Cartereau A , Taillebois E , Selvam B , Martin C , Graton J , Le Questel JY , Thany SH .

	FIGURE 1. Phylogenetic tree showing relationships of alpha 7 nAChR subunit protein sequence of the cockroach P. americana and its orthologs in several insect species and human. The bootstrap support value (%) based on 1,000 replicates are shown when higher than 50%. The D. melanogaster GABAA subunit (accession number AAA28556.1) was used as outgroup. Accession sequence identifiers are as follows: Anopheles gambiae: Agamα1 (AAU12503.1), Agamα2 (AAU12504.1), Agamα3 (XP_310786.3), Agamα4 (XP_566274.3), Agamα5 (XP_314691.2), Agamα6 (XP_308042.3), Agamα7 (XP_309153.3), Agamα8 (XP_311925.3), Agamα9 (XP_310203.3), Agamβ1 (XP_309158.3); Aphis gossypii: Agosα1 (AAM94383.1), Agosα2 (AAM94382.1), Agosα3 (ABR21379.1), Agosα4 (ABR21380.1), Agosα7 (AFM78640.1), Agosα8 (BBA21164.1), Agosβ1 (AAM94384.1); Apis mellifera: Amelα1 (NP_001091690.1), Amelα2 (NP_001011625.1), Amelα3 (NP_001073029.1), Amelα4 (NP_001091691.1), Amelα5 (AJE70263.1), Amelα6 (NP_001073564.1), Amelα7 (AJE70265.1), Amelα8 (NP_001011575.1), Amelα9 (NP_001091694.1), Amelα10 (XP_392070.3), Amelβ1 (NP_001073028.1), Amelβ2 (NP_001091699.1); Bactrocera oleae: Boleα2 (XP_014100082.1), Boleα7 (XP_014102300.1), Boleβ2 (XP_0111204502.1), Boleβ3 (XP_014090987.1); Bombyx mori: Bmorα1 (ABV45511.1), Bmorα2 (ABV45512.1), Bmorα3 (ABV45513.1), Bmorα4 (ABV45514.1), Bmorα5 (ABV45516.1), Bmorα6 (NP_001091830.1), Bmorα7 (ABV45520.2), Bmorα8 (ABV45521.1), Bmorα9 (ABV45523.1), Bmorβ1 (NP_001166819.1), Bmorβ2 (NP_001103400.1), Bmorβ3 (NP_00110341.1); Ceratitis capitata: Ccapα2 (XP_004536261.1), Ccapα7 (JAB87466.1), Ccapβ1 (XP_012156453.1), Ccapβ2 (XP_012162675.1); Drosophila melanogaster: Dmelα1 (CAA30172.1), Dmelα2 (NP_524482.1), Dmelα3 (CAA75688.1), Dmelα4 (CAB77445.1), Dmelα5 (AAM13390.1), Dmelα6 (NP_723494.2), Dmelα7 (CAD86936.1), Dmelβ1 (P04755.1), Dmelβ2 (CAA39211.1), Dmelβ3 (NP_525098.1); Musca domestica: Mdomα2 (ABD37617.1), Mdomα5 (ABY40460.1), Mdomα6 (ABJ09669.1), Mdomβ1 (XP_005180169.1), Mdomβ2 (XP_005185796.1), Mdomβ3 (ABY40465.1); Myzus persicae: Mperα3 (CAB52297.1), Mperβ1 (XP_022165274.1), Mperβ2 (XP_022167599.1); Nasonia vitripennis: Nvitα1 (ACY82683.1), Nvitα2 (ACY82684.1), Nvitα3 (ACY82685.1), Nvitα4 (ACY82686.1), Nvitα5 (ACY82688.1), Nvitα6 (ACY82689.1), Nvitα7 (ACY82692.1), Nvitα8 (ACY82693.1), Nvitα9 (ACY82694.1), Nvitα10 (ACY82695.1), Nvitα11 (ACY82696.1), Nvitα12 (ACY82697.1), Nvitβ1 (ACY82698.1), Nvitβ2 (ACY82699.1), Nvitβ3 (ACY82700.1); Nilaparvata lugens; Nlugα1 (AAQ75737.1), Nlugα2 (AAQ7574101), Nlugα3 (AAQ75739.1), Nlugβ2 (AAQ75742.2); Periplaneta americana: Pameα1 (AKV94620.1), Pameα2 (AKV94621.1), Pameα3 (AKR16132.1), Pameα4 (AFA28129.1), Pameα6 (AKV94622.1), Pameα7 (MK790056), Pameα8 (AFA28130.1), Pameβ1 (AKV94624.1); Tribolium castaneum: Tcasα1 (ABS86902.1), Tcasα2 (ABS86903.1), Tcasα3 (ABS86904.1), Tcasα4 (ABS86905.1), Tcasα5 (ABS86907.1), Tcasα6 (ABS86908.1), Tcasα7 (ABS86911.1), Tcasα8 (ABS86912.1), Tcasα9 (ABS86913.1), Tcasα10 (ABS86914.1), Tcasα11 (ABS86915.1), Tcasβ1 (ABS86916.1); Homo sapiens: Hsapα2 (AAB40109.1), Hsapα3 (AAA59942.1), Hsapα4 (AAA64743.1), Hsapα5 (AAA58357.1), Hsapα6 (AAB40113.1), Hsapα7 (CAA49778.1), Hsapα9 (CAB65091.1), Hsapα10 (CAC20435.1), Hsapβ2 (CAA37320.1), Hsapβ3 (CAA47851.1), Hsapβ4 (CAA48336.1); Ctenocephalides felis: Cfelα1 (ABB42999), Cfelα2 (ABB43000), Cfelα3 (ABB43001), Cfelα4 (ABB43003), Cfelα7 (ABB43004), Cfelα8 (ABB43002), Cfelβ1 (ABB43005).
