Insect nicotinic receptor interactions in vivowith neonicotinoid, organophosphorus, andmethylcarbamate insecticides and a synergistXusheng Shaoa,b,1, Shanshan Xiab, Kathleen A. Durkinc, and John E. Casidaa,1

aEnvironmental Chemistry and Toxicology Laboratory, Department of Environmental Science, Policy, and Management, and cMolecular Graphics andComputation Facility, College of Chemistry, University of California, Berkeley, CA 94720; and bSchool of Pharmacy, East China University of Science andTechnology, Shanghai 200237, China

Contributed by John E. Casida, September 3, 2013 (sent for review July 26, 2013)

The nicotinic acetylcholine (ACh) receptor (nAChR) is the principalinsecticide target. Nearly half of the insecticides by number andworld market value are neonicotinoids acting as nAChR agonists ororganophosphorus (OP) and methylcarbamate (MC) acetylcholines-terase (AChE) inhibitors. There was no previous evidence for in vivointeractions of the nAChR agonists and AChE inhibitors. The nitro-methyleneimidazole (NMI) analog of imidacloprid, a highly potentneonicotinoid, was used here as a radioligand, uniquely allowing fordirect measurements of house fly (Musca domestica) head nAChR invivo interactions with various nicotinic agents. Nine neonicotinoidsinhibited house fly brain nAChR [3H]NMI binding in vivo, correspond-ing to their in vitro potency and the poisoning signs or toxicity theyproduced in intrathoracically treated house flies. Interestingly, ninetopically applied OP or MC insecticides or analogs also gave similarresults relative to in vivo nAChR binding inhibition and toxicity, butnow also correlating with in vivo brain AChE inhibition, indicatingthatACh is the ultimateOP- orMC-inducednAChRactive agent. Thesefindings on [3H]NMI binding in house fly brain membranes validatethe nAChR in vivo target for the neonicotinoids, OPs and MCs. Asan exception, the remarkably potent OP neonicotinoid synergist,O-propyl O-(2-propynyl) phenylphosphonate, inhibited nAChR invivo without the corresponding AChE inhibition, possibly via a re-active ketene metabolite reacting with a critical nucleophile in thecytochrome P450 active site and the nAChR NMI binding site.

The nicotinic nervous system has two principal sites of in-secticide action, the nicotinic receptor (nAChR) activated by

acetylcholine (ACh) and neonicotinoid agonists (1–6), and ace-tylcholinesterase (AChE) inhibited by organophosphorus (OP)andmethylcarbamate (MC) compounds to generate andmaintainlocalized toxic ACh levels (Fig. 1) (7). The nAChR and AChEtargets have been identified in insects by multiple techniques butnot by direct assays of the ACh binding site in the brain of poi-soned insects. Here we use the outstanding insecticidal potencyof the nitromethyleneimidazole (NMI) analog of imidacloprid(IMI) (8) as a radioligand (9), designated [3H]NMI, to directlymeasure the house fly (Musca domestica) nAChR not only in vitrobut also in vivo, allowing us to validate by a previously unde-scribed method the neonicotinoid direct and OP/MC indirectnAChR targets (Fig. 2). This approach also helped solve the in-triguing mechanism by which an O-(2-propynyl) phosphorus com-pound strongly synergizes neonicotinoid insecticidal activity (10)by dual inhibition of cytochrome P450 (CYP) (11–13) and thenAChR agonist site (described herein). Insecticide disruption atthe insect nAChR can now be readily studied in vitro and in vivowith a single radioligand allowing better understanding of theaction of several principal insecticide chemotypes (Fig. 3).

