The Group That Carries the Selectivity: Neonicotinoids and What Metabolism Removes

1. Introduction: selectivity with an address

This journal has written about several insecticide classes and asked of each where its safety margin comes from. For most the answer is diffuse. For this one it is a specific chemical group meeting a specific amino acid.

1.1 The first for which that was the reason

Rather than the first to be selective at all.⁵

1.1b And the class is not marginal to this trade

Compounds in this group appear in termite and ant products, in veterinary spot-ons and collars applied to animals living in the buildings we service, and in the agricultural products used on the land surrounding this city.

1.2 What this article argues

That a margin located in one binding interaction is a margin one metabolic step can remove, that this is the second time we have found that shape, and that the same residues carrying the selectivity carry the resistance. Sections 10, 12 and 21 are the case.

2. Why the class exists

Which is a sentence our resistance articles have been building toward.

These chemicals were first introduced in the early 1990s as alternatives to organophosphates, pyrethroids and carbamates which were losing effectiveness due to insect resistance.⁷

2.0b Which dates the class precisely

Early 1990s, which makes it the newest of the major structural classes and roughly contemporaneous with fipronil.⁷

2.1 A class introduced because three others were failing

Which is the sequence our article on rotation and mixtures described as the nonstrategy benchmark: a new mode of action arriving when the previous one runs out.⁷

2.1b Which makes the class a resistance artefact as much as an invention

The commercial opening existed because three earlier classes were failing, so the compounds were selected against a problem defined by what had already stopped working rather than by what would work best in the abstract. That framing is ours.

2.2 And they were developed as systemics

Moving within the plant, which is the property that placed them in the pollinator debate and which our exposure article approached from a different direction.⁷

3. What they do

At the receptor.

Imidacloprid is an agonist at nicotinic acetylcholine receptors and shows selective toxicity for insects over vertebrates, with both alpha and non-alpha subunits contributing to the interaction.⁶

3.0b And the receptor is shared between the taxa

Both insects and vertebrates use these receptors for fast cholinergic transmission, which means the class is not selective by virtue of the target being absent in one of them. The target is present in both and differs in detail.⁴

Which distinguishes it from the glutamate-gated chloride channels our fipronil article described as observed only in invertebrates, where selectivity comes from a target one side does not have.

3.1 An agonist rather than a blocker

It activates the receptor rather than preventing acetylcholine from doing so, which puts it alongside pyrethroids as a compound that adds signal rather than removing it.

3.2 And the receptors are the fast cholinergic ones

Ligand-gated ion channels mediating fast cholinergic synaptic transmission in insect and vertebrate nervous systems, which is why both taxa have them.⁴

4. The claim that makes them unusual

Quoted in §1.

Earlier insecticides were selective. What is claimed here is that this class is the first whose selectivity is attributable to affinity at the target itself.⁵

4.1 And the qualification in front of it matters

Not the first to target these receptors, and not the only selective insecticides, which is a narrow and careful claim rather than a sweeping one.⁵

5. Which is worth separating from the others

Our framing, drawing on articles we have already written.

An organophosphate can be selective because insects activate it and mammals detoxify it faster. A bait can be selective because only the target eats it. A crack and crevice placement can be selective because only the target goes there.

5.0b Which is not a criticism of the older kinds

Selectivity by placement is the basis of the crack and crevice category and of every bait in this journal, and it works. The distinction being drawn is about where the margin lives rather than about how good it is.

5.1 None of those is a property of the binding event

They are properties of the route, the behaviour or the metabolism surrounding it.

5.2 This one is a property of the molecule meeting the protein

Which is a cleaner kind of selectivity and, as §10 argues, a more concentrated one.

6. The electrostatic mechanism

The insect form of nAChR contains a unique positively charged amino acid residue that favors the binding of neonicotinoids containing a negatively charged nitro or cyano group.⁷

6.0b The mechanism is stated as electronic rather than structural

What is described is charge attracting charge, not a shape fitting a pocket, which is why a single residue substitution changes the outcome so completely.¹

6.1 With the location identified

Basic residues in loop D of the binding site play a key role in electronic interactions with the nitro or cyano group.¹

6.2 And the compound's own contribution described

Electrostatic interactions of the nitroimine group and the bridgehead nitrogen with particular receptor residues are likely to have key roles in determining selective toxicity, supported by chemical calculation of atomic charges and by site-directed mutagenesis.⁶

7. And the vertebrate side of it

Which is the other half of a difference.

