Ito R , Kamiya M , Takayama K , Mori S , Matsumoto R , Takebayashi M , Ojima H , Fujimura S , Yamamoto H , Ohno M , Ihara M , Okajima T , Yamashita A , Colman F , Lycett GJ , Sattelle DB , Matsuda K .

	Figure 1. Neonicotinoids, their targets (nAChRs) and the functional expression of An. gambiae nAChRs in X. laevis oocytes, with the aid of cofactors AgRIC-3, AgUNC-50 and AgTMX3, measured by their responses to the neurotransmitter ACh. (a) Structure of neonicotinoids in IRAC group 4A. Imidacloprid and thiacloprid possess an ethylene bridge (E-bridge), while others have no E-bridge. (b) Top left edge and side-views of a nAChR structure where helices, loop and sheets are coloured cyan, magenta and red, respectively. The figure was illustrated by PyMol software (Schrödinger, USA) using the protein data base file 2BG9. The orthosteric site (ACh and neonicotinoid binding domain) is arrowed. (c) Schematic representations of the orthosteric sites formed at α/non-α and α/α subunit interfaces. Loops A, B, C, D, E, F and G involved in the interactions with ACh and neonicotinoids are shown. Basic residues (arginines) in loops D and G underpinning electrostatic interactions with the nitro or cyano groups (see panel A for the functional groups) are highlighted. (d) Responses to 100 µM ACh recorded from X. laevis oocytes injected with the subunit cRNAs together with the cofactor cRNAs. (e) Current amplitude of the responses 100 µM ACh of X. laevis oocytes injected with the subunit and cofactor cRNAs. Each box plot represents the 75 and 25% percentiles of data and horizonal line in each box indicates the median of data (n = 10 oocytes, from two frogs). Asterisks * and ** indicate that the differences are significant at levels of p < 0.05 and < 0.01, respectively (one-way ANOVA, Kruskal–Wallis test). The Agβ1 subunit is essential for the functional expression and the Agα1 subunit enhanced the amplitude of the ACh-induced response. (f) Heatmap representation of pEC50 values of ACh for the 13 An. gambiae nAChRs. White area means that the value could not be determined because the nAChR was not robustly expressed in the oocytes. The expressed nAChRs display diverse ACh sensitivity.
	Figure 2. Concentration–agonist activity relationships for ACh and neonicotinoids (imidacloprid, thiacloprid, clothianidin, acetamiprid, dinotefuran and nitenpyram) tested on 13 An. gambiae nAChRs expressed in X. laevis oocytes and analyses of factors governing agonist activity indices pEC50 and Imax. (a) Concentration-agonist activity relationships for ACh and neonicotinoids. Each data plot represents the mean ± standard error of the mean (n = 5). Curves were fitted by nonlinear regression analysis. (b,c) Two dimensional clustering of pEC50 (b) and Imax (c) values of the neonicotinoids for the 13 An. gambiae nAChR subtypes expressed in X. laevis oocytes. Imidacloprid and thiacloprid containing the E-bridge were paired, while acetamiprid, clothianidin and nitenpyram form a separate group. Dinotefuran, showing unique binding features, forms an outgroup with ACh. Thus, the E-bridge contributes to enhancing the affinity of neonicotinoids. For subunit combinations, neonicotinoids exhibited the highest affinity for the Agα3/Agβ1 and Agα3/Agα8/Agβ1 nAChRs with no Agα2 subunit, indicating that the Agα2 subunit has an affinity reducing effect. (d) Principal component scores for the neonicotinoids. Combined analyses of pEC50 and Imax pointed to unique features of dinotefuran which was plotted alone in the second quadrant. (e) Correlation of the agonist potency indices with the nAChR subunits and the neonicotinoids. The blueish colour in pEC50 and reddish colour in Imax of the Agα2 subunit indicated that neonicotinoids have a lower affinity for those subtypes which include Agα2, while increasing the efficacy. The Agα3 subunit increases the affinity while it has no clear effect on efficacy. For Imax of compounds, imidacloprid and thiacloprid generally showed lower efficacy than clothianidin, dinotefuran and nitenpyram.