ResultsnAChR–Neonicotinoid Interactions. The high nAChR affinity probe[3H]NMI provided the possibility of examining nAChR–insecticideinteractions in vitro and in vivo. IMI and clothianidin (CLO)were selected as two distinct neonicotinoid chemotypes (i.e.,

chloropyridinyl imidazole and chlorothiazolyl acyclic, respec-tively). These chemotypes gave in vitro IC50 values (concen-tration or dosage for 50% inhibition of [3H]NMI binding) of28 and 18 nM, respectively, and some or all of the IMI and CLOremained bound on in vivo assays, yielding intrathoracic IC50values of 4.2 and 0.5 μg/g, respectively (Fig. 4).The toxicological relevance of the nAChR–neonicotinoid inter-

actions was tested by determining dose–response and structure–activity relationships for nAChR binding inhibition versus poisoningsigns. The results for dose–response (Fig. 5A) and structure–activity (Fig. 5B) both established nAChR binding inhibitionclosely related to poisoning signs (i.e., the [3H]NMI assay is fora toxicologically relevant site in neonicotinoid poisoning). Thus,the findings with nine neonicotinoids confirmed a common targetleading to the insecticidal activity.

nAChR–OP/MC Interactions. Paraoxon (PAR) and chlorpyrifos (CHL)were chosen to represent the direct-acting and bioactivated AChEinhibitors, respectively (7). As expected, neither one of these OPsinhibited nAChR binding of [3H]NMI in vitro, even at 20,000 nM,but surprisingly they were both potent nAChR inhibitors in vivowith IC50 values of 5.1 and 0.4 μg/g. respectively (Fig. 6A). Thestudies, therefore, proceeded to test if there was a general re-lationship between nAChR binding inhibition and toxicity by ex-amining varied doses of OP and MC compounds and assayingother insecticides.Topically applied PAR and CHL established a clear dose–

response relationship for nAChR binding inhibition and poi-soning signs with a similar correlation for the two compounds(Fig. 6B). The nAChR binding inhibition–poisoning sign re-lationship was more firmly established with nine OPs and car-bamates (Fig. 7 A and B). This process established that nAChRbinding inhibition was associated with and possibly causal to thepoisoning but the total lack of potency in vitro meant the OP

Significance

The insect nicotinic receptor is the direct or indirect target forneonicotinoids, organophosphorus compounds, and methyl-carbamates, which make up about 45% of the insecticides bynumber and world market value. In this study, the house flybrain nicotinic receptor in vivo interactions with neurotoxicantsare revealed by a unique radioligand reporter assay, providingdirect in vivo proof of events at the receptor level leading topoisoning by these three major insecticide chemotypes.

Author contributions: X.S. and J.E.C. designed research; X.S., S.X., and K.A.D. performedresearch; and X.S. and J.E.C. wrote the paper.

The authors declare no conflict of interest.1To whom correspondence may be addressed. E-mail: shaoxusheng@ecust.edu.cn or ectl@berkeley.edu.

This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1316369110/-/DCSupplemental.

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was metabolically bioactivated (an unlikely event for PAR) orthat another binding inhibitor was formed such as elevated AChlevels from AChE inhibition.

AChE–OP/MC Interactions. The possible relationship between AChEinhibition and nAChR binding inhibition was examined and acorrelation established with varying doses of PAR or CHL (Fig.6 C and D) and with nine OP and MC insecticides of widelyvarying structures (Fig. 7 C and D). Under the conditions of invivo poisoning, enough ACh apparently accumulates locally inthe nAChR to block [3H]NMI binding and disrupt nAChRfunctioning.

Lack of nAChR Interactions with Other Insecticides. The followingdiverse types of insecticides and mechanisms of toxicity (1) werefound to be inactive in vitro (1,000 nM) and in vivo (100 μg/gintrathoracic injection, 1 h posttreatment) assayed for nAChRbinding inhibition with [3H]NMI as above: the nicotinic blockingagent cartap (14); the nicotinic activator spinosad (15); theGABA/glutamate receptor modulators fipronil, endosulfan, andavermectin; and the voltage-gated Na+ channel modulators del-tamethrin, allethrin, and DDT (1).