Serine with the ability to form a hydrogen bond in loop C of some insect alpha subunits, and glutamate with a negative charge at the corresponding position in vertebrate receptors, may contribute to enhancing and reducing the neonicotinoid actions respectively.¹

7.0b Two loops doing two jobs

Loop D is named for the electronic interaction with the compound's nitro or cyano group, and loop C for the hydrogen bonding and charge difference between taxa. The binding site is assembled from several loops and the selectivity argument draws on at least two.¹

7.1 So the same position carries opposite charges in the two taxa

Attracting the compound in one and repelling it in the other.¹

7.2 With the authors noting the evidence is incomplete

Stating that there is no clear evidence what loop C properties underpin the target site actions of neonicotinoids, which is why they ran the experiment in §8.¹

8. The experiment behind that

Reported in the same work.

A mutation of serine at position 221 to glutamate in a named motif in loop C of a fruit fly subunit markedly reduced the agonist action of imidacloprid and thiacloprid, pointing to a contribution of that serine to selective action. The explanation offered is repulsion of the negative nitro group of imidacloprid and cyano group of thiacloprid by the electronegative glutamate.¹

8.0b The design is a transplant rather than a comparison

Instead of measuring an insect receptor against a vertebrate one and attributing the difference to whatever distinguishes them, the experiment changes one residue and holds the rest constant, which is what allows the effect to be assigned to that position.¹

8.1 Which is the vertebrate residue installed in an insect subunit

Producing a receptor the compound acts on poorly.¹

8.2 And two further substitutions behaved differently

Replacing the same serine with alanine hardly affected affinity or efficacy for acetylcholine or imidacloprid, and only slightly reduced efficacy for thiacloprid at receptors with a higher proportion of the second subunit.¹

9. Which is a hybrid receptor

A methodological point worth recording.

The receptors tested were an insect alpha subunit combined with a vertebrate beta subunit, expressed in frog oocytes, because insect receptors have been difficult to express functionally.¹⁴

9.1 So the measurements are on a chimera

Part insect and part bird or rat, which is a standard workaround and is not the receptor the compound meets in an animal.

That caution is ours, though the literature is open about the construction.⁴

9.1b Which is a familiar compromise in this journal

Our articles on registration testing and on resistance bioassays both found measurements made on convenient preparations standing in for conditions of use, and this is the receptor-level version of it.

9.2 And the workaround is recent

One paper reports that a cofactor enables robust functional expression of insect receptors in oocytes, which is presented as a development rather than as long-standing practice.³

10. So the selectivity rests on one feature

Our statement of what §§6 to 8 amount to.

A negatively charged substituent on the molecule, meeting a positively charged residue in the insect receptor and a negatively charged one in the vertebrate receptor.¹⁷

10.0b With one qualification from the sources themselves

Both alpha and non-alpha subunits are said to contribute to the interaction, and the bridgehead nitrogen is named alongside the nitroimine group, so the binding is not a single contact even though the charge difference is the feature doing the selective work.⁶

10.1 Remove the substituent and the argument goes with it

Which is not a hypothetical, and §12 is what happens.

11. Which is an unusual place to put a safety margin

Our assessment.

A margin resting on many small differences degrades gradually when one of them changes. A margin resting on a single interaction is intact or it is not.

11.0b The contrast with a metabolic margin is worth stating

Where selectivity comes from a mammal detoxifying faster than an insect, the margin is a rate difference that varies with dose, individual and enzyme induction, and it degrades rather than disappearing. A charge interaction either holds or does not.

11.1 And a chemical group is a removable thing

Metabolism acts on functional groups, which is what metabolism is.

11.2 So the question is not whether the group can be lost

It is what the molecule is once it has been.

