Molecular basis of
insecticide resistance in
vectors
Overview
■
Many vector species of public health importance
including mosquitoes have developed resistance
to one or more insecticides.
■
The emergence of insecticide resistance in a
vector population is a natural/evolutionary
phenomenon.
■
Resistance genes appear through random
mutations in which individuals are born "resistant”.
Cont.
■
Molecular genotyping of resistance is the
identification of the underlying genes that confer
the inherited resistance.
■
It provides understanding of both the degree of
resistance expressed in individual insects with
the resistance gene and the frequency of such
insects in the population.
Cont.
■
The magnitude of the problem can be appreciated
as the fact that, in 1946 resistance to insecticides
was reported in only 2 species of insects of public
health importance, in 1962 the number rose to 81
species & in 1997 to over 500 arthropods.
■
A knowledge of insecticide resistance is important
from the point of view of proper selection of
insecticides.
Insecticides
■
■
■
■
Insecticides are organized into classes—
organophosphates (OPs), carbamates (Carbs),
pyrethroids (Pys), neonicotinoids, etc.— that share a
common chemical structure and mode of action (MOA).
MOA is the specific process by which an insecticide kills
an insect, or inhibits its growth.
The target site of action is the exact location of inhibition,
such as interfering with the activity of an enzyme within a
metabolic pathway.
MOA and target site of action are often used
interchangeably in practice and are combined as MOA in
this learning module.
Insecticide resistance
■
When insect population can no longer be
controlled by a dose of insecticide which used to
provide control of them this is termed as
insecticide resistance.
■
However, for it to be considered true resistance,
the resistant insects must be able to pass on
the ability to resist the insecticide to their
offspring.
Definition of insecticide resistance
WHO’s definition (1957)
■“The development of an ability in a strain of insects to
tolerate doses of toxicants which would prove lethal to
the majority of individuals in a normal population of
the same species”.
■It’s
about a pest population NOT an individual:
Reduction in the susceptibility of a population to
pesticides.
■It’s
genetically based: heritable reduction in
susceptibility.
Cont.
Insecticide resistance Action Committee
(IRAC) defines resistance as:
“The selection of heritable characteristic in an
insect population that results in the repeated
failure of an insecticide product to provide the
intended level of control when used as
recommended”.
This definition is based on operational
performance of the insecticide
The champions
Green peach aphid
Whitefly
Colorado potato beetle
spider mite
Diamondback moth
cotton bollworm mosquito
Time until development of
resistance
■
■
■
■
■
■
Organophosphates – 14 years
Organochlorines (DDT) – 7 years
Carbamates – 5 years
Pyrethroids – 4 years
Neonicotinoids?
Formulations – Cockroach Gel Baits, Ear Tags
Types of resistance
1. Cross resistance:
❑resistance to one insecticide leads to
resistance to another yet unused insecticide.
❑Usually the two insecticides belong to the
same class and share identical or similar mode
of action.
2. Multiple resistance:
❑resistance to multiple insecticides of different
classes by multiple mechanisms.
❑Consequence of sequential application of
insecticides (Pesticide treadmill).
1. Cross-resistance
■
Cross-resistance often occurs between
insecticide class that have the same mode of
action for killing vectors.
■
For example, if a resistance gene creates a
change in target site in a vector, it is likely to
affect any other insecticides that attack the same
target sites, thus conferring the cross-resistance.
Cont.
■
Similarly, an alteration to an enzyme that affects
susceptibility to one insecticide may result in
cross-resistance to another.
■
Eg, In metabolic resistance, cross resistance
between PY and carbamates associated with
mutations in cytochrome P450 enzymes
detected.
2. Multiple resistance
■
Occurs in insect populations that resist two or
more insecticide classes with unlike modes of
action.
■
Insects develop this type of resistance by
expressing multiple resistance mechanisms.
■
Localized populations of Colorado potato beetle
are notorious for multiple resistance to more than
50 insecticides with various modes of action.
Cont.
■
Example: Knock down resistance gene (kdr)
confers resistance to DDT and pyrethroids.