nAChR–PPP Synergist Interactions. PPP (prop-2-yn-1-yl propylphenylphosphonate) (Fig. 8) is a remarkably effective CYP in-hibitor and synergist for neonicotinoid insecticides, leading to thequestion of what is so unusual about this compound. PPP is nota direct inhibitor of Musca nAChR binding or AChE, with littleor no inhibition of either target in vitro at 10,000 nM. PPP in vivoat 100 μg/g inhibits nAChR binding without inducing poisoningsigns, in contrast to all of the other OPs and carbamates exam-ined. PPP inhibits nAChR binding in vivo but not in vitro, and isa less-effective AChE inhibitor in vivo than the other OPs. Thisfinding leads to the hypothesis that PPP, with its unusualO-propynyl substituent, undergoes bioactivation not only to a

CYP inhibitor, but also to a nAChR binding inhibitor acting ata site that does not lead to nAChR activation or produce prom-inent poisoning signs. As a first step in understanding PPP me-tabolism, it was administrated to mice and the urine was examinedfor a propionic acid metabolite formed via a ketene intermediate(Fig. 8). The propionic acid metabolite was identified by LC/MScomparison with a synthetic standard (m/z = 272.66) and cochro-matography (Rt = 23.92 min), as described in SI Text. The PPPketene is therefore a candidate activated intermediate in thenAChR binding inhibition and synergist action (16).

Discussion[3H]NMI as a Reporter on nAChR Status in Vivo. This high-affinityradioligand with a rapid association and slow dissociation rate(9) can be used to record the in vivo binding site of the MuscanAChR, either normal or altered, by toxic action (Fig. 2). It isapplicable to neonicotinoid agonists and OP and MC AChEinhibitors. No disruption was found for nicotinic agents cartapand spinosad acting at other sites and with a variety of insecti-cides toxic by other mechanisms.

Nicotinic Agonist–nAChR Interactions. The Musca nAChR assaywith [3H]NMI is very sensitive to neonicotinoids in vitro andin vivo. The potencies of the neonicotinoids depend on the bal-ance of their nAChR association and dissociation rates. Theneonicotinoid potency in vitro is generally a good predictor ofthe in vivo effects.

Anticholinesterase–nAChR Interactions. A great variety of OP che-motypes and some MCs are potent nAChR binding inhibitors invivo but not in vitro. Our earlier studies showed desnitro-IMI is

Fig. 1. The insect nicotinic receptor is the direct or indirect target forneonicotinoids, organophosphorus compounds and methylcarbamates,which make up about 45% of the insecticides by number and world marketvalue (2, 7).

Fig. 2. In this study,Musca nicotinic receptor in vivo interactions with majorinsecticide chemotypes are revealed by a [3H]NMI radioligand reporter assay.*Position of tritium label.

Fig. 3. Two neonicotinoid nicotinic agonists and two anticholinesteraseinsecticides.

Fig. 4. Neonicotinoids IMI and CLO inhibit nAChR binding of [3H]NMI invitro related to concentration (A) and in vivo related to dose (B).

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a nAChR binding inhibitor in vivo in mouse brain (17). As anexception, physostigmine is a direct nAChR binding inhibitor inMusca but at a different site in the receptor (18). Poisoning byanticholinesterases, both OPs and MCs, is easily monitored byAChE inhibition in the head of poisoned Musca (19). In thepresent study the MC carbofuran was highly effective in bothAChE and nAChR binding inhibition. Freshly decapitatedMusca are similar to normal Musca in OP sensitivity, indicatingthat action at a site other than the brain plays a role in poisoning(20). PPP lacks the AChE-associated toxic effects of other OPsindicating that, in this case, the brain is more an indicator thana direct target of the insecticidal action. In any case, the in vivonAChR binding inhibition by most OPs and MCs is undoubt-edly a result of AChE inhibition (19), and accumulation of the

endogenous ACh agonist (21) that serves as a potent inhibitor inthis type of binding assay (22).

