1. Insecticides 632 Lecture 6 - Application of cellular neuroscience to a practical problem

2. Cellular Neuroscience - Revision nResting potential nAction potential nChannels: u voltage gated, u ligand gated, ionotropic & metabotropic nChemical synaptic transmission

3. Aims of lecture nto know problems of effective application of insecticides nto know the main types of insecticides nto know their site(s) of action npossible mechanisms of resistance

4. Reading Matters nWeb see http://biolpc22.york.ac.uk/632 nBook: u Tomlin, CD S (1997) The pesticide manual nPapers u Narahashi T (1996) Neuronal ion channels as the target sites of insecticides Pharmacology & Toxicology 79: 1-14 u ffrench-Constant, RH et al (1998) Why are there so few resistance- associated mutations in insecticide target genes? Phil.Trans. R. Soc. B 353 1685-1693 u Matsuda et al (2001) Neonicotinoids: insecticides acting on insect nicotinic acetylcholine receptors Trends pharm. 11:573-580

5. Delivering insecticide effectively? nrapidity nspecificity u to target species u side effects nstability u light & air (oxygen) u not too persistent nsolubility ncheap

6. Main targets ndevelopment u ecdysis [moulting] specific to insects u cuticle specific to insects nrespiration nCNS

7. Why Knockdown nresting insects have low metabolic demand u unlike mammals u general respiratory or muscular poisons not so good? nknockdown insecticides u disable insect quickly u OK to kill slowly u target CNS

8. Main classes norganochlorine (1940s) ncyclodiene norganophosphorus npyrethroids (1975-) nImidacloprid (1990s) nphenyl pyrazoles

9. Organophosphorus nexample: malathion ncarbamates have similar action nmore toxic to insects nphosphorylate acetylcholinesterase nraises [ACh], so use atropine as antidote if humans are poisoned

10. Organophosphorus nphosphate group, with two CH 3 / C 2 H 5 and one longer side chain noften S replaces O malathion

11. Carbamates also related noriginally derived from calabar beans in W Africa naldicarb LD 50 5mg/kg

12. nInsects u OP  oxidase  much more toxic F cytochrome P450 oxidase in mitochondria, etc nVertebrates u OP  carboxyesterase  non-toxic More toxic to insects

13. Phosphorylate acetylcholinesterase nactive site of enzyme has serine - OH nactive site binds P from phosphate u half like very long (80 min) acetylcholine maloxon

14. Cyclodiene ne.g. Dieldrin, Lindane n once widely used n like other organochlorines, very lipid soluble

15. Cyclodiene mode of action naffects GABA A which carry Cl- currents u binds to picrotoxin site u not GABA site u enhances current u faster desensitisation dieldrin GABA induced Cl - current

16. Cyclodiene sensitivity ninsects are more sensitive to GABA A insecticides because u receptor is a pentamer  the  -subunit binds the insecticide  insect homooligomer  3 receptors  mammals have heterooligomer 

17. Phenyl pyrazoles nfipronil u also targets GABA A receptors u same site as Lindane

18. Organochlorine nDDT nlow solubility in water, high in lipids nat main peak of use, Americans ate 0.18mg/day u human mass 80kg nNa Channel effect nmore toxic to insects

19. DDT nsymptoms of poisoning are bursty discharges

20. Na current effect nNa current is slower to end in DDT orange bar marks stimulus

21. Pyrethroids nvery quick knockdown nneed an oxidase inhibitor nphotostable and effective u 30g/hectare (1% of previous insecticides\)

22. Pyrethroids nmajor current insecticide nderived from chrysanthemum nNa channel effect nmore toxic because of differences in Na sequence nmay also have other effects ? n typically esters of chrysanthemic acid

23. typical pyrethroids... aromatic rings & Cl or Br contribute to toxicity Deltamethrin most toxic nNo CN nhyperexcitation nconvulsions nCN next to ester bond nhypersensitive nparalysis

24. Na channel effect n Sodium current lasts longer u Voltage clamp n Note tail current controltetramethrin single voltage voltage series

25. Na channel effect - ii n Unitary sodium current lasts longer u patch clamp u type II open even less often but for even longer

26. more toxic because nof differences in Na channel sequence nrat mutant isoleucine  methionine in intracellular loop of domain 2 (I874M)

27. other effects ? nPyrethroids have been reported to affect u calcium channels u GABA, ACh, glutamate receptors

28. Imidacloprid nnewer nicotinic nbinds to ACh receptor

29. Imidacloprid ii stimulate nerve and record EPSP apply carbamylcholine

30. Summary so far nNa + channels targets of DDT, pyrethroids nAChEsterase targets of OPs nACh receptor target of Imidacloprid nGABA A receptor target of cyclodienes & fipronil

31. Problem of Resistance nresistance means that the insects survive! nsome species never develop, u e.g. tsetse flies - few offspring nmost very quick u e.g. mosquitoes - rapid life, many offspring ncross resistance, e.g. to DDT and pyrethroids because same target is used. n[behavioural resistance]

32. Resistance mechanisms norganophosphates norganochlorine ncyclodiene npyrethroids nsee Ann Rev Entomology 2000

33. Organophosphates ncarboxylesterase genes amplified u e.g. in mosquito, Culex, up to 250 x copies of gene/cell ncarboxylesterase gene mutated u higher kinetics and affinity ( Tribolium ) n detoxified by glutathione-S- transferases (i.e. addition of glutathione)

34. Organochlorine nDDT detoxified by glutathione-S- transferases (i.e. addition of glutathione) nNa channel resistance

35. Cyclodiene ntarget site change known as Rdl u resistance to dieldrin nGABA A receptor u alanine 302  serine [or glycine] u change affects cyclodiene, picrotoxin binding u and reduces desensitisation

36. Pyrethroids nnon-target resistance P450 oxidase u more transcription giving more expression u leads to cross-resistance to organophosphates & carbamates ntarget resistance Na + channel

37. Na + channel n kdr : leucine  alanine (L1014F) u 9 Musca strains nsuper-kdr : methionine  threonine (M918T)

38. Effect on currents M918T blocks current completely

39. Comparative mutations

40. Conclusions nCellular neuroscience helps understand many insecticide actions nbinding to channel proteins u ligand-gated u voltage gated nmutation leads to resistance u target site u enzymatic degradation nWeb page u http://biolpc22.york.ac.uk/632/