Nicotinic modulation insecticides act on diverse receptor subtypes with distinct subunit compositions

Insect nicotinic acetylcholine receptors (nAChRs) are ligand gated ion channels mainly expressed in the central nervous system of insects. They are the directed targets of nicotinic modulation insecticides including neonicotinoids, the most widely used insecticides in the world. However, the resistance development from pests and the negative impacts on the pollinators affect their applications and create demand for the alternatives. Thus, it is very important to understand the mode of action of these insecticides at the molecular level, which is actually unclear for more than 30 years. In this study, we systematically examined the susceptibility of ten Drosophila melanogaster nAChR subunits mutants against eleven nicotinic modulation insecticides. Our results showed that there are several subtypes of nAChRs with distinct subunits compositions that are responsible for the toxicity of different insecticides, respectively. At least three of them are the major molecular targets of seven structurally similar neonicotinoids in vivo. On the other hand, the spinosyns may exclusively act on the α6 homomeric nAChR but not any other heteromeric pentamers. Behavioral assays using thermogenetic tools further confirmed the bioassay results and support the idea that receptor activation rather than inhibition leads to the insecticidal effects of neonicotinoids. The present findings reveal native nAChR subunit interactions with various insecticides and have important implications for resistance management and the development of novel insecticides targeting this important ion channels. Author Summary The neonicotinoids and spinosyns make up about 27% of the insecticides by world market value. Novel insecticides like sulfoxaflor, flupyradifurone and triflumezopyrim are developed as alternatives due to the negative effects of neonicotinoids on pollinators. Although all act via insect nicotinic acetylcholine receptors, the mode of action is unclear. Our work shows that these insecticides act on diverse receptor subtypes with distinct subunit compositions. This finding could lead to the development of more selective insecticides to control pests with minimal effects on beneficial insects.

recent paper also generated a 1 R81T Drsosophila and found that it has serious 161 defects in reproduction and locomotion [13], however, the 1 mutant we made 162 here did not show any significant fitness cost ( Figure S2). The sequences of 5, 163 6 and 7 are very close and show high similarity to the vertebrate nAChR 7 164 subunit. They can also form heteromecic ion channels in vitro with different 165 combinations like 5/6, 5/7 and 5/6/7 [14]. Since only the 6 mutant 166 showed resistance to spinetoram, we then wonder whether there is a genetic 167 redundancy among these evolutionarily conserved gene. However, the 5/7 168 double mutant was still sensitive to spinetoram (Table S11), indicating that the 6 169 homomeric channel could be the solo target for spinosyns. The way insects react when they are exposed to neonicotinoids, sulfoxaflor, 174 flupyradifurone and spinosyns are similar. The early-onset behaviors including 175 hyperactivity, convulsion, uncoordinated movements, leg extension and tremors. 176 At higher doses, these excitatory symptoms can induce severe tremors and 177 complete paralysis that lead to death [15][16][17]. We then wondered whether artificial activation of nAChR-expressing neurons wound induce insecticides-like 179 poisoning symptoms. Thus, we used the thermosensitive cation channel 180 Drosophila TRPA1 to acutely hyper-stimulate these neurons with all available 181 nAChR KI-Gal4 strains [18]. We found that expressing trpA1 in nAChR1 2A-  and nAChR2  183 neurons strongly induced hyperactivity behavior at 32 ℃, and eventually led to 184 paralysis ( Figure 3A, Video 1), which is similar to the above-mentioned 185 symptoms. However, activation of nAChR3 2A-GAL4 neurons did not show any 186 behavioral defects . These results parallel the above bioassay data that the 187 deletion of 1, 2, 3, 6 and 2 caused medium to high resistances to these 188 insecticides respectively. Therefore, thermogenetic activation of some nAChR-189 expressing neurons in a short time window phenocopies the action of 190 insecticides in target pests, which demonstrates that in vivo pharmacological 191 activation of these subunits-containing nAChRs leads to toxicity and finally death.

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The poisoning symptoms associated with triflumezopyrim is distinct from 193 other nicotinic modulation insecticides since it inhibits rather than activates insect 194 nAChRs. There is no any neuro-excitatory symptoms after treatment of 195 triflumezopyrim, on the contrary, it induces lethargic poisoning characterized by slow but coordinated leg movements and insects became less responsive to 197 stimuli over time [19]. Thus, we chose to use UAS-Shibire ts to inhibit nAChR-198 expressing neurons [20]. As expected, nAChR1  nAChR2 2A-GAL4 neurons produced a "sluggish" behavior rather than hyperactivity  207 We confirmed that the KO coding regions were not detected or barely 208 detectable with real-time PCR quantification ( Figure S2). There was no big 209 difference of expression levels of each subunits in these mutant flies, except that 210 the 3 was relatively less transcribed than other genes. For the 1 heterozygous 211 mutant, the mRNA levels of all subunits were almost same as the wild type 212 control.

Discussion
The Insecticide Resistance Action Committee (IRAC) classifies neonicotinoids, 216 sulfoximines, butenolides and mesoionics into sub-groups 4A, 4C, 4D and 4E 217 respectively, according to their chemical similarity relations. However, our results clearly showed that sulfoxaflor and flupyradifurone may specifically act on the 219 same nAChR subtype which consists of 3 and 1 subunits ( Figure 4A), albeit 220 their big differences in chemical structures. More importantly, we found that the 221 neonicotinoids act on distinct nAChR subtypes and such selectivity is not 222 dependent on the aromatic heterocyclic (A), or the electron-withdrawing nitro or 223 cyano moiety (X-Y) which is considered the key toxophore. Interestingly, the ring    Despite the widespread use of neonicotinoids for almost four decades, the 245 first and only field-evolved target-site resistance mutation (R81T in nAChR1) 246 was reported in 2011 and it has only been found in two species to date [11,12].

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Such unusual phenomenon can be partially explained by our findings that the 248 seven neonicotinoids have at least three distinct molecular targets in vivo. To  alternatives for neonicotinoids, however, their risk to bees is controversial [34][35][36]. Therefore, it is critical to understand the mode of action of these insecticides 311 inside bees. Since most Drosophila nAChR subunit genes (except 5 and 3) 312 have one-to-one orthologs in the honeybee and bumblebee genomes [7], the 313 expression and assembly of receptors could be conserved between flies and 314 bees, suggesting that our results will enable further studies about the 315 ecotoxicology and risk assessment for these nAChR modulators.

Figure S1
The crossing schemes to establish the nAChR1 R81T knock-in line.
The HDR event was isolated by imidacloprid selection and confirmed by PCR.
The vas-Cas9 (3XP3 RFP) was removed by the absence of red fluorescence in eyes.