Jim's questions:

"Is 2% the "measured residual in the Suchail paper"?"

Yes.

"How do we know that all of the measured residual actually did bind to neurons, anyway?"

Because it remained in tissues with ACh receptors, and where there are ACh receptors, the IMI will bind to them.  Stoichiometry tells us there will always be some unattached molecules as well as attached ones, but since the compound localized, most of it was bound.  As Randy and I too pointed out, the residual amount lasted longest in the bee thorax.  The compound in the other tissues, including the head, cleared more rapidly after the *acute* application.

"Can someone walk us through an English description of the process by which the
dose is fed, digested, and moved around in the bee, and why all (or any!) of
it would collect in the brain, and then bind to specific neurons?"

In the C14 paper, the bees were deliberately fed an acute dose of IMI dissolved in sugar water after 2 hours of withholding food.  Then, the bees were allowed to freely consume ordinary sugar water without restriction.  Thus, they knew exactly how much each bee had consumed of an acute dose of IMI that was labeled with C14 along its carbon backbone, and they could track it within the bee.  The IMI first has to pass through the gut.  Cells in the gut wall have a detoxification mechanism called the "cytochrome P450 oxidase system" (read http://en.wikipedia.org/wiki/Cytochrome_P450 for more info) which metabolizes a lot of the IMI right away, before it can get into the body to contaminate the nervous system.  The C14 starts to accumulate in the rectum, showing that metabolism is indeed happening.  However, in a loose analogy to the situation with blood sugar in diabetics, who "spill" sugar from the blood into the urine when their proximal renal tubules are overloaded.....too much IMI can overwhelm the gut detoxification metabolism and get into the hemolymph (blood) of the bee.  Apparently, some is directly absorbed too...perhaps right through the lining of the mouth and esophagus...the route by which it bypasses the gut system is unclear but the evidence proves that it does.  Studies have shown contact toxicity to neonics, thus they do directly absorb it. To further aggravate the problem, it turns out that the IMI metabolites are also AChR agonists and they, too, overwhelm the gut and some get into the hemolymph.  Now these AChR agonists circulate everywhere and of course, localize in the synapses where they irreversibly bind to AChRs.  This binding is specific because neurotransmitter binding is specific.  Not all neurons have AChR, however insect neurons have a very large proportion of the AChR type.  So this IMI collects in the nervous tissue of the bee, most of which is in the head and thorax, and it also collects along motor end plates, of which there are many in the thorax because it's full of very large muscles.

I'm unhappy to think that there is a "scorecard" here, as I'm not trying to win anything except some clarity for folks who are interested and, like me, are trying to understand some of the complexity facing the bee situation.

But to address Jim's last questions, the "missing 30%" was in the other paper having to do with acute vs. chronic effects of IMI.  Suchail et al say that the missing 30% of the dose they administered was probably "hidden" as unknown metabolites that they weren't tracking.  In that study they didn't use C14, instead, they were quantifying the individual metabolites of the IMI, and they suggest that perhaps they didn't follow all of them.

Now to turn to Randy's comments and questions:

"So I sought the opinion of a world expert on neonics, Dr. John Casida of
Berkeley, who emphatically told me that the binding was not irreversible.
 This opinion has been experimentally supported by Cresswell, and by anyone
who has watched an insect recover from IMI intoxication."

Dr. John Casida of Berkely is a noted biochemist whose lab works (among other things) on dissociated membranes to determine the binding properties of different receptors, such as AChR.  As you so often say to us, Randy, we are interested in "field realistic doses".  And, as I have tried to convey to you, the binding properties of receptors in test tubes are not physiologically "field realistic".  It's very useful information, but it's not the same as the living animal.  In the lab, they can reversibly bind IMI and other neonics to AChR in isolated membrane preps by using even more powerful agonists or by using unrealistic concentrations of competitive agonists.  This competitive "reversible binding" is not possible in the nerve synapse of the living bee, where the IMI is not displaced by a stronger agonist, and once it binds to the AChR, it stays there a very long time.  Thus, as far as "us physiologists" are concerned, the binding in the living bee is functionally irreversible...I think this explains the apparent contradictions in the papers you cite.

How does an insect recover from acute IMI intoxication?  Well, it does.  But how?  Turns out that if you keep watching that exact same insect, it would die early, in about 7-10 days.  Why does it die early?  Because it was poisoned and the poison takes time to kill affected neurons and ultimately the bee.  But you are right that the acute effects disappear (twitching, strange body postures, etc) to be replaced by lethargy.  Poisoned bees "recover" from the acute effects but do not act normally after that.  Lethargy, less work, less productivity, foraging confusion, etc have all been described.  I don't know how to explain the reduction of the acute effects except to surmise that the cytochrome P450 mechanism can siphon off some of the IMI....but it doesn't get it all, as shown by Suchail and others.  I haven't read whether anyone has determined if one acute event is better or worse for a hive than a chronic "sublethal" event, but I would guess that the acute event is less bad, because as new bees are born that weren't exposed, the hive can right itself more easily than if there is always a slow dribble of poison coming in that affects every new bee as well as the old soldiers.

"It makes no difference whether it degrades to CO2 or into something else."

But it does matter, because the degradation products include at least two metabolites that are even stronger agonists than the IMI parent compound.  That matters.

"....the elimination half-life of radioactivity,
which represents the sum of radioactivity of all detected compounds in all
honeybee compartments, was about 25 h.   Christina, please refer to
Suchail's plate 1.  It clearly shows how the radioactivity in the thorax
(the main binding site) rapidly disappears."

Half of the compound was gone after 25 hours.  Plate 1 shows that the highest amount (of the total administered) in the thorax was 20%, and after 70 hours, 5% was still there.  That means it took 70 hours for 3/4 of what was originally distributed to the thorax to disappear, leaving 1/4 still in place.

"does not Suchail's study and subsequent
research suggest to you that even if IMI "irreversibly" binds, that its
biological/neurological  effect must rapidly diminish as the compound and
its metabolites disappear from the bee's body?"

Well, you say you already did the calculations that I worked out earlier today and shared with the list.  So what did your work indicate?  Mine says that 0.5 ng of IMI...consumed chronically as bees do when foraging on treated crops for days and then consuming the product in the hive....is enough to cause delayed mortality.

Randy, you bring up the cigarette smoker again.  You were very certain in telling me, a few months ago, that smokers would all be dead if nicotine were toxic.  I tried to explain the concept of adaptation to you then, and now that you seem to "get it", I'm glad.  But please keep in mind that nicotine is not as strong an AChR agonist to your neurons as IMI is to bee neurons.

Nice study out recently, someone sent to me...http://www.ncbi.nlm.nih.gov/pubmed/23443944...it shows that bees adapt to neonics by up-regulating acetylcholinesterase!  That's something I was waiting to find out and I hope the work in this new study is confirmed by others, as it offers us yet another piece of the story.  I'm betting that someone will figure out that bees also recycle AChR, and that they can slowly get rid of irreversibly-bound AChR that way.  Maybe some strains of bee can do these counterbalancing things more efficiently than others, helping to explain the variable susceptibility we are wondering about.

Christina

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