Hello James,

I very much enjoyed your thoughtful, engaged response to the issue of whether or not neonics are "irreversible" in their binding affinities.

You provided us with multiple references.

I looked at most of them.   On the topic of "irreversibility", what is said in your references is as follows:

1.SELECTIVE TOXICITY OF NEONICOTINOIDS ATTRIBUTABLE TO SPECIFICITY OF INSECT AND MAMMALIAN NICOTINIC RECEPTORS:  Annual Review of Entomology,  Tomizawa and Casida.

These authors reference previous work and do not prove in this paper that neonicotinoids are rapidly unbound from the acetylcholine receptors.  In referencing previous work, they only state that neonics are highly selective for invertebrates.  That observation is true, based on several studies.  Neonics are much more selective for invertebrate than vertebrate ACh receptors.  Reversible binding is not mentioned.

2.  Nauen, R., Ebbinghaus-Kintscher, U., Elbert, A., Jeschke, P., Tietjen, K., 2001. Acetylcholine receptors as sites for developing neonicotinoid insecticides. In: Ishaava, I. (Ed.), Biochemical Sites of Insecticide Action and Resistance. Springer, Berlin, Heidelberg, pp. 77-105.

The authors do not state anything about voltage clamping or rapidly reversible binding.  The principle author (Nauen) in fact says differently in another paper:

http://www.ncbi.nlm.nih.gov/pubmed/11464788

3.  Jeschke, P., Nauen, R., 2005. Neonicotinoid insecticides. In: Gilbert, L.I., Iatrou, K.,Gill, S.S. (Eds.), Comprehensive Molecular Insect Science. Elsevier B.V., Oxford, pp. 53-105.

This paper looks at different levels of resistance to Imidacloprid (IMI) among barn flies.  For some of the strains, the resistance mechanism was known.  This paper has nothing to do with either voltage clamping or ACh receptor binding affinities.  Most of the resistance had to do with either metabolism or alteration of receptor characteristics.  It was interesting to learn that there is a wide range of susceptibility to IMI among different strains of the same fly in nature as well as among "lab fly" strains.

4.  Liu, M.Y., Casida, J.E., 1993. High-affinity binding of [3H] Imidacloprid in the insect acetylcholine receptor. Pestic. Biochem. Physiol. 46, 40-46

Quoting from the results:  "Preliminary studies on 3H-IMI binding in house fly membranes indicated very rapid and high specific binding.  Optimization of the system involved determination of binding site stability and the effects of temperature and detergent concentration.  Frozen heads and membranes retain full activity for several weeks at -70 degrees C adn there is no loss in binding to membranes within 4 hr at 22C.  Under the standard assay condition but without Triton X-100, the binding varied with temperature as follows:  76% at 0C, 100% at 22C, 48% at 37C, 35% at 47C, and 7% at 57C. ....Specific binding accounts for about 95% of total binding....The rapid phase of the association for about 90% of the binding within a few seconds is followed by a slow phase for the remaining 10%."

Note that "rapid and high specific binding" does not mean "rapidly reversible binding".  It means the binding is very fast and very specific.  Later, they tell us that there are two binding phases (usually this tells biochemists something about the nature of the binding site(s) on the receptor).  They conclude that there is essentially one binding site in the insect version of AChR.  They say that the strong binding affinity of insect receptors for IMI and the relatively weak affinity of vertebrate AChR for IMI makes this a good candidate pesticide.

5.  Nauen, R., 1995. Behaviour modifying effects of low systemic concentrations of imidacloprid on Myzus persicae with special reference to an antifeeding response. Pestic. Sci. 44, 145-153

Quoting the abstract:

"From the results of these experiments and the observed symptomatology it is possible to postulate two different and dose-dependent modes of action of imidacloprid on M. persicae: (1) the well-known mode of action with visually obvious irreversible symptoms (paralysis, tremor, uncoordinated leg-movement) at field rates, and (2) the reversible starvation response as an antifeedant effect, which is not coupled with typical symptoms of neuronal disorder, at lower concentrations."

It is important to realize they are talking about metabolism and recovery after removing the aphids from the source of IMI.  This paper does not say that AChR binding is reversible, it says that the animals recover by starving after removing them from the IMI source.  Maybe it's most relevant to beekeeping to say that by removing the source of IMI after drinking it for awhile, the aphids didn't die (although they were clearly far from healthy thereafter).  But please don't confuse this with receptor binding affinities.

6. Schmuck, R.  Effects of a Chronic Dietary Exposure of the Honeybee Apis mellifera (Hymenoptera: Apidae) to Imidacloprid

Schmuck says in his introduction that "Nauen et al. (2001) demonstrated that the binding of imidacloprid and its insecticidally active metabolites to the nAChR was fully reversible."  So of course I went to that article:

Nauen R, Ebbinghaus-Kintscher U, Schmuck R (2001) Toxicity and nicotinic acetylcholine receptor interaction of imidacloprid and its metabolites in Apis mellifera (Hymenoptera: Apidae). Pest Manag Sci 57:577–586

We see that Schmuck was the senior author on this one.

Interestingly, they point out that honeybees fed a lower dose of IMI in sucrose solutions end up imbibing more of it.  They say this is probably due to the fast neurological effects of the higher concentration (acute dose), that prevents the bees from continuing to feed.  They found that the LD50 at 48 hours was about the same as the acute oral/contact dose.

This tells me that IMI builds up in the system as the bees ingest it over time.

"IC50 values for the displacement of [3H]imidacloprid by several metabolites such as olefine, 5-OH-, 4,5-OH-imidacloprid, urea and 6-CNA were 0.45, 24, 6600, > 100,000, and > 100,000 nM, respectively. Displacement of [3H]imidacloprid by imidacloprid revealed an IC50 value of 2.9 nM, thus correlating well with the observed acute oral toxicity of the compounds in honeybees."  It means that IMI binding is highly specific and very strong...unlikely to dissociate from the AChR under normal conditions.  In my opinion, they are being misleading when they say that the binding is fully reversible.  In the lab, yes.  In nature???  Not likely, given that the displacement kinetics are completely unnatural.  Those metabolites aren't going to be found in a normal synapse, and there isn't anything else except ACh in there to compete with IMI.

Thanks for a very thorough and challenging set of papers to read.  I didn't continue with the several additional Schmuck papers you cited, these were the ones dealing with field-relevant effects.  I'll get to them another time. I don't agree with you that there is no proof of cumulative effects.  I do agree that "sublethal" means that bees don't die outright, that they continue to fly, bring home food, and raise the family.

Christina

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