year Intrepid
2nd Year Avaunt, SpinTor, Danitol or Bt
Strategy-II
1st Year Intrepid
Avaunt, SpinTor,
Danitol or Bt
2nd Year
Avaunt, SpinTor,
Danitol or Bt
Intrepid
Resistance insecticides Insecticides in IRM
Guthion
Imidan
Intrepid
Spintor
Avaunt
Danitol
Bacillus thuringiensis
Example: Tufted Apple Bud Moth
32

 These inhibitory compounds we called synergists
allow the insecticide to function normally only by
blocking their inhibitory enzymes and do not in
reality enhance the potency of the insecticides.
Ex: Piperonylcyclone when mixed with DDT prevents
its detoxification to DDE in the resistant strain
house flies and these keeps them susceptible to
DDT.
 As control agents, synergists can potentially
render resistant populations susceptible and or
prevent the development of resistance.
2. Use of Synergists:
Synergist Insecticide
DMC
DDT
(Dehydrochlorinas
e)
PBO Pyrethroids
Methylene
dioxyphenyl
Carbamates,
Pyrethrin, DDT
Propyl paraoxon
Malathion,
Parathion
Gupta, 2001 33

 Synergists are among the most straightforward tools for overcoming metabolic
resistance because they can directly inhibit the resistance mechanism itself.
 It specifically interact with resistance associated with phase I metabolic
enzymes (MFO).
Use of PBO along with synthetic pyrethroids can delay the development of
resistance in DBM and mosquitoes (Kumar et al., 2002).
NOTE: The WARF of the USA has developed a substance WARF antiresistant
that can block DDT Dehydrochlorinase (the enzyme that detoxifies the DDT in
houseflies ) and thus prevent the conversion of DDT to DDE and retain the insects
susceptibility to this insecticide.
34

(Curtis et al., 1993)
35

If an insect is treated with a synergist a few hours prior to exposure to
an insecticide, it allows time for the synergist to cross the insect cuticle and
inhibit those enzymes involved in metabolic resistance.
 It creates sensitivity or hyper sensitivity in insects.
It reduces the insecticide dose to be subsequently applied.
It can be achieved by
- Split application of synergist and insecticide
- Use of formulation technologies which allow a differential time
release of synergist and insecticide
Panini et al., 2016 36
Temporal synergism concept

 These increase the level of target pest
control and broadening the range of pests
controlled
 Insecticide mixtures may offer benefits
for IRM when appropriately incorporated
into rotation strategies with additional
mode(s) of action, but generally a single
mixture should not be relied upon alone.
3. Use of Mixtures:
37

Considerations:
a) Should be compatible
b) Individual insecticides selected for use in mixtures should be highly effective and
be applied at the rates at which they are individually registered for against the
target species.
c) Mixtures with components having the same IRAC mode of action Classification are
not recommended for IRM.
d) When using mixtures, consider any known cross- resistance issues ‐ between the
individual components for the targeted pest/s
e) Mixtures become less effective if resistance is already developing to one or both
active ingredients, but they may still provide pest management benefit
38

Ex: A BHC – DDT mixture against malaria mosquitoes (BHC is for DDT
resistant survivors and DDT is for BHC resistant survivors)
Note:
 A mixture of two insecticides and independent action has been
suggested as a counter measure for the resistance.
 However in such cases it takes the a much shorter time for the
insects to turn the resistant to both the compounds than what it
would take if they are used separately
39

 Resistance to one insecticide leads to the enhanced
susceptibility to another insecticide.
EX: 1. Cyclodiene resistant boll weevils are found to be
susceptible to Malathion
2. DDT resistant houseflies also susceptible to Malathion
 Such combinations are discovered and used
4. Negatively correlated insecticides:
40

 Just as pheromones modulate insect
behaviour, hormones regulate growth
and reproduction in insects.
 Both these processes can be interfered
with by providing exogenous hormones at
wrong times that is when they are not
needed by the insect system .
5. Use of insect hormones in IRM:
41

 It should be a continuous process to have
more and more substitutions or alternates.
 There should be a constant attempt to
search for newer insecticides
 This will make a large number of
insecticides available for substitution when
particular insecticides fails to kill
6. Developing newer compounds:
42

 By treating border rows only (for pests that
migrate into your planting from outside) and
localized "hot spots" where pest numbers are
over the action threshold.
 You leave large areas unsprayed for
susceptible pests to survive.
 These areas can be treated at a later date,
if thresholds are reached.
7. Spot spraying:
43

 Pheromones regulate the insect behaviour.
 By using of the sex pheromones insects
could be driven to poison baits and they
will die.
 By providing the aggregation pheromones
they could be driven to the wrong host
plants where they would starve and die.
 To evaluate the insecticide resistance in
the field
8. Pheromones Role:
44

An integrated approach to the control of insect pests will reduce the application of the
insecticides which in turn will lower insecticides pressure on the insects under such a condition
genes governing resistance may not get activated or may take a longer time to do so and keep
resistance postponed for some time
9. INTEGRATED APPROACH:
45

