Insecticide modes of action

1
Insecticide modes of action

• The processes, properties and major compound
  classes that underpin crop protection

2
Stages involved in determining insecticidal
efficacy

• Delivery formation of insecticide deposit
• Contact of a deposit by the target pest
• Bioavailability dose transfer
• Penetration through the insect integument
• Distribution to the tissues
• Metabolism
• Excretion
• Interaction at the site of action its
  consequences

3
Classification of these stages

• Physical processes
• delivery to a target or intermediate surface
• form of deposit its bioavailability
• Biological physiological processes
• effect of target behaviour on interception dose
  transfer
• pharmacokinetics
• penetration, tissue distribution, metabolism,
  excretion
• pharmacodynamics

4
Conventional formulations

• Insecticides are applied to crops using
  conventional formulations such as ECs and WPs,
  but new formulations are now being developed
  based on new technologies
• Conventional formulations are retained on the
  intermediate plant surface and spread before
  drying but tend to provide an incoherent deposit
• Formulations can also be applied directly to the
  surface of the target insect

5
Delivery of insecticides to target crop surfaces

• Delivery is normally achieved using water based
  (high volume) or oil based (low volume) sprays
• The number and size distributions of the
  insecticide droplets or particles deposited can
  vary substantially with profound implications for
  persistence, encounter subsequent transfer to
  the target organism

6
Pesticide deposits on crop surfaces

• An amorphous pesticide deposit
• spreads and dries
• remains in intimate contact with the surface
  waxes and plant epidermis
• comprises many adhered insecticidal particles or
  droplets

7
Deposit form

• The form of the surface deposit changes with time
  after application
• Its final appearance, the number, size and
  distribution of the component particles or
  droplets is a function of
• its rate of drying and
• the nature of the formulation in which it was
  delivered

8
EC formulations

• The pyrethroid ?-cypermethrin is often marketed
  as an emulsifiable concentrate or EC
• Syngentas ?-cypermethrin EC, for example, is
  marketed under the product name Karate

9
?-cypermethrin EC on glass

• When sprayed onto a surface such as glass,
  ?-cypermethrin ECs dry to form incoherent
  residues of concentrated micro droplets

10
?-cypermethrin EC on glass

• The range of micro droplet sizes can be very
  large
• Moreover, the form of the deposit may change with
  time after application

11
?-cypermethrin EC on glass

• The retained deposit dries to form an incoherent
  crystallising liquid
• What is the biological efficacy of such a
  deposit?
• How much dose is transferred?

12
Encounter, Dose Transfer and Pharmacokinetics
13
Dried ?-cypermethrin EC

• Surface cover 6

14
Dried polymer formulation

• Unlike EC oil based ULV formulations, polymer
  deposits of alphamethrin can be coherent

15
Results - a polymer formulation

• Surface cover 94

16
Oil based ULV formulations

• Like Ecs, involatile oil based ULV formulations
  are similarly comprised of discrete droplets of
  a.i., although the droplet size distribution will
  usually be more tightly controlled
• Because of the low vapour pressure of the oil
  carrier, these formulations remain as liquids and
  can flow during dose transfer

17
Pick up and re-deposition of oils from cabbage
leaf surfaces
18
Mathematical model of the dose transfer process

• The proportion of a deposit placed on a cabbage
  leaf surface that is transferred to a contacting
  mustard beetle is given by the expression
  pt.e-prN Rf
• where pt is the proportion picked up per contact
  available for redeposition, pr is the
  proportion redeposited per contact, Rf is the
  fraction retained N is the number of
  contacts following the initial encounter

19
Pick up and re-deposition of oils from cabbage
leaf surfaces
20
Pick up and re-deposition of polymer formulations

• EC and Oil based ULV formulations may have high
  initial bioavailability as a result of rapid flow
  from leaf to insect to result in large values of
  pr,
• but the exponent, pr, may also be large !
• Polymeric formulations can have high longer term
  bioavailability because re-deposition of a.i. is
  reduced leading to high values for the fraction
  retained, Rf