12. What metabolism does to it

The metabolite desnitro-imidacloprid has a strongly reduced potency on insect nAChRs, but in turn an increased affinity for mammalian nAChRs, and studies in mice suggested a higher toxicity for it than for the parent compound.²

12.0b Which is what the electrostatic account predicts

If the negative group is what attracts the insect residue and repels the vertebrate one, then removing it should cost affinity on one side and relieve repulsion on the other. The metabolite behaves as §6 says it should.¹²

That connection is ours, and it means the metabolite result is not an anomaly but a consequence of the mechanism the class is built on.

12.1 Reduced for insects and increased for mammals

Both halves of the selectivity moving, in opposite directions, from one change.²

12.2 Which the source frames as expected

Noting that previous knowledge of the metabolism shows a shift in the bioactivity spectrum can occur.²

13. The comparison drawn

And it is the line that stops a reader.

Binding assays using mammalian receptors have shown that the metabolite has an affinity similar to the high-affinity ligand nicotine, with nicotine described as a well-known neurotoxicant and developmental neurotoxicant for vertebrates including man.²

13.0b And the direction of travel is worth stating plainly

The design problem these compounds solved was to keep nicotine's mode of action while losing its mammalian affinity. The metabolite reverses that achievement.²

13.1 Which is the parent compound's own ancestor

The class is named for its relation to nicotine, and the metabolite has arrived back at nicotine-like affinity for the receptors nicotine acts on.

That observation is ours.

14. And it has been found in people

Which moves this from pharmacology to exposure.

The finding is described as consistent with detection of the desnitro metabolite and another named metabolite in human urine samples analyzed in a recent biomonitoring study.²

14.0b And the second metabolite named is a different one

An olefin metabolite appears alongside the desnitro compound in the same detection, and our sources say nothing about its receptor properties.²

14.1 We have the citation and not the study

So we know the metabolites were detected and nothing about concentrations, population or source.²

14.2 And detection is not a dose

Modern analytical methods detect a great many things at very low concentrations, and our articles on statistic provenance would want the number before drawing anything from it.

15. Which is the same shape as our fipronil finding

And we noticed it only on writing this article.

That article reported a photoproduct of fipronil described as equipotent with the parent at the insect receptor and around tenfold more potent at the mammalian one, so that degradation retained insecticidal activity while removing part of the safety margin.

15.0b With one difference between the two cases

The fipronil transformation was photochemical, happening on a treated surface in sunlight. This one is metabolic, happening inside an organism that has absorbed the compound.²

15.1 Here the degradation product loses insect potency and gains mammalian affinity

Which is a different arrangement of the same problem.²

16. A pattern worth naming

Our generalisation, offered with the caveat that two cases are not a pattern.

In both classes, the selectivity of the registered compound has been assessed and the selectivity of what it becomes has been assessed separately or not at all, and in both cases the transformation moved the margin the wrong way.

16.0b And there is a reason the parent figures circulate

A selectivity ratio for a named active ingredient is a single memorable number that travels into labels, training material and articles like this one. A statement about the shifting properties of four metabolites does not travel at all.

16.1 Which is a question about what a registration covers

Our efficacy data article established that registration assesses a product. Whether it assesses the metabolites of that product's active ingredient at the same depth is a question we raise and cannot answer.

16.2 And it is not a claim that anybody overlooked it

Metabolite toxicology is a standard part of a registration package. What we can say is that the figures reaching us as the selectivity of these compounds are figures for the parent molecules.

17. The target is not one target

One study shows that a third alpha subunit co-exists with four others in the same neurons of adult fruit flies, thereby expanding the possible nAChR subtypes in these cells alone from 4 to 12.³

17.0b Which is what a subunit family allows

Five interchangeable parts assembled five at a time into a pentamer produces a large number of distinct receptors, each with its own pharmacology, from a small number of genes.³

17.1 Twelve possible receptors in one cell population

Assembled from a handful of interchangeable parts.³

17.1b Which bears on every potency figure in this article

A measurement on one subunit combination is a measurement on one of the twelve, and nothing guarantees the others behave the same way.³

17.2 And the authors note the work is unfinished

Stating that other subunits from the family remain to be explored.³

18. What that does to a mode-of-action label

Our reading.

Saying that a compound acts at the nicotinic acetylcholine receptor names a family rather than a site, and the compound's effect depends on which members of that family a given neuron has assembled.