 Growers are requested to plant a non-Bt refuge around each plot of Bt
cotton
- Refuge must be at least 5 rows wide
- The non-Bt refuge must be at least 20% of the total cotton plot
 The company supplies non-Bt seeds in a package attached to the Bt cotton
seeds.
- For each 450g Bt cotton seeds, 120g non-Bt cotton seeds
 Frequent monitoring of insect susceptibility is conducted
II. Bt Crops:
Refugia strategy
46

47

 Spatially separated applications of different compounds against the same
insect constitute a “mosaic” approach to resistance management.
 By using two insecticides in different dwellings within the same village.
 This creates the potential for insects within a single generation to come into
contact with both insecticides, and would reduce the rate of resistance
selection, provided that multiple resistance within the vector population was
extremely rare.
 If such a fine scale mosaic is to be used, careful records of which
insecticide was used in each house are essential.
III. Vectors and household pests:
1. Fine scale mosaic strategy
48

49

‘Control of pest population achieved by
releasing large no. of sterile male insects, which
will compete with normal males and reduce the
insect population in subsequent generations’.
 E. F. Knipling (1937): Screwworm fly
 Methods of sterilization
1. Chemosterilants
a. Alkylating agents-TEPA, Chloro ethylamine
b. Antimetabolites- Amithopterin, 5-Fluororacil
2. Irradiation: X-rays, Neutrons, Co60
2. Sterile Insect Release (SIRM) method:
50

 After a decade of fighting for regulatory approval and public acceptance, Oxitec (A Firm from UK) released
genetically engineered mosquitoes into the open air in the United States for the first time.
 Tests a method for suppressing populations of wild Aedes aegypti mosquitoes.
Vector: Zika, dengue, chikungunya and yellow fever.
Aim: Mate with the wild female population, responsible for biting prey and transmitting disease.
 The GM males carry a gene that passes to their offspring and kills female progeny in early larval stages.
 Male offspring won’t die but instead will become carriers of the gene and pass it to future generations.
 As more females die, the Aedes aegypti population should dwindle.
Methodology:
 They placed boxes containing Oxitec’s mosquito eggs at six locations in three areas of the Keys.
 The first males are expected to emerge within the first two weeks of May (12000 males/week)
 Within 16 weeks: 20 million male mosquitoes will release to environment
 Oxitec mosquitoes carry a fluorescent marker gene
(Nature, 2021)
51

Novel approaches in IRM
Genome editing
II. Genome editing in plants for
resistance against insect pests
1. Knocking down susceptible genes
2. Modification of plant volatile
blends
3. Changing foliage colour
I. Editing in insects for susceptibility
toward plants/ insecticides
1. Modification of Cry protein binding
receptors
2. Knockdown of detoxification enzymes.
3. Editing of genes to disrupt chemical
communication and mating partner
identification
4. Knock out of developmental genes
Tyagi et al., 2020
52

53

I. Genome editing in insects
 It can be successfully employed in a two-step strategy involving the modification
of target insects, and their subsequent release into the natural environment’
1. Modification of Cry protein binding receptors
 Knockdown of cadherin receptors that are genetically linked to Cry1Ac toxin
resistance: Evidence for successful genome editing in H. armigera.
 Technique - CRISPR/Cas9 system
 Target gene- HaCad
 This strategy can also be adapted to modify target sites in midgut receptors
responsible for resistance development against BtICPs or insecticidal proteins.
Wang et al., 2016
54

2. Knockdown of detoxification enzymes
A. Knock-Down of Gossypol-Inducing Cytochrome P450 Genes Reduced Deltamethrin Sensitivity in
Spodoptera exigua.
 Gossypol-fed beet armyworm larvae showed higher 7-ethoxycoumarin-O-deethylase (ECOD) activities and
exhibited enhanced tolerance to deltamethrin.
 Meanwhile, gossypol-induced P450s exhibited high divergence in the expression level of two P450 genes:
CYP6AB14 and CYP9A98 in the midgut and fat bodies contributed to beet armyworm tolerance to deltamethrin.
 Knocking down of CYP6AB14 and CYP9A98, via double-stranded RNAs (dsRNA) in a controlled diet, rendered
the larvae more sensitive to the insecticide.
 These data demonstrate that generalist insects can exploit secondary metabolites from host plants to enhance
their defense systems against other toxic chemicals.
 Impairing this defense pathway by RNA interference (RNAi) holds a potential to eliminate the pest’s tolerance
to insecticides
Hafeez et al., 201955

B. CYP6AE gene cluster knockout in Helicoverpa armigera reveals role in detoxification
of phytochemicals and insecticides
 Technique: CRISPR/Cas9
 CYP6AE cluster contributes to the tolerance of two phytochemicals (xanthotoxin and 2-
tridecanone) and two insecticides (fenvalerate and indoxacarb).
Fig. Responses to phytochemical toxins and insecticides
Subfamily of CytP450
Wang et al., 2018
The CRISPR-Cas9- based reverse genetics
approach in conjunction with in vitro metabolism can
rapidly identify the contributions of insect P450s in
xenobiotic detoxification and serve to identify
candidate genes for insecticide resistance.
56