21
Rape leaf surface revealing wax blooms (ca. 1mm
diameter)
22
Light micrograph of polymer deposit boundary
(1w/v)
23
EC Leaf Transects Polymer
24
?-cypermethrin Field Screen P. cochleariae on
oil seed rape
mortality
90 control
g ai/ha
University of Portsmouth
25
Pharmacokinetics - penetration

• Once an insecticide has been encountered
  transferred to the target, it must penetrate
  through the insect integument and enter the
  insect body where the site of action is located
• The factors determining the rate and extent of
  the insecticide penetration process can be
  investigated using diffusion cells

26
Static diffusion cell
27
Penetration profiles
28
Insecticide flux across isolated cuticles of
Spodoptera littoralis

• Flux increases inversely
• with molecular weight (MW)
• with log P
• Lag times increase
• with increasing dipolar character of a molecule

29
Relationship between lag time and dipole moment
30
Loading unloading the cuticle

• During penetration, the cuticle accumulates
  penetrant as steady state conditions are attained
• The loaded material is retained by the cuticle
  and can prove difficult to remove
• The cuticle can therefore act as a depot
• reducing the amount of insecticide available to
  reach the site of action, e.g. imidacloprid

31
Recovery of Imidacloprid in successive extractions
32
Interpretation of penetration results

• Flux is determined by
• partition across the interface between the thin
  epicuticular waxes and the more polar region
  beneath
• the rate of diffusion across the thick integument

33
Interpretation of penetration results

• Lag time is determined by
• the time taken to load up the wet endocuticle
  which has a large capicitance for polar molecules

34
Practical consequences

• Small, polar molecules move rapidly across the
  cuticle surface, but a large proportion may be
  retained in the wet endocuticle
• Larger, non-polar molecules have lower fluxes but
  shorter lag times
• If, as with the pyrethroids, the intrinsic
  activity is very high, lag time rather than flux
  may determine speed of action

35
Tissue distribution of a nicotinoid insecticide
36
Elimination of a nicotinoid insecticide
37
Practical consequences

• For most tissue compartments, detoxication is
  slow and steady state tissue equilibria are often
  established
• The major route of elimination of the applied
  insecticide is from the hind gut as faeces
  (frass)
• A second route, regurgitation is observed
  whenever the dose reaches levels of intoxication
• In vivo metabolic degradation does occur can also
  occur

38
Tissue distribution

• Large differences in the concentration of
  compounds accumulating in the various tissues are
  often observed
• compound dependent
• time dependent
• tissue dependent

39
Tissue composition

• The ratio DW/(WW-DW) provides a measure of the
  relative amounts of organic material and water in
  a tissue
• This tissue partition coefficient can be used
  to predict the tissue concentration of a putative
  insecticide at steady state

40
Tissue composition and compound distribution
41
Tissue distribution

• There is an approximately 10-fold change of
  tissue concentration for a 105-fold change in
  logP
• Tissues range in composition
• from ca. 10 times as much water as organic
  material (haemolymph)
• to ca. 3 times as much organic material as water
  (nerve cord)

42
Movement of radio-label

• Labelled material applied topically to the
  external surface of the cuticle
• moves through the cuticle into the haemolymph,
  gut wall gut contents is then eliminated in the
  faeces
• tissues bathed in haemolymph are exposed to label
  which accumulates to reach a steady state
• non-polar materials remain in the tissue even
  after the levels in the haemolymph may have fallen

43
Mammillary model of pharmacokinetics
44
What is an insecticide site of action?

• A site of action is macromolecular structure to
  which the insecticide binds in order to exert its
  toxic action
• Sites of action vary depending on the nature of
  the interacting ligand and the macromolecule to
  which it binds
• These interactions may involve protein receptors,
  enzymes or components of the insect integument