PPP Synergist–nAChR Interactions. PPP is far more effective thanthe most important commercial insecticide synergist piperonylbutoxide in enhancing neonicotinoid toxicity, and at the doseused here it increases the topical potency of several neon-icotinoids by 50- to 100-fold (10). PPP is an exception to theproposed OP phosphorylation of AChE, leading to ACh accu-mulation and nAChR binding inhibition. PPP is a highly potentinhibitor of CYP in mammalian liver, acting on several insecti-cides and substrates (13). Several 2-propynyl compounds areactivated by CYP oxidation to reactive ketene derivatives (23).The proposed activated form of PPP is the ketene, which alsoserves as the intermediate to the propionic acid derivative ob-served here as a metabolite and identified by synthesis and LC/MS (Fig. 8). PPP inhibits nAChR binding in vivo but not in vitro,and without the associated AChE inhibition found with otherOPs [i.e., it may be that the combined CYP and nAChR bindinginhibition lead to the exceptional potency (10) of this synergist].The possibility was considered that because PPP-ketene and

NMI each have an aryl substituent and electronegative tip, theymight bind at the same general nAChR site. To elucidate thistheory, we built a homology model of the homomeric Muscaα5α5 dimer interface. Musca α5 (uniprot A9XFY4) has highsequence homology with both the Drosophila melanogaster α5(uniprot Q8T7V5, 99% identity for 200 residues in ligandbinding domain) and α7 (uniprot A4GXC7, 98% identity for 200residues in ligand binding domain) (24). These sequences areknown to form functional homomeric ion channels inMusca (25)and Drosophila (26), so are useful structures for investigations.

Fig. 5. Neonicotinoid inhibition of nAChR [3H]NMI binding in vivo pre-dicts poisoning signs for varied doses of IMI and CLO (A) and for variedneonicotinoids injected at 0.2 μg/g (B). ACE, acetamiprid; CYC, cycloxaprid;DIN, dinotefuran; NIT, nitenpryam; THIA, thiacloprid; TMX, thiamethoxam.

Fig. 6. OPs PAR and CHL inhibit nAChR binding of [3H]NMI in vivo relatedto dose (A) and predictive of poisoning signs (B) because of AChE inhibition(C and D). The varied topical doses of CHL or PAR in A and C were 0.1, 0.3, 1,3, and 10 μg/g.

Fig. 7. Various OPs and MCs do not inhibit nAChR binding in vitro (A) butdo inhibit in vivo (B), usually predictive of poisoning signs and because ofAChE inhibition (C and D). *PPP is an exception to these relationships andis not included in the correlation lines. CAR, carbofuran; CAI, carbaryl; CA2,N-propylcarbaryl; OPI, ethoprophos; OP2, profenofos; OP3, tribufos; PAR,paraoxon. The varied compounds were at discriminating topical doses of3 μg/g for CHL and PAR, 10 μg/g for CAR and OP2, and 100 μg/g for CA1, CA2,OP1, OP3, and PPP.

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On docking in the Musca α5α5 and related Myzus α2β1 (27)models, NMI and PPP-ketene adopted the known neonicotinoidorientation found for IMI seen in the 3C79 structure with theelectronegative tip of the ligands oriented toward a charge sta-bilizing region in the active site [in 3C79, these are: Gln-57E,Ser-189A, Cys-190A, Tyr-55E; Myzus: Asn, Val (backbone), Cys,Trp; Musca: Lys, Asn, Cys, Trp]. In Musca, this places the ketenemoiety near a lysine nucleophile for possible derivatization (Fig.9 and SI Text). This proposal for nAChR binding inhibition bythe PPP-ketene is somewhat analogous to inhibition of phos-photriesterase by alkynyl phosphate esters, involving a keteneintermediate acylating a histidine nucleophile (16).

Concluding RemarksThat the nicotinic receptor plays a critical role in insect control isnow even more evident with [3H]NMI as a reporter on in vivoevents leading to poisoning (Figs. 1 and 2). Neonicotinoids suchas NMI and IMI, with exceptional potency and specificity, havea slow nAChR off rate (22), probably associated with a confor-mational change, allowing retention at the binding site in thehead of a poisoned insect during quick freezing in liquid nitro-gen, membrane preparation at 4 °C, and assay at 22 °C. OP- andMC- induced AChE inhibition and ACh accumulation in thesynapse during poisoning apparently give a nAChR conforma-tional change sufficient to inhibit [3H]NMI binding assayed asindicated. The present study emphasized Musca but the radio-ligand probe and methodology are also applicable to many otherinsects in studies of resistance mechanisms and species specificity.