3. Editing of genes to disrupt chemical communication and mating partner
identification
A. Drosophila melanogaster
 Fruit flies are attracted by a diversity of odors that signal the presence of food, potential mates,
or attractive egg-laying sites.
 In the case of Drosophila, a mutation in Or83b gene disrupted the selection of the egg-laying site
(host) and impaired olfactory detection capacity.
 Or83b Encodes a Broadly Expressed Odorant Receptor Essential for Drosophila Olfaction
 Dendritic localization of conventional odorant receptors is abolished in Or83b mutants.
 Or83b mutation disrupts behavioural and electrophysiological responses to many odorants.
 Or83b therefore encodes an atypical odorant receptor that plays an essential general role in
olfaction.
Larsson et al., 2004
57

B. Spodoptera litura
 Orco gene is an essential OR partner for both host and mate detection in
Lepidoptera.
 Knock out of the Orco (olfactory receptor coreceptor) gene in Spodoptera litura
caused distraction in mating partner selection and loss of identity toward host
plants leaving them anosmia.
 Orco knockout caused defects in plant odor and sex pheromone olfactory
detection in homozygous individuals
 Technique: CRISPR/Cas9 genome editing
 The CRISPR/Cas9 system induced Orco mutations at very high efficiency (89.6%)
80% of mutated individuals transmitted mutation to their progeny with up to 43.3%
germline transmission efficiency.
Koutroumpa et al., 2016
58

C. Helicoverpa armigera
 In insects, female adults release pheromones and convey males, the status of their
maturity prior to mating.
 Males access the pheromone signals and select mature females.
 Knock out of odorant receptor 16 (OR16) in H. armigera made males unable to
receive pheromone signals from mature females.
 Results in mating with immature females that subsequently led to dumping of
sterile eggs.
 Technique: CRISPR/ Cas9
 Knock out of OR16 receptor in lepidopteran pests can therefore be a novel and
effective strategy to regulate mating time for pest management in agricultural
crops.
Sun et al., 2017
59

4. Knock out of developmental genes
 Knocking out developmental genes such as abd-A (Abdominal A) gene, a transcriptional factor involved
in downstream regulation of various target genes that are extensively involved in development.
 Loss of function mutants through CRISPR/ Cas9 resulted in the generation of abd-A mutant
phenotypes in various agricultural pests like Spodoptera litura, Spodoptera frugiperda and Plutella
xylostella.
 Insects thus produced showed deformity in body segments, disarmed prolegs, anomalous gonads, and
embryonic lethality indicating the success of gene editing tools.
Target insect Target gene Reference
Spodoptera litura Slabd-A Sun et al., 2017
Spodoptera
frugiperda
Sfabd-A Wu et al., 2018
Plutella xylostella abdominal-A Sun et al., 2017 60

Background:
Editing plants for insect pest management has been less
exploited so far (Tyagi et al., 2020).
II. Genome editing in Plants
Base:
Most polyphagous pests recognize host plants using the plant’s own
volatile blends, visual appearance, gustatory clues, oviposition sites, and
their interactions are coevolved.
61

1. Knocking down susceptible genes
 Emerging as a reliable strategy
 Insects rely on important chemical components from plants for their
development, immunity, and behavior.
 In rice, CRISPR/Cas9-based knock down of CYP71A1 gene encoding
tryptamine 5- hydroxylase which catalyzes the conversion of
tryptamine to serotonin resulted in reduced growth in brown plant
hopper.
 The study was hypothesized on the fact that serotonin, a
neurotransmitter from plants is essential for larval immunity and
behavior.
Lu et al., 2018
62

2. Modification of plant volatile blends
Studies have demonstrated that changes in volatile blends retract insects from
host plants.
 In transgenic Arabidopsis thaliana plants, aphid (Myzus persicae) infestation
leads to release of a sesquiterpene hydrocarbon (E)-β-farnesene (Eβf) which
retracts feeding by other host populations and attracts a parasitic wasp
Diaeretiella rape that controls aphid population.
Note:
Care are should be taken such that the modification will not lead to deleterious
effects toward beneficial insect population.
Beale et al., 2006
63

3. Changing foliage colour
 Visual appearance of plants plays a prominent role in
the ability of insects to recognize and attack host
plants.
 Alteration in plant pigmentation has been found to
modify insect host preferences.
 Red leaf tobacco: Transgenic- Overproduction of
anthocyanin pigmentation- red coloration of leaves
 This change in leaf color proved to be acting as a
deterrent to the herbivores, Spodoptera litura and
Helicoverpa armigera confirming the importance of
leaf color and appearance on host recognition in
insects.
Upon feeding
S. litura- Delayed pupation
H. armigera- Increase in larval
mortality
Malone et al., 2009
64

Challenges to Managing Resistance
 Do not have adequate tools
 Not enough registered products to permit rotation
 The program may not be as effective
 The program may be more expensive
 Products with resistance risk for one pest are also used for others
65

66

Novel Approaches in Insecticide Resistance Management (IRM)

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