45
What is an insecticide site of action?

• Different insecticidal classes have different
  pharmacodynamic modes of action depending on
  chemical structure and the resulting molecular
  properties
• These must complement those of the macromolecule
  closely for tight binding high insecticidal
  activity
• This requirement can be illustrated using
  G-protein coupled receptors as an example

46
What are GPCRs?

• Activate 2nd messengers via conformational
  change cAMP, cGMP, IP3

• G Protein-Coupled Receptors are 7 Trans-Membrane
  Helices (7TMs)

47
Sequence and Property Data

• 47 inward-facing amino acids
• 3 Properties
• 47 x 3 141 variables x properties

48
Sites for ligand binding

• Different ligands bind to different receptor
  pockets
• Each pocket is constructed of a set of amino acid
  side chains whose local surface properties match
  those of the ligand

49
Molecular surface properties

• These ParaSurf representations show the location
  of three such properties on a pyrethroid a
  receptor sidechain
• ionisation potential (red), electron affinity
  (green) polarisability (blue)
• For tight binding, these must be complementary
  lie within critical distances of each other
• Furthermore, their local hydration surfaces must
  be complementary

50
Molecular surface properties

• These ParaSurf representations show the location
  of three such properties on a pyrethroid a
  receptor sidechain
• ionisation potential (red), electron affinity
  (green) polarisability (blue)
• For tight binding, these must be complementary
  lie within critical distances of each other
• Furthermore, their local hydration surfaces must
  be complementary

51
Molecular surface properties

• These ParaSurf representations show the location
  of three such properties on a pyrethroid
  receptor sidechains
• ionisation potential (red), electron affinity
  (green) polarisability (blue)
• For tight binding, these must be complementary
  lie within critical distances of each other
• Furthermore, their local hydration surfaces must
  be complementary

52
Molecular surface properties

• These ParaSurf representations show the location
  of three such properties on a pyrethroid a
  receptor sidechain
• ionisation potential (red), electron affinity
  (green) polarisability (blue)
• For tight binding, these must be complementary
  lie within critical distances of each other
• Furthermore, their local hydration surfaces must
  be complementary

53
Molecular surface properties

• These ParaSurf representations show the location
  of three such properties on a pyrethroid a
  receptor sidechain
• ionisation potential (red), electron affinity
  (green) polarisability (blue)
• For tight binding, these must be complementary
  lie within critical distances of each other
• Furthermore, their local hydration surfaces must
  be complementary

54
Development of insecticides with different modes
of action

• Insecticides have probably been used by man since
  soon after the development of agriculture
• Initially organic plant products or inorganic
  materials would have been used
• Development of these crop protectants was
  initially slow and by the outbreak of World War
  1, the known insecticide classes in common use
  included the arsenicals, pyrethroids, derris and
  nicotine

55
Insecticide pharmacodynamic modes of action

• These groups had characteristic features, but
  their modes of action were unknown
• During the 1930s, Bayer became engaged in
  developing nerve poisons for military purposes
• A group based on organophosphate compounds proved
  to be effective insecticides

56
Organophosphate insecticides
profenofos
diazinon 
dichlorvos
parathion
57
Organophosphate insecticides
thiometon
acephate

• These OP compounds are now known to act as
  inhibitors of the enzyme acetyl cholinesterase
  which is responsible for breaking down the
  neurotransmitter acetyl choline
• Non-phosphate esters may be oxidised to the more
  insecticidal phosphates within the insect body
  (metabolic activation)
• The more polar OPs, e.g acephate, show systemic
  activity

58
Organochlorine insecticides
DDT
?-HCH

• A second group of compounds were developed during
  and after world war II based on halogenated
  hydrocarbons
• These included groups with different sites of
  action, but all had a common feature of long
  persistence in the environment
• ?-HCH has high vapour pressure and can act as a
  fumigant

59
Organochlorine insecticides
dicofol

• OCls are nerve poisons acting on the axon
  membrane
• Some, e.g. DDT and its analogue dicofol, have
  since been shown to act at the voltage-gated
  sodium channel which is similar in structure to a
  GPCR