18.0b And it is not a criticism of the classification scheme

Grouping by target family is the only practical basis for rotation guidance, since nobody can enumerate subunit compositions per species per tissue. The point is about how much confidence the grouping supports, not about whether to use it.

18.1 Which is the objection our fipronil article made in a different form

That mode-of-action classification does less predictive work than resistance management guidance assumes, because the label is coarser than the biology.

18.2 And it explains why potency varies between species

Which is §19.

19. The pollinator receptor result

Reported plainly, and it is uncomfortable.

Three named neonicotinoids exhibited agonist actions on some receptors of the fruit fly, the honeybee and the bumblebee, with more potent actions on the pollinator nAChRs.³

19.0b With the qualifier the paper attaches

On some receptors, which matters given §17: a statement about particular subunit combinations rather than about every receptor the animals possess.³

19.1 More potent on the bees than on the pest model

Which is a receptor-level statement rather than an exposure one.³

19.2 And the paper frames the class's non-target effects as the motivation

Opening on the statement that adverse effects on non-target insects are of serious concern.³

20. Which bears on our exposure article

Where we examined how perimeter and barrier treatment reaches pollinators.

That article was about routes: what gets into nectar, how far material drifts, what happens when a lawn is mown. This adds that the receptor at the end of those routes is more sensitive than the one in the pest the product was designed against.

20.0b And the comparison is within insects rather than across taxa

The selectivity argument in §6 is about insects against vertebrates and says nothing about which insects. A within-insect difference of this kind sits entirely outside it.³

20.1 So exposure and susceptibility both point the same way

Which is a worse combination than either alone.

20.2 With the usual caution about hybrid receptors

Section 9 applies to this measurement as much as to the others.³

21. Resistance at the same residues

A substitution in loop D of aphids reduces the affinity of neonicotinoids, resulting in resistance.¹ A point mutation at a different position was identified in the brown planthopper and associated with target-site resistance.⁴

21.1 Which is what a well-located mechanism implies

If one interaction carries the selectivity, that interaction is also the cheapest thing for selection to alter, since a single substitution there does more than a change anywhere else.

22. A prediction that came true

Which is rare enough in this literature to be worth dwelling on.

The aphid substitution was first predicted then shown to reduce affinity and confer resistance.¹

22.0b And the prediction was of a specific substitution

Not merely that resistance would arise, but that it would arise by replacing a particular charged residue at a particular position, which is a far narrower thing to get right.¹

22.1 Predicted from the electrostatic model

If the selectivity depends on a positively charged residue attracting a negatively charged group, then replacing that residue with an uncharged one should reduce binding, and an animal carrying that change should be resistant.

22.2 Which is the model being tested by nature

And passing, which raises confidence in §6 considerably more than any further binding study would.

That assessment is ours.

23. The stranger result

From a separate experiment.

A substitution at the planthopper position, introduced into the receptor and co-expressed with a rat subunit, produced a receptor that imidacloprid bound with high affinity but on which it did not show agonist actions. Further study demonstrated that it acted as an antagonist, blocking acetylcholine responses, slowly reversibly.⁴

23.0b And the construct carried a rat subunit

Which is the hybrid arrangement §9 describes, so the receptor on which the compound turned antagonist was part planthopper and part rat.⁴

23.1 Described as a first

The first time a point mutation in that loop of an insect alpha subunit had been identified that changes the mode of interaction between neonicotinoid insecticides and the receptor.⁴

24. What an antagonist insecticide would mean

Our reasoning, offered speculatively.

An agonist kills by overstimulating. An antagonist would kill, if it killed, by silencing cholinergic transmission instead, which is a different physiology and would be expected to produce different symptoms and a different dose response.

24.0b Which complicates the usual resistance account

Target-site resistance is normally described as the compound no longer binding well. Here binding is retained at high affinity and what changes is the consequence of binding, which is a distinct mechanism that a binding assay alone would not detect.⁴

24.1 And the compound still binds

With high affinity, which means a resistance mechanism of this kind is not an escape from binding but a change in what binding does.⁴

24.2 Whether the animal survives is not something we can say

The experiment measured receptor behaviour in oocytes, not insect mortality, and we found no statement either way.⁴

25. The subgroup split

Since the class divides.