	Figure 3. Crystal structure of the Q55R mutant of Ls-AChBP in complex with dinotefuran. The mutation was made to mimic insect nAChR basic residues located in loop D of the β1 subunits [15,25,27,28,30,32,34,71]. (a) Top and (b) side views of the crystal structure showing that Ls-AChBP assembles to form a homo-pentamer and that dinotefuran bound to all the five orthosteric sites. (c) Expanded view of the interactions of dinotefuran with key amino acids at the binding site. Main chains of principal and complementary proteins are coloured pale yellow and pale cyan, respectively. Dinotefuran and the key amino acids are represented as sticks, and carbons, nitrogens, oxygens and sulfur are coloured white/grey, blue, red and yellow, respectively. A water molecule involved in the hydrogen bond networks is represented as a sphere and coloured marine blue. Hydrogen bonds and electrostatic interactions represented as dotted lines are coloured cyan and orange, respectively. The CH-N interactions are represented as a white dashed line. The X-ray crystal structure revealed that the nitro group interacted with Lys34 in loop G and Arg55 in loop D of the complementary subunit, while its guanidine group stacked with Tyr185 in loop C. Uniquely, the tetrahydrofuran ring interacts with nitrogen of Trp143 loop C by CH-N interactions which are not seen in the AChBP complexed with imidacloprid, clothianidin, thiacloprid and the nitromethylene analogue of imidacloprid [32].
	Figure 4. Progress of knockdown of neonicotinoids for adult females of An. gambiae mosquitoes (An. gambiae s.l. (N'gousso strain An. coluzzi)) and the features of neonicotinoids. (a) Time-dependent development of knockdown following treatment with the neonicotinoids. (b) Relationship of log k (k is rate of progress of knockdown symptom) and log P (P is 1-octanol/water partition coefficient). (c) Correlation of the predicted and measured log k values. The high correlation of the predicted and measured values suggests a prominent role for the Agα1/Agα2/Agα8/Agβ1 nAChR in determining the rate of progress of the knockdown symptom in adult females of An. gambiae.
	Figure 1. . Neonicotinoids, their targets (nAChRs) and the functional expression of An. gambiae nAChRs in X. laevis oocytes, with the aid of cofactors AgRIC-3, AgUNC-50 and AgTMX3, measured by their responses to the neurotransmitter ACh. (a) Structure of neonicotinoids in IRAC group 4A. Imidacloprid and thiacloprid possess an ethylene bridge (E-bridge), while others have no E-bridge. (b) Top left edge and side-views of a nAChR structure where helices, loop and sheets are coloured cyan, magenta and red, respectively. The figure was illustrated by PyMol software (Schrödinger, USA) using the protein data base file 2BG9. The orthosteric site (ACh and neonicotinoid binding domain) is arrowed. (c) Schematic representations of the orthosteric sites formed at α/non-α and α/α subunit interfaces. Loops A, B, C, D, E, F and G involved in the interactions with ACh and neonicotinoids are shown. Basic residues (arginines) in loops D and G underpinning electrostatic interactions with the nitro or cyano groups (see panel A for the functional groups) are highlighted. (d) Responses to 100 µM ACh recorded from X. laevis oocytes injected with the subunit cRNAs together with the cofactor cRNAs. (e) Current amplitude of the responses 100 µM ACh of X. laevis oocytes injected with the subunit and cofactor cRNAs. Each box plot represents the 75 and 25% percentiles of data and horizonal line in each box indicates the median of data (n = 10 oocytes, from two frogs). Asterisks * and ** indicate that the differences are significant at levels of p < 0.05 and < 0.01, respectively (one-way ANOVA, Kruskal–Wallis test). The Agβ1 subunit is essential for the functional expression and the Agα1 subunit enhanced the amplitude of the ACh-induced response. (f) Heatmap representation of pEC50 values of ACh for the 13 An. gambiae nAChRs. White area means that the value could not be determined because the nAChR was not robustly expressed in the oocytes. The expressed nAChRs display diverse ACh sensitivity.