MethodsChemicals. CYC and NMI were synthesized at the East China University ofScience and Technology (Shanghai, China). [3H]NMI (9) (60 Ci/mmol) wasprepared by Shanghai Ruxu Radiochemicals (Shanghai, China). PPP propionicacid metabolite was made by permanganate oxidation of the butynyl ana-log of PPP (Fig. 8 and see SI Text). All other compounds were from com-mercial sources. Structures for the neonicotinoids, OPs, and MCs are given inSI Text.

nAChR Binding Assays in Vitro. Musca adults were frozen with liquid nitro-gen, shaken to break them into body parts, and the heads isolated by

sieving. The frozen heads were homogenized in 0.32 M sucrose, 0.1 mMEDTA, 100 mM sodium phosphate (pH 7.4) using a ground-glass pestle tissuegrinder. The homogenate was filtered although four layers of 64-μm-meshnylon screen and centrifuged at 500 × g for 30 min. The supernatant wascollected and again filtered through four layers of 64-μm-mesh nylon screenand centrifuged at 25,000 × g for 60 min at 4 °C. The pellet was resuspendedin binding buffer consisting of 50 mM sodium chloride and 10 mM sodiumphosphate (pH 7.4). These membranes were used fresh or stored at −80 °Cfor up to several weeks without loss of binding activity. The reaction mix-ture, containing 1 nM [3H]NMI, 300–500 μg membrane protein (28) andvarying concentrations of unlabeled inhibitor in a final volume of 500 μL,was incubated for 60 min at 22 °C. Bound [3H]NMI was quantitated by fil-tration through Whatman GF/B glass-fiber filters prewetted by bindingbuffer with a Brandel M-24R Harvester, followed by two rinses each with5 mL of ice-cold binding buffer and liquid scintillation counting with nor-malization relative to protein levels. Nonspecific binding was determinedwith 20 μM unlabeled NMI. Specific binding was 80–90% of total binding.The percentage inhibition of nAChR binding was determined from two orthree separate experiments, each with triplicate samples. IC50 data werecalculated by nonlinear regression analysis for logarithm of inhibitor con-centration versus probit percentage inhibition.

nAChR Binding Inhibition in Vivo. nAChR in vivo inhibition 1 h after treatmentwas determined by in vitro assays on [3H]NMI binding in brain membranes.Thus, Musca adults anesthetized with carbon dioxide for 10 min were in-dividually treated with the test neonicotinoids dissolved in water or 10%

Fig. 8. CYP bioactivation of PPP synergist (R1 = C6H5; R2 = n-C3H7O) toa proposed ketene intermediate active as a postulated inhibitor of CYP ac-tivity and nAChR binding. Nuc refers to histidine (16) or another nucleophile.The identified propionic acid metabolite, synthesized from a 1-butynyl an-alog, established the PPP-ketene intermediate (SI Text).

Fig. 9. Proposed binding site interactions of ACh, NMI, and PPP-ketene atthe Musca nAChR model.

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(vol/vol) DMSO by intrathoracic injection (0.4 μL for each Musca) or with testOPs or MCs in acetone (0.4 μL) applied topically to the ventrum of the ab-domen. Twenty-five Musca were treated for each dosage with duplicatesamples. One hour after treatment and observation of the poisoning signs(see below), the Musca were frozen with liquid nitrogen and after shaking asabove the heads were picked out from the other body parts. Twenty headswere homogenized in 1 mL binding buffer at 4 °C using a Polytron (Brink-mann Instruments) for 20 s. Each homogenate was centrifuged at 700 × g for30 min at 4 °C. The supernatant was filtered through four layers of 64-μm-mesh nylon screen and then 800 μL was recovered for each binding assay bythe in vitro procedure detailed above. Inhibition values were based on two orthree independent experiments and adjusted for protein concentration.