Imidacloprid is a nitroguanidine and acetamiprid a cyanoamidine, and the new generation of nicotine-related insecticides possess either a nitromethylene, nitroimine or cyanoimine group.⁷⁵

25.0b Which are two ways of carrying a negative charge

A nitro group and a cyano group differ chemically while both presenting the electronegative feature the mechanism in section 6 requires, which is why both subgroups belong to one class.⁵

25.1 And structure matters for the mammalian side

A 1999 paper title states that minor structural changes in nicotinic insecticides confer differential subtype selectivity for mammalian nicotinic acetylcholine receptors.⁸

25.2 Which we have as a title and not a result

Taken from a regulator-affiliated fact sheet's reference list rather than from the paper itself.⁸

26. What we take from this

Three things.

The selectivity is real and well characterised. Better characterised than for any other class this journal has covered, down to named residues and charges.¹⁷

And it is carried by one removable feature. Section 12.²

Which is the second time we have found a transformation product moving the margin the wrong way. Section 15.

26.1 And one thing we are not saying

That the class is unsafe at the exposures structural use produces. Nothing here measures that, and §27 records the limit.

27. Our own position

The disclosure.

Products in this class are used in structural work, in termite and ant treatment and in veterinary products our clients apply to animals in the same buildings, and this article reports a metabolite concern without establishing that it matters at the exposures those uses produce.

27.0b And the article was not written to reach that conclusion

We came to fill a gap in this journal's chemistry coverage and found the metabolite result while reading about the receptor, which is the order worth recording since the finding is the kind that invites a stronger claim than the evidence supports.

27.1 Which is the honest limit of what we have

Receptor affinity is not risk, and §14.2 says detection is not dose.

28. The Manitoba position

28.1 The agricultural context is immediate here

This province is a major producer of the crops these compounds have been used on, which means the regulatory arguments about them are local arguments and not distant ones.

28.1b And the veterinary route is the closest one to a household

Spot-on and collar products applied to animals living indoors put the compound on a surface people touch daily, which is a different exposure geometry from a soil or perimeter application and is the one we are asked about most often.

28.2 What we could not find

Any Canadian biomonitoring data for these metabolites, any Manitoba figure for structural or veterinary use volumes, and any local study of residues in or around treated buildings.

28.3 And we have deliberately not covered the regulatory dispute

Which concerns agricultural use, is politically contested, and would take a different article with different sources.

29. Limitations and open questions

Most of the electrophysiology is on hybrid receptors. Insect alpha subunits with vertebrate beta subunits in frog oocytes, which §9 records and which applies to the selectivity experiments, the resistance experiments and the pollinator comparison.¹³⁴

That is the most important limitation because §§8, 19 and 23 all rest on constructs rather than on native receptors, and the papers themselves treat functional expression of insect receptors as a recent development.³

The metabolite evidence reaches us through one paper's introduction. The comparisons with nicotine, the mouse toxicity and the urine detection are all summaries of other work cited in a single article we read as an extract.²

We have no potency figures. Strongly reduced, increased affinity and similar to nicotine are the terms available, with no ratios, and §13's force depends on a comparison we cannot quantify.²

One key review is behind a paywall. The statement in §1 that this is the first class whose selectivity is attributable to target site affinity comes from an abstract and preview text rather than the full review.⁵

And two sources are reference lists. The 1999 mammalian subtype paper and several supporting studies are known to us as citations in a fact sheet rather than as documents.⁸

Sections 5, 10, 11, 13.1, 15, 16, 18, 20, 22.2 and 24 are our reasoning. The comparison with other kinds of selectivity, the argument that a single-feature margin is a concentrated one, the pattern across two chemical classes, the consequence for mode-of-action labelling and the reading of the antagonist result are ours rather than sourced positions.

30. Conclusion

Neonicotinoids are described as the first insecticides whose selectivity for insects can be attributed to higher affinity at the target site rather than to metabolism, placement or dose.⁵ The mechanism reported for that is electrostatic and specific: the insect receptor carries a positively charged residue that favours binding of compounds bearing a negatively charged nitro or cyano group, while vertebrate receptors carry a negatively charged residue at the corresponding loop C position which repels the same group.¹⁷ Installing the vertebrate residue into an insect subunit markedly reduced the compound's agonist action.¹ So the margin is carried by one chemical feature meeting one amino acid, which is the most precisely located safety margin this journal has examined.

