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Insecticide modes of action. The processes, properties and major compound classes that underpin crop protection. Stages involved in determining insecticidal efficacy. Delivery & formation of insecticide deposit Contact of a deposit by the target pest Bioavailability & dose transfer
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Insecticide modes of action The processes, properties and major compound classes that underpin crop protection
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
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
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
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
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
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
EC formulations • The pyrethroid -cypermethrin is often marketed as an emulsifiable concentrate or EC • Syngenta’s -cypermethrin EC, for example, is marketed under the product name Karate
-cypermethrin EC on glass • When sprayed onto a surface such as glass, -cypermethrin ECs dry to form incoherent residues of concentrated micro droplets
-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
-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?
Dried -cypermethrin EC • % Surface cover = 6
Dried polymer formulation • Unlike EC & oil based ULV formulations, polymer deposits of alphamethrin can be coherent
Results - a polymer formulation • % Surface cover = 94
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
Pick up and re-deposition of oils from cabbage leaf surfaces
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
Pick up and re-deposition of oils from cabbage leaf surfaces
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
-cypermethrin Field Screen: P. cochleariae on oil seed rape % mortality 90 % control g ai/ha Universityof Portsmouth
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
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
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
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
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
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
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
Tissue distribution • Large differences in the concentration of compounds accumulating in the various tissues are often observed • compound dependent • time dependent • tissue dependent
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
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)
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
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
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
G Protein-Coupled Receptors are 7 Trans-Membrane Helices (7TMs) Activate 2nd messengers via conformational change: cAMP, cGMP, IP3 G-Proteins bind to intracellular loops What are GPCRs?
Conserved Asp 03:13 Sequence and Property Data • 47 inward-facing amino acids • 3 Properties • 47 x 3 = 141 variables x properties
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
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
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