AChE Assay. Musca head AChE activity was assayed by the Ellman procedure(29) comparing topically treated insects with untreated controls in eachexperiment. Five heads (8.5 mg) were homogenized in 2 mL 100 mM sodiumphosphate buffer (pH 8.0) using a Polytron, as above, for 15 s. The ho-mogenate was then centrifuged at 700 × g for 30 min at 4 °C and the su-pernatant passed through four layers of 64-μm-mesh nylon screen. Eachincubation mixture contained 30–40 μg protein (28) in 200 μL 100 mM po-tassium phosphate buffer, to which were added 5,5′-dithiobis-2-nitrobenzoicacid and acetylthiocholine (10- and 75-μM final concentration, respec-tively) and incubated at 25 °C to monitor the absorbance change at 405 nmfor 30 min.

Poisoning Signs. Poisoning signs for 50 flies were scored 1 h after toxicantinjection or topical application using the following rating system for each fly:0 for normal, 1 for impaired movement (unable to walk or fly), and 2 for dead(failure to move when prodded). The values in the blinded scoring werecalculated by the following equation: number of impaired flies plus twice thenumber of dead flies (i.e., all impaired was rated 50 and complete mortalitywas scored 100).

CYP Metabolism. Male albino Swiss Webster mice administered PPP (100 mg/kg,i.p.) in DMSO (1 μL/g body weight) were kept in glass metabolism cages.This treatment gave no apparent adverse effects. All mouse experimentswere approved by the UC Berkeley Animal Care and Use Committee(ACUC). Urine was collected for 24 h after treatment and stored at −80 °C

until LC/MS analysis. Thawed urine (200 μL) was evaporated to dryness undernitrogen at 25 °C. Residues were resuspended in 25% (vol/vol) acetonitrile(ACN)/0.1% formic acid aqueous solution (300 μL) and filtered through 0.2-μm nylon for LC/MS analysis. PPP urinary metabolites were analyzed by LC/MS with electrospray ionization in the positive mode and a PhenomenexLuna 5μ-C18 column (250 × 2.0 mm, 5 μm) using a mobile phase of ACN and0.1% formic acid beginning with 5% (vol/vol) ACN for 3 min and increasingto 100% (vol/vol) by 25 min at a flow rate of 0.2 mL/min. A final 10-min washwith 5% ACN/ 0.1% formic acid aqueous solution eluted interfering mate-rials. PPP-propionic acid was assigned as a urinary metabolite based on a m/z272.6 peak at tR 23.9 min absent in control urine and cochromatography ofthe 272.6 peak with added authentic compound (SI Text).

Calculations. Musca α5α5 homology models were built and refined with thePDB structures 4AQ5 (30) and 3SQ6 as templates. These structures re-spectively are nAChRs from Torpedo and Lymnaea with 43% and 39%identities and 68% and 61% positives in homology with 205 residues in theligand binding domain of the Musca α5 structure. We then used an InducedFit protocol pairing Glide and Prime software (Glide, v5.8, Prime, v3.1;Schrödinger) to dock ACh, NMI and the PPP-ketene in the known neon-icotinoid binding pocket of our well-refined α2β1 homology model forMyzus (27) and our new Musca models, including a bridging water as seenin the Aplysia structure 3C79 (31). (See SI Text for refinement anddocking details.)

ACKNOWLEDGMENTS. We thank Amanda Ly for the acetylcholinesteraseactivity assays; Breanna Morris and Tami Swenson for the cytochrome P450metabolism experiments; Nanyang Chen for help in the synthesis of prop-2-yn-1-yl propyl phenylphosphonate (PPP) derivatives; Ellen Key and Yan Xufor assistance in experiments and manuscript preparation; and ErnestHodgson, Robert Hollingworth, and David Soderlund for helpful commentson the manuscript. This study was supported in part by the National HighTechnology Research Development Program of China (2011AA10A207), KeyProjects of the National Science and Technology Pillar Program of China(2011BAE06B05), and the National Natural Science Foundation of China(21002030) (for funding the synthesis of [3H] nitromethyleneimidazole, cyclo-xaprid, nitromethyleneimidazole, and PPP derivatives); and a Shanghai Edu-cation Committee Fellowship (to X.S.).

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