■
Multiple resistance is less common than crossresistance but is potentially a greater concern
because it drastically reduces the number of
insecticides that can be used to control the
insect in question.
Insecticide tolerance
■
■
■
In contrast to resistance, insecticide tolerance is a
natural tendency and is not a result of selection
pressure.
Mature caterpillars are more tolerant to many
insecticides than younger ones of the same
species due to differences in body size,
exoskeleton thickness, and the ability to
metabolize a poison.
These differences are identified as tolerance or
natural resistance rather than true insecticide
resistance.
Evolution of Insecticide Resistance:
How does it occur?
R
Individuals in a population are never equally susceptible to
an insecticide. Although initially rare, insecticide-resistant R
genotype is present. Frequency is 1/12 = 0.083
Evolution of Insecticide resistance
R
An insecticide is used, leaving insecticide-resistant individuals
(their R genotype) and some susceptible individuals (S genotype)
Cont.
R
R
R
R
If R genotype reproduces as well as S genotype, in the
next generation the frequency of R will be the same as
the survivors in the preceding generation, i.e. 0.333
R genotype frequency
Cont.
Generations
Evolution of insecticide resistance
❑ When LD50 ratio ≥ 10, resistance
occurs.
❑ Resistance: occur through
insecticide selection.
❑ Selection acts on genetically-based
variation in susceptibility which
arise from:
– Mutation, the source of all new genetic
variation.
– Genetic recombination that rearranges
genetic variation.
– Gene flow from populations having
different allelic frequencies.
Factors influencing evolution of
resistance
1. Genetic factors
1. Biological factors
1. Operational (application) factors
1. Genetic Factors influencing
evolution of resistance
❑ Number of R gene: quicker
RR
❑ Dominance of R alleles:
quicker if R allele is
dominant.
❑ Initial frequency of R
alleles: the higher the
quicker.
❑ Fitness of R alleles:
quicker if no fitness cost.
% mortality (Probit Scale)
if one major gene.
SS
SS
RS
RS
RR
Log dose
2. Biological factors that promote
resistance
❑ Short generation time.
❑ High fecundity.
❑ No (or little) migration occurs between
populations.
❑ The species is highly mobile, increasing the
possibility of exposure to insecticides.
❑ Low economic threshold (ET).
❑ Use the same insecticide every
generation.
❑ A large geographical area is
treated.
❑ High dose.
❑ No refuge exists.
❑ Long persistent insecticide or
slow-release formulations.
❑ Use insecticides related to one
used earlier.
Pest density
3. Operational factors that promote
resistance
Spray
ET
Weeks
Insecticide Resistance
Mechanisms (IRMs)
❑
1.
IRMs are grouped into four categories:
Metabolic resistance.
1.
Target-site resistance.
1.
Physiological resistance.
1.
Behavioural resistance.
Mechanisms of Insecticide
Resistance
Activated
Insecticide
Target sites
Detoxified
Sequestrated
Excreted
Intoxication
Death
1. Metabolic Resistance:
■
This is the most common insecticide resistance
mechanisms in insects and it is based on
enzyme systems.
◻
■
RR individuals have more enzymes or more efficient
enzymes.
Insects can become resistant to insecticides,
when the enzymes which break down unwanted
molecules are either significantly increased in
amount or modified to become more efficient at
breaking down the insecticide.
Cont.
■
It occurs when increased or modified activity of
an enzyme system prevent an insecticide from
reaching its intended site of action.
■
The three main detoxifying enzyme systems are:
esterases (CarE), mono-oxygenases
(Cytochrome P450 monoxygenases (P450)
and glutathione S-transferases (GST).
Cont.
❑ Esterases: involved in resistance to
organophosphates (OP), carbamates (Carb), and
Pyrethroids (Py).
❑ P450: Cytochrome P450 monooxygenases,
involved in metabolism and resistance to all
classes of insecticides.
❑ GST: involved in resistance to DDT, OP and Py.
Cont.
■
Nearly all of the strains of Culex quinquefasciatus which
resist a broad range of OP insecticide have been found
to posses multiple copies of a gene for esterases,
enabling them to overproduce this type of enzyme.