It is also the most concentrated one, because a chemical group is the kind of thing metabolism removes. The desnitro metabolite is reported to have strongly reduced potency at insect receptors and increased affinity at mammalian ones, with mouse studies suggesting higher toxicity than the parent and binding assays giving an affinity similar to nicotine, itself described as a known neurotoxicant and developmental neurotoxicant in humans. It and another metabolite have been detected in human urine.² We have the summaries and not the numbers, and detection is not dose. But this is the second time we have found a transformation product moving a safety margin the wrong way, after the fipronil photoproduct, and in both cases the selectivity figures in circulation describe the parent molecule.

Two further things complicate the picture. The target is not a target: one subunit shown to co-exist with four others in the same neurons expands the possible receptor subtypes in that cell population alone from four to twelve, and three neonicotinoids showed agonist actions on fruit fly, honeybee and bumblebee receptors with more potent actions on the pollinators.³ And the residues carrying the selectivity are where resistance appears: an aphid substitution in loop D was predicted from the electrostatic model before it was found in the field, which is the model passing a harder test than any binding assay could set it.¹ One related change did something stranger still, leaving the compound binding with high affinity but no longer acting as an agonist, blocking acetylcholine instead.⁴

References

1. The mechanism of loop C-neonicotinoid interactions at insect nicotinic acetylcholine receptor alpha1 subunit predicts resistance emergence in pests. Open-access journal article, read as abstract and introduction. Source for the statement that neonicotinoids selectively modulate insect nicotinic acetylcholine receptors and that serine able to form a hydrogen bond in loop C of some insect alpha subunits and glutamate with a negative charge at the corresponding position in vertebrate receptors may contribute to enhancing and reducing neonicotinoid actions respectively; for the authors' statement that there is no clear evidence what loop C properties underpin the target site actions; for the experimental design substituting alanine and glutamine at position 221 of a fruit fly subunit and testing imidacloprid and thiacloprid on hybrid receptors with a chicken beta subunit expressed in frog oocytes, with the alanine substitution hardly affecting affinity or efficacy for acetylcholine or imidacloprid while slightly reducing efficacy for thiacloprid at receptors with a higher proportion of the beta subunit; for the earlier finding that substituting glutamate at the same position markedly reduced the agonist action of both compounds, with the explanation that the electronegative glutamate repels the negative nitro group of one compound and cyano group of the other; for the statement that basic residues in loop D play a key role in electronic interactions with the nitro or cyano group; and for the account that a named substitution in loop D of aphids was first predicted and then shown to reduce the affinity of neonicotinoids, resulting in resistance. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7200709/
2. Acute effects of the imidacloprid metabolite desnitro-imidacloprid on human nicotinic acetylcholine receptors relevant for neuronal signalling. Open-access journal article, read as an extract of its discussion, in which the statements below are summaries of other work rather than the paper's own results. Source for the statement that previous knowledge of imidacloprid metabolism shows a shift in the bioactivity spectrum can occur; for the report that the desnitro metabolite has strongly reduced potency on insect nicotinic receptors but increased affinity for mammalian ones; for the note that studies in mice suggested a higher toxicity of the metabolite compared with its parent compound; for the report that binding assays using mammalian receptors have shown the metabolite to have an affinity similar to the high-affinity ligand nicotine, with nicotine described as a well-known neurotoxicant and developmental neurotoxicant for vertebrates including man; and for the statement that this is consistent with findings of the desnitro metabolite and an olefin metabolite in human urine samples analysed in a recent biomonitoring study. https://pmc.ncbi.nlm.nih.gov/articles/PMC8536575/
3. Functional impact of subunit composition and compensation on fruit fly nicotinic receptors, targets of neonicotinoids. Open-access journal article, read as abstract. Source for the statement that neonicotinoid insecticides target insect nicotinic acetylcholine receptors and that their adverse effects on non-target insects are of serious concern; for the report that a cofactor enables robust functional expression of insect receptors in frog oocytes; for the finding that three named neonicotinoids exhibited agonist actions on some receptors of the fruit fly, honeybee and bumblebee, with more potent actions on the pollinator receptors; and for the finding that a third alpha subunit co-exists with four other named subunits in the same neurons of adult fruit flies, expanding the possible receptor subtypes in those cells alone from four to twelve, with other subunits from the family remaining to be explored. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9934367/
