1. Volume 27, Issue 11, Pages 1610-1615.e3 (June 2017)
A Pheromone Antagonist Regulates Optimal Mating Time in the Moth Helicoverpa armigera 
Hetan Chang, Yang Liu, Dong Ai, Xingchuan Jiang, Shuanglin Dong, Guirong Wang 
Current Biology 
Volume 27, Issue 11, Pages e3 (June 2017)
DOI: /j.cub
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2. Current Biology 2017 27, 1610-1615.e3DOI: (10.1016/j.cub.2017.04.035)
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3. Figure 1
Effect of Age on the Ratio of the Sex Pheromone Components Z11-16:OH and Z9-14:Ald in the Pheromone Glands of Helicoverpa armigera Females
(A) Total ion current chromatograms from single pheromone gland extracts of females at different ages.
(B) Relative ratios of Z11-16:OH (left) and Z9-14:Ald (right) compared with the major component Z11-16:Ald. The relative ratios of pheromone components were calculated by comparing with the peak area of the most prevalent component, Z11-16:Ald, as 100%. N = 10. The data are shown as means ± SD and were analyzed by Student’s t test: ∗∗∗p < 0.001; NS, p > 0.05.
See also Table S1.
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4. Figure 2 Targeted Mutation of the HarmOR16 Gene Using CRISPR/Cas9
(A) The graphical representation of the two-segment system for inducing double-strand breaks. The single guide RNA (sgRNA) comprised a 20 bp sequence (marked in green) complementary to a genome target adjacent to the PAM position of NGG (marked in red) and stem loops from the tracrRNA-meditated binding to the Cas9 protein. Another significant segment, the dual NLS-tagged codon-optimized Cas9 protein, is indicated in blue. Both segments were initially synthesized as RNA with in vitro transcription directed by the T7 promoter.
(B) Schematic diagram of the sgRNA targeted in exon 3 of HarmOR16 (top) and the genotype of OR16 mutants in the four F0 moth genotypes (OR161, OR162, OR163, and OR164; bottom). The target site is shown in yellow and includes a restriction enzyme site (Nco I), the PAM sequence is marked in red, and the triangle represents the cleavage site. Deleted bases are shown in dashes, and inserted bases are indicated with lowercase letters. The net changes in length are labeled at the right of each genotype (+, insertion; −, deletion).
(C) Genotyping via restriction enzyme assays in F1 moths. The upper row represents uncleaved bands that indicate that the samples contained mutations, and the two lower rows represent the expected size of cleaved fragments derived from wide-type samples. The wide-type, heterozygote, and homozygote individuals are marked at the top of the graph.
(D) A flow diagram of the crossing scheme employed in this study. The red region represents the autosomes with disrupted HarmOR16.
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5. Figure 3
The Effect of the OR16 Receptor on Z11-16:OH- and High-Concentration Z9-14:Ald-Evoked Electrophysiological and Avoidance Behavioral Responses
(A) Diagram of an OR6 neuron, labeled in violet, and an OR16 neuron, labeled in green, housed within type-C sensilla trichodea.
(B) The representative traces of spontaneous activity in OR6 and OR16 neurons in wild-type (WT) and mutant males. The action potentials of OR6 and OR16 neurons are marked.
(C) Quantification of the mean spontaneous activities for the experiment shown in (B). Error bars indicate the SEM. N = 12–13. Data were analyzed by Student’s t test: ∗∗p < 0.001; NS, p > 0.05.
(D) Representative traces of OR6 and OR16 neurons activated by 1 mg pheromone components, including Z11-16:Ald, Z9-16:Ald, Z9-16:OH, Z9-14:Ald, and Z11-16:OH in WT and mutant males. The red bold line represents the 0.3 s odor stimulation. The red pane represents the amplification part of the response. The violet circle represents OR6 neurons and the green circle represents OR16 neurons.
(E) Quantification of the mean responses to the indicated stimulus for the experiment shown in (D). Error bars indicate the SEM. Data were analyzed by Student’s t test. N = 18–20. The response values to specific odour stimuli were calculated as the differences in spike numbers observed between 1 s before and 1 s after stimulus delivery.
(F) The percentage of males that made the final choice of orientating to the standard pheromone blend arm (I) or the experimental blend arm (II or III). N = 3–4. Each replication contained 30 male individuals. I, 97% Z11-16:Ald + 3% Z9-16:Ald; II, 97% Z11-16:Ald + 3% Z9-16:Ald + 10% Z11-16:OH; III, 97% Z11-16:Ald + 3% Z9-16:Ald + 5% Z9-14:Ald. The data are shown as the mean ± SEM and were analyzed by Chi-square test: ∗p < 0.05; ∗∗∗p < 0.001; NS, p > 0.05.
See also Figure S1 and Table S2.
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6. Figure 4 Mating with Mature Female Benefits Reproduction Outputs
(A) Distribution of mating rates of WT (black) and OR16 mutant (red) males with WT females at different ages after emergence.
(B) The mating rates of WT males with WT females painted with 2 μL paraffin oil (as a control), Z11-16:OH (100 ng/μL) at day 3. For both two experiments, data were analyzed by Student’s t test. Error bars indicate the SEM. ∗∗∗p < 0.001; ∗∗p < N = 3
(C) Comparison of various mate ages on the reproduction output of WT pairs. We randomly picked eight WT pairs of each mate age, allowed females to lay eggs on gauze, and counted the total number of offspring. The data were analyzed by one-way ANOVA. Error bars indicate the SEM. N = 3.
(D) The comparison of overall reproduction output of WT and mutant pairs. We selected a total of 24 WT or mutant groups (eight for each day: 2, 3, and 4) and recorded the total number of offspring. Error bars indicate the SEM. Data were analyzed by Student’s t test: ∗∗∗p < N = 3.
See also Table S3.
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