■
In contrast strains of malathion resistant Anopheles have
been found with non-elevated levels of an altered form
of esterase the specifically metabolizes the OP
malathion at a much faster rate than that in susceptible
individuals.
■
Metabolic resistance mechanisms have been identified
in vector populations for all major classes of insecticides.
Molecular mechanisms of resistance
Esterase in
mosquitoes
green peach aphid
P450, GST, esterase
esterase, Target sites
2. Target-Site Resistance:
■
Target-site insensitivity: the 2nd most common
mechanisms, called altered target-site
resistance.
■
Insecticides generally have a specific site of action
within an insect (the nervous system), this is
usually a receptor protein.
■
When a mutation occurs in the genetic code for the
receptor protein, it can modify the shape of the
protein and prevent the insecticide from interacting
at the site of action and thus confers resistance.
Reduced target sensitivity
❑ AchE insensitivity: resistance to OP and Carb.
❑ Sodium Channel insensitivity: knockdown
resistance (kdr) to DDT and Py. Super kdr
❑ GABA receptor (Cl- channel) insensitivity:
resistance to cyclodiens, fipronil, and avermectins.
❑ AchR insensitivity: resistance to nicotin,
neonicotinoids and spinosad
Cont.
❑
Occurs when the site of action of an insecticide is
modified in a resistant strains, such that the
insecticide no longer binds effectively.
❑
Result: the insect is unaffected or less affected by
the insecticide. Kdr = DDT & PY, Ace-1 = OP and
carbamates.
❑
Reduced susceptibility to PYs conferred by kdr
mutations has been confirmed in An. gambiae in
West, Central and East Africa.
3. Physiological Resistance:
❑
A mutation may cause a physical adaptation of
the insect which helps it to protect against the
insecticide, such as a thicker cuticle, extra
waxy covering, or faster excretion of waste.
❑
Reduces uptake of insecticide due to the
modification of the insect cuticle that prevent or
slow absorption of insecticides by:
❑
Reduced penetration
❑ Increased Sequestration/seizure
❑ Increased direct excretion
Reduced penetration
❑ RR individuals have a pen
gene that reduces
penetration of insecticides 2
to 3 fold due to:
– Modified composition
– Modified structure
Insecticides
Epicuticle
Endocuticle
❑ Protects pests from a
wide range of contact
insecticides.
❑ Usually present along
with metabolic and/or
target mechanisms to
amply resistance.
Exocuticle
Penetration
Hemolymph
Target site
Increased Sequestration
Epicuticle
• Integument
• Fatbody
Exocuticle
Endocuticle
Increased direct excretion
Excretion
Insecticides
Example: Tobacco hornworms
Cont.
■
At present there are 40 malaria-endemic
countries reporting physiological resistance to
insecticides, most to pyrethroids.
■
E.g. A single gene mutation (knock down
resistance gene or kdr) has mutated the Na-K
Pump of An. gambiae.
4. Behavioural Resistance:
■
Although not common, there are examples where
mutations have altered the natural behaviour of
the insect, reducing exposure of the insect to the
insecticide and allowing it to survive (detect and
avoid the toxin).
■
Any modification in insect’s behaviour that helps to
avoid the lethal effects of insecticides.
■
Changes in vectors resting and feeding behaviour
to minimize contacts with insecticides.
Cont.
❑ 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.
❑ An. funestus now bites late in the
evening rather than at midnight.
❑ Houseflies: avoid treated
surface.
❑ Cockroaches: avoid treated
surface and baits.
❑ Diamondback moth: avoid
permethrin.
Run from insecticides
Factors that promote resistance
development
1.
Frequency of application: many applications
over a large geographic area quicken the
development of resistance.
❑
1.
Overuse and misuse
Persistence of residues: long persistence of
the insecticides for indoor residual spraying
(IRS) and Long-lasting insecticidal nets
(LLINs) have a strong selection effect leading
to quicker resistance.
Cont.
3.
Rates of reproduction: short life cycle and
high rate of reproduction speeds resistance
development.
◻
3.
Mosquitoes have short life cycle and high fecundity
Population isolation: open population allows
migration of susceptible individuals which will
have diluting effect.
END
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