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 • Syngenta’s -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 (1%w/v)

23. EC Leaf Transects Polymer

24. -cypermethrin Field Screen: P. cochleariae on oil seed rape % mortality 90 % control g ai/ha Universityof 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. 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?

47. Conserved Asp 03:13 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