Charvet CL , Guégnard F , Courtot E , Cortet J , Neveu C .

P40 OD010440 NIH HHS

	Fig. 1. Motility modulation of H. contortus L3 larvae exposed to levamisole, pyrantel or ivermectin. The automated larval migration assay (ALMA) was used to determine dose-dependent paralysis effect of Lev, Pyr or Ivm on the H. contortus L3 from Weybridge (A; C and E) or Kokstad isolate (B; D and F). Representative recording traces of the real-time fluorescence counting relative to the L3 migration during 5 min exposed to Lev (A and B); Pyr (C and D) or Ivm (E and F). Each trace corresponds to the mean data from 3 runs performed with 7500 L3 larvae. The controls correspond to untreated L3 larvae.
	Fig. 2. Motility modulation of H. contortus L3 larvae exposed to nicotine, nornicotine or anabasine. The automated larval migration assay (ALMA) was used to determine dose-dependent paralysis effect of Nic, Nor or Ana on the H. contortus L3 from Weybridge (A; C and E) or Kokstad isolate (B; D and F). Representative recording traces of the real-time fluorescence counting relative to the L3 migration during 5 min exposed to Nic (A and B); Nor (C and D) or Ana (E and F). Each trace corresponds to the mean data from 3 runs performed with 7500 L3 larvae. The controls correspond to untreated L3 larvae.
	Fig. 3. Determination of the dose response relationships for levamisole (A); pyrantel (B); ivermectin (C); nicotine (D); nornicotine (E) and anabasine (F) on H. contortus L3 larvae using the automated larval migration assay (ALMA). Results are shown as the mean ± se from 3 distinct ALMA assays performed with 7500 H. contortus L3 larvae from the Weybridge isolate (in black) or Kokstad isolate (in red). IC50 values are indicated on the graphs. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)
	Fig. 4. Effects of nicotine, nornicotine or anabasine on the motility of C. elegans. Paralysis assays were performed on N2, acr-16 (ok789) and lev-8 (ok1519) C. elegans strains in agar plate containing 31 mM of nicotine (A), nornicotine (B) or anabasine (C) after 15, 30, 45 and 60 min drug exposure. Paralysis was scored based on the absence of worm's movement in response to prodding. Data are the mean ± SEM of n = 12, ****p < 0.0001, ***p < 0.001, **p < 0.01 and *p < 0.05, one way ANOVA with Bonferroni post-hoc test between N2 and the mutant strains.
	Fig. 5. Amino-acid alignments of ACR-16 subunit sequences from Caenorhabditis elegans, Haemonchus contortus and Parascaris equorum. acr-16 deduced amino-acid sequences were aligned using the MUSCLE algorithm (Edgar, 2004) and further processed using GeneDoc. Predicted signal peptide sequences are shaded in grey. Amino acids conserved between all the ACR-16 sequences are highlighted in dark blue. Amino acids specifically shared by ACR-16 homologs from parasitic species are highlighted in red. Amino acids specifically shared by Clade V nematode species (C. elegans and H. contortus) are highlighted in light blue. The cys-loop, the four transmembrane regions (TM1–TM4) and the primary agonist binding (YxCC) are indicated above the sequences. Cel (Caenorhabditis elegans), Hco (Haemonchus contortus), Peq (Parascaris equorum). (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)
	Fig. 6. Concentration-response relationships of acetylcholine and nicotine derivatives on the P. equorum N-AChR expressed in Xenopus oocytes. Representative current traces for single oocytes perfused with acetylcholine (ACh), nicotine (Nic), anabasine (Ana) and nornicotine (Nor). The concentration of agonist (μM) is indicated above each trace.
	Fig. 7. Concentration-response curves of acetylcholine and nicotine derivatives on the C. elegans (A) and P. equorum (B) N-AChRs expressed in Xenopus oocytes. The N-AChRs were challenged with acetylcholine (ACh), nicotine (Nic), anabasine (Ana) and nornicotine (Nor). All responses are normalized to 1 mM Ach. Results are shown as the mean ± se.
	S1Fig. tif. H. contortus L3 larvae migration assay using auto-fluorescence quantification. Correlation between the fluorescence counting (counts/sec) and the number of L3 larvae that migrated to the recording chamber during 5min. Migration assays were performed using 1000, 2000, 3500, 5000 and 7500 L3 larvae respectively. Each data point represents mean ± SE of three independent runs
	S2Fig. tif. Comparison of the motility reduction of H. contortus L3 from the Weybridge vs Kokstad isolates exposed to Levamisole (Lev), Pyrantel (Pyr), Ivermectin (Ivm), Nicotine (Nic), Nornicotine (Nor), Anabasine (Ana) as determined by ALMA assays. Mean data of the twenty last fluorescence measures ± SEM. Welsh two sample t-test, Wey vs Kok. ****p < 0.001, ***p < 0.001, **p < 0.01
	S3Fig. tif. 1. Maximum likelihood tree showing relationships of ACR-16 acetylcholine receptor (AChR) subunits from C.elegans, H. contortus and P. equorum with other C. elegans AChR subunits. Tree was built upon an alignment of AChR subunit sequences excluding the predicted signal peptide and the variable region between TM3 and TM4. The tree was rooted with DEG-3 group subunit sequences. Scale bar represents the number of substitution per site. Boostrap values > 80% are indicated on branches. Accession numbers for sequences used in the phylogenetic analysis are provided in Material and Methods section. C. elegans AChR subunit groups are named as proposed by Mongan et al. (Mongan et al., 2002). Cel, Hco and Peq refer to Caenorhabitis elegans, Haemonchus contortus and Parascaris equorum respectively
	S4Fig. tif. Tentative expression of Hco-ACR-16 subunit in Xenopus oocytes. Representative recording traces from a single oocyte challenged with 1 mM ACh and 1 mM Nic. Experiment repeated in three independent batches of oocytes.
	S5Fig. tif. Representative recording traces showing the effect of 100 μM acetylcholine (ACh), nicotine (Nic), anabasine (Ana) and nornicotine (Nor) on Xenopus oocytes expressing the C. elegans L-AChR (A), the H. contortus L-AChR-1 (B) and the the H. contortus L-AChR-2 (C)

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