60
Organochlorine insecticides
chlordane
endosulfan

• ?-BHC and the cyclodienes chlordane endosulfan
  act as antagonists of the GABA receptor-chloride
  channel complex
• Cyclodienes such as aldrin dieldrin are now
  banned because of their ecotoxicity persistence

61
More recent insecticide developments

• Concerns over the environmental behaviour
  toxicity of these classes resulted in intense
  research after World War II
• As a result, a new generation of safer, less
  persistent environmentally friendlier
  insecticides were developed

62
More recent insecticide developments

• The OPs were further refined to result in new
  chemical classes, eg. carbamates, acting at the
  same site of action but with improved properties
• species specificity, optimised persistence, low
  mammalian toxicity, a range of physico-chemical
  properties giving a variety of uses as fumigants,
  soil insecticides, systemic compounds

63
Carbamates
bendiocarb
fenobucarb

• They inhibit acetyl choline esterase, acting
  primarily as contact stomach poisons with
  systemic action
• their MWs, water solubility, dipolar character
  log Ps are appropriate for penetration across the
  cuticle and gut wall

64
Development of synthetic pyrethroids

• The structure of the natural pyrethroids (left)
  had finally been established in the late 1940s,
  leading to the synthesis in 1949 of the first
  synthetic pyrethroid, allethrin later resolved as
  bioallethrin (right)
• This synthetic compound had both knockdown and
  killing action, but was too photolabile for use
  in crop protection
• To overcome this, the alcohol moiety was replaced
  by a m-phenoxybenzyl ring (phenothrin)
  different substituents were placed at the
  terminal sp2 carbon (permethrin)

65
Development of synthetic pyrethroids
phenothrin
permethrin

• The first photostable pyrethroid, permethrin,
  suitable for use in crop protection was developed
  by Elliott and Janes in 1973
• The recognition that this class acted at a known
  receptor, the sodium channel, led to rapid
  development of new compounds
• with improved environmental properties and an
  extended range of applications

66
Development of synthetic pyrethroids
cypermethrin
deltamethrin

• Later that decade, Elliott, Janes and Pullman
  synthesised new Class II pyrethroids, such as
  cypermethrin deltamethrin, containing an
  ?-cynao group subtended by the benzylic carbon
  atom
• restricting motion about the ester bond and
  enhancing killing, but diminishing knockdown
  activity

67
Insect Growth Regulators
diflubenzuron
lufenuron

• IGRs act at a site within the cuticle disrupting
  cuticle formation, the process of ecdysis hence
  growth, development metamorphosis
• They inhibit chitin synthetase, acting primarily
  as stomach poisons
• their MWs, water solubility, dipolar character
  log Ps are inappropriate for cuticular
  penetration, but allow passage across the gut wall

68
Neonicotinoids
imidacloprid
thiamethoxan

• Developed from unstable nitromethylene compounds
• Act as agonists at the nicotine receptor (a GPCR)
• Affect cholinergic transmissions in the insect
  central nervous system.
• Are generally more polar than OPs, carbamates and
  pyrethroids
• act as stomach contact poisons with systemic
  properties

69
Fermentation products

• Microbial products produced as a result of
  industrial scale fermentation
• Include complex organic molecules such as the
  avermectins (above) which stimulate the release
  of GABA, an inhibitory neuro transmitter, thus
  causing paralysis

70
Fermentation products

• Also includes a group of toxic proteins produced
  by the microbial insect pathogens Bacillus
  thuringiensis, B. subtilis B. sphaericus
• Spores or extracted protein endotoxin act as an
  insecticide with stomach action
• Following ingestion, the crystals of endotoxin
  are solubilised the epithelial cells of the gut
  are damaged, insects stop feeding and eventually
  starve to death

71
Properties of representative insecticides for
major classes
72
Acknowledgements

• University of Portsmouth
• David Salt
• Richard Greenwood
• Bob Loveridge
• Nasir Chowdhury
• David Livingstone
• Brian Hudson