4. Imidacloprid acts as an antagonist on insect nicotinic acetylcholine receptor containing a named mutation. Bibliographic record and abstract in a biomedical literature database. Source for the description of nicotinic acetylcholine receptors as ligand-gated ion channels mediating fast cholinergic synaptic transmission in insect and vertebrate nervous systems; for the note that a point mutation had been identified in the brown planthopper associated with target-site resistance to neonicotinoids; for the experimental finding that introducing a related substitution and co-expressing with a rat beta subunit in frog oocytes produced a receptor which imidacloprid bound with high affinity, though lower than the unmutated version, but on which it showed no agonist actions; for the demonstration that it instead acted as an antagonist blocking acetylcholine responses with a stated inhibitory constant and slow reversibility; and for the authors' statement that this is the first time a point mutation in that loop of an insect alpha subunit has been identified that changes the mode of interaction between neonicotinoid insecticides and insect receptors. https://pubmed.ncbi.nlm.nih.gov/18824080/
5. Neonicotinoids: insecticides acting on insect nicotinic acetylcholine receptors. Review article in a pharmacological sciences title, read as abstract and preview text rather than in full, which we flag because a central claim of this article comes from it. Source for the statement that imidacloprid, other nitroimines and the nitromethylenes are not the first insecticides to target insect nicotinic receptors nor the only insecticides to exhibit selectivity for insects, but are the first for which this selectivity can be attributed to a higher affinity for the target site; for the description of the new generation of nicotine-related insecticides as possessing either a nitromethylene, nitroimine or cyanoimine group; and for the account of early work expressing a locust receptor alpha subunit as a homo-oligomer in frog oocytes, which displayed a pharmacology characteristic of certain native insect receptors and responded to nicotine and a named nitromethylene with rapid depolarising responses. https://www.sciencedirect.com/science/article/abs/pii/S0165614700018204
6. Overview entry on imidacloprid in a scientific reference platform, compiled from published chapters and articles. A secondary compilation rather than a primary source. Source for the statement that imidacloprid is an agonist at nicotinic acetylcholine receptors showing selective toxicity for insects over vertebrates; for the statement that studies using binding assays, molecular biology and electrophysiology suggest both alpha and non-alpha subunits contribute to the interaction; and for the statement that electrostatic interactions of the nitroimine group and bridgehead nitrogen with particular receptor amino acid residues are likely to have key roles in determining selective toxicity, supported by chemical calculation of atomic charges and by a site-directed mutagenesis study. https://www.sciencedirect.com/topics/pharmacology-toxicology-and-pharmaceutical-science/imidacloprid
7. In vitro metabolism of imidacloprid and acetamiprid in rainbow trout and rat. Open-access journal article, read as an extract of its introduction. Source for the statement that the insect form of the nicotinic acetylcholine receptor contains a unique positively charged amino acid residue favouring the binding of neonicotinoids containing a negatively charged nitro or cyano group; for the identification of imidacloprid as a nitroguanidine and acetamiprid as a cyanoamidine; for the statement that these chemicals were first introduced in the early 1990s as alternatives to organophosphates, pyrethroids and carbamates which were losing effectiveness due to insect resistance; and for the statement that they were originally developed as systemic insecticides with higher selectivity factors based on stronger affinity for the insect than the mammalian receptor. https://pmc.ncbi.nlm.nih.gov/articles/PMC9822985/
8. Imidacloprid technical fact sheet, archived version, published by a pesticide information centre operated in cooperation with a federal regulator. Read for its reference list rather than its body text. Source for the existence of a 1999 pharmacology paper whose title states that minor structural changes in nicotinic insecticides confer differential subtype selectivity for mammalian nicotinic acetylcholine receptors, of a 1998 paper on the effects of the alpha subunit on imidacloprid sensitivity of recombinant receptors, and of a 2005 annual review on neonicotinoid insecticide toxicology and mechanisms of selective action. https://npic.orst.edu/factsheets/archive/imidacloprid.html