	FIGURE 2. Alignment of nAChR α7 subunit protein sequences of the cockroach P. americana with its orthologs in several insect species and human. The loops (LpA-F) involved in ligand binding and transmembrane motifs (TM1-4) forming the ion channel are indicated. Sites of cysteine residues involved in the Cys-loop are marked with asterisk; the vicinal cysteine residues characteristic of alpha-type and the key residues are shown in frame. Alignment was done with drosophila sequence as reference, identical residues (dark gray shading) and similar residues (light gray shading) are indicated.
	FIGURE 3. Expression pattern of Pameα7 subunit in the cockroach nervous system. (A) Expression level of Pameα7 using semi-quantitative RT-PCR experiments. Data are shown as expression ratio normalized with actin. Expression pattern was determined in the nerve chain (NC), antennal lobes (AL), mushroom bodies (MB) and optical lobes (OL). Significant differences are designated by different letters. (B) Electrophoresis showing Pameα7 and actin (used as quantitative and positive control). Dara are the mean of four independent experiments. Significant difference was determined using one-way ANOVA and Bonferroni post hoc test. All statistical analyses were added in Supplementary Figure S1C. (C) In situ hybridization of Pameα7 mRNA on cockroach adult brain. Expression is detected in the antennal lobes (C1), the non-compact Kenyon cells of the mushroom bodies (C2) and the optic lobes (C2).
	FIGURE 4. Effect of nicotine (Nic) and acetylcholine (ACh) on cockroach α7 nAChRs expressed in Xenopus laevis oocytes. (A) Typical example of nicotine- and acetylcholine-induced currents. Currents are recorded at 10 mM nicotine or acetylcholine, respectively. (B) Dose-response curve are represented for nicotine and acetylcholine. Data are normalized to 10 mM nicotine and each point represents a mean ± S.E.M of n = 12 tested oocytes. (C,D) Nicotine (10 mM) evoked currents recorded after the expression of cockroach α7 subunit alone or with rat RIC-3. Each histogram represents mean ± S.E.M of n = 10 oocytes. NS = no significant difference using one-way ANOVA and Bonferroni post hoc test.
	FIGURE 5. Comparative effect of nicotine on different experimental conditions. (A) Typical examples of nicotine-induced currents on not injected oocytes (native oocytes), oocytes with only water, oocytes with pGem vector and oocytes with α7 cDNA. Bars indicate 10 s application of nicotine at 1, 5 or 10 mM. (B–D) Histograms summarize applications of nicotine at different experimental conditions. Each histogram represents n = 12 oocytes, *p < 0.05 using One-way ANOVA and Bonferroni post hoc test.
	FIGURE 6. Effect of nAChR antagonists on nicotine evoked currents. (A) 5 min pretreatment with 10 μM α-Bgt, (B) 10 μM MLA, (C) 5 μM mecamylamine (MEC), and (D) 10 μM d-TC. Bars indicate application of 10 s nicotine at 10 mM. Histograms illustrate the effect of each antagonist on nicotine-evoked currents. In each case, data are mean ± S.E.M of n = 12 oocytes, *p < 0.05 using one-way ANOVA and Bonferroni post hoc test. NS = no significant.

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