	Figure 2. . Concentration–agonist activity relationships for ACh and neonicotinoids (imidacloprid, thiacloprid, clothianidin, acetamiprid, dinotefuran and nitenpyram) tested on 13 An. gambiae nAChRs expressed in X. laevis oocytes and analyses of factors governing agonist activity indices pEC50 and Imax. (a) Concentration-agonist activity relationships for ACh and neonicotinoids. Each data plot represents the mean ± standard error of the mean (n = 5). Curves were fitted by nonlinear regression analysis. (b,c) Two dimensional clustering of pEC50 (b) and Imax (c) values of the neonicotinoids for the 13 An. gambiae nAChR subtypes expressed in X. laevis oocytes. Imidacloprid and thiacloprid containing the E-bridge were paired, while acetamiprid, clothianidin and nitenpyram form a separate group. Dinotefuran, showing unique binding features, forms an outgroup with ACh. Thus, the E-bridge contributes to enhancing the affinity of neonicotinoids. For subunit combinations, neonicotinoids exhibited the highest affinity for the Agα3/Agβ1 and Agα3/Agα8/Agβ1 nAChRs with no Agα2 subunit, indicating that the Agα2 subunit has an affinity reducing effect. (d) Principal component scores for the neonicotinoids. Combined analyses of pEC50 and Imax pointed to unique features of dinotefuran which was plotted alone in the second quadrant. (e) Correlation of the agonist potency indices with the nAChR subunits and the neonicotinoids. The blueish colour in pEC50 and reddish colour in Imax of the Agα2 subunit indicated that neonicotinoids have a lower affinity for those subtypes which include Agα2, while increasing the efficacy. The Agα3 subunit increases the affinity while it has no clear effect on efficacy. For Imax of compounds, imidacloprid and thiacloprid generally showed lower efficacy than clothianidin, dinotefuran and nitenpyram.
	Figure 3. . Crystal structure of the Q55R mutant of Ls-AChBP in complex with dinotefuran. The mutation was made to mimic insect nAChR basic residues located in loop D of the β1 subunits [15,25,27,28,30,32,34,71]. (a) Top and (b) side views of the crystal structure showing that Ls-AChBP assembles to form a homo-pentamer and that dinotefuran bound to all the five orthosteric sites. (c) Expanded view of the interactions of dinotefuran with key amino acids at the binding site. Main chains of principal and complementary proteins are coloured pale yellow and pale cyan, respectively. Dinotefuran and the key amino acids are represented as sticks, and carbons, nitrogens, oxygens and sulfur are coloured white/grey, blue, red and yellow, respectively. A water molecule involved in the hydrogen bond networks is represented as a sphere and coloured marine blue. Hydrogen bonds and electrostatic interactions represented as dotted lines are coloured cyan and orange, respectively. The CH-N interactions are represented as a white dashed line. The X-ray crystal structure revealed that the nitro group interacted with Lys34 in loop G and Arg55 in loop D of the complementary subunit, while its guanidine group stacked with Tyr185 in loop C. Uniquely, the tetrahydrofuran ring interacts with nitrogen of Trp143 loop C by CH-N interactions which are not seen in the AChBP complexed with imidacloprid, clothianidin, thiacloprid and the nitromethylene analogue of imidacloprid [32].
	Figure 4. . Progress of knockdown of neonicotinoids for adult females of An. gambiae mosquitoes (An. gambiae s.l. (N'gousso strain An. coluzzi)) and the features of neonicotinoids. (a) Time-dependent development of knockdown following treatment with the neonicotinoids. (b) Relationship of log k (k is rate of progress of knockdown symptom) and log P (P is 1-octanol/water partition coefficient). (c) Correlation of the predicted and measured log k values. The high correlation of the predicted and measured values suggests a prominent role for the Agα1/Agα2/Agα8/Agβ1 nAChR in determining the rate of progress of the knockdown symptom in adult females of An. gambiae.

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