Orco knockout by CRISPR/Cas9 and verification in Pieris brassicae.

(A), gene structure of Orco. Yellow blocks indicate exons and blue block indicates target area in the second exon. Black solid line indicates intron. Black arrows indicate the designated cleavage site of the Cas9 protein. The target knockout area is magnified to show the sequence. Blue letters indicate base pairs in the second exon segment, purple letters indicate mutation sites. Protospacer adjacent motif (PAM) sequences are in blue bold. (B), predicted transmembrane structure of Orco. The left and right panels represent wildtype Orco and mutated Orco transmembrane domains, respectively. Orange blocks indicate ORN membrane, extracellular (Extra) and intracellular (Intra) are shown. White circles indicate amino acids of Orco, blue circles on the left panel indicate the target mutation area. Cyan circles and the brown circle on the right panel indicate the mutated area and the early stop codon, respectively. Orco-positive ORNs and larval antennal center. P. brassicae larval antennae and larval antennal center (LAC) staining. (C), Odorant receptor neurons (ORNs) were stained (green cells, several are indicated by white arrows) in wildtype (WT) larval antennae. (D), ORNs were not stained in Orco knockout (KO) larval antennae. (E), An ORN was stained (green cell, indicated by white arrow) in WT larval palps. (F), ORNs were not stained in KO larval palps. (G), glomeruli in the LAC of a WT brain. (H), glomeruli in the LAC of a KO brain. Glomeruli are indicated by white dashed circles in (G) and (H). (I), number of glomeruli counted in WT caterpillar brain (orange) and Orco KO caterpillar (blue) brain. Significant difference was detected by Wilcoxon rank-sum test.

Pieris brassicae butterfly electrophysiological responses to chemical compounds and mating and oviposition dynamics.

(A), electroantennogram (EAG) response of male butterflies (n = 18 for both WT and KO). (B), EAG response of female butterflies (n = 15 for WT and n = 17 for KO). Left panels represent wildtype (WT) butterfly EAG responses, and right panels represent are knockout (KO) butterfly EAG responses. Significant differences between WT and KO butterflies were identified using Student’s t-test when the data was normally distributed and the variances were equal or using a Kruskal-Wallis rank-sum test when these criteria did not apply. Significant difference levels are indicated by asterisks, ns (P > 0.05), * (0.01 < P < 0.05), ** (0.001 < P < 0.01), *** (P < 0.001), - (no response recorded from WT butterfly antennae). Butterfly antennal responses (mV) to the tested chemicals are indicated by a color scale from navy (0 mV) via yellow to red (1.1 mV). (C), mating frequency of P. brassicae butterflies. The boxplots indicate the number of spermatophores in WT female butterflies (n = 11) and in KO butterflies (Orco-/-) (n = 14), the difference was tested by using Wilcoxon rank-sum test. (D), P. brassicae wildtype (WT) and Orco-/- knockout butterfly oviposition dynamics. The orange line indicates the number of eggs per female (mean ± SE) laid by wildtype butterflies (n = 11), the blue line indicates the number of eggs laid by Orco knockout butterflies (n = 14). Differences were analyzed using a GLM with negative binominal distribution, difference test results are shown in the line chart.

Pieris brassicae caterpillar growth and foraging behavior.

(A), P. brassicae caterpillar growth on cabbage plants, y axis shows weight (g) of WT and KO caterpillars after 10 days of feeding, n = 17. (B), caterpillar behavioral choices in a Petri dish. The numbers of caterpillars that chose one of the two discs are shown in the respective bars (n = 54 -73). Schematic drawing shows behavioral setup. Petri dish diameter was 188 mm, disc diameters were 13 mm. Discs in the Petri dish represent cabbage, paper and tomato leaf discs. Grey bars indicate paper disc choices, light green indicates cabbage leaf disc choices and orange indicates choices for tomato leaf discs. (C), caterpillar behavioral choices in Y-tube olfactometer without parasitoid wasps. Schematic drawing shows the Y-tube olfactometer. The dashed line indicates a black metal Y wire in the center of glass Y-tube olfactometer. The main arm of the Y-olfactometer is 200 mm length, the lateral arms are 275 mm length, the angle between the lateral arms is 80 °. Light green, dark green and grey bars represent choices for healthy plant, infested plant and no plant respectively. Healthy plant, plants were not treated; no plant, an empty jar without any insect or plant; infested plant, plants were infested by early L3 caterpillars. (D), caterpillar behavioral choices in Y-tube olfactometer with parasitoid wasps. Different treatments are in different colors. Dark green, infested plant - wasps; magenta, infested plant + wasps. Significant differences were tested between wildtype (WT) and knockout (KO) caterpillars or between two discs by Chi-square test in panel B-D, P-values are shown on the right side of each bar. (E), P. brassicae caterpillar growth when exposed to disarmed C. glomerata female parasitoids, y axis shows weight (g) of caterpillars after 10 days of feeding, n = 8. Schematic drawing shows disarmed female C. glomerata (ovipositor removed). (F), P. brassicae caterpillar survival rate when exposed to healthy C. glomerata female wasps, n = 7. Significant differences in development and survival rate were assessed using a one-tailed Student’s t-test, P values are indicated above boxplots. Schematic drawing shows a healthy female C. glomerata (unmanipulated). In both panels orange boxplots indicate wildtype caterpillars (WT), blue boxplots indicate Orco knockout caterpillars (KO).

Overview of caterpillar-(Pieris brassicae) and parasitoid wasp-(Cotesia glomerata) associated volatile compounds.

(A), PCA (Principal Component Analysis) two-dimensional score plot of five treatment groups: Cg, Cotesia glomerata female parasitoid wasps (n = 12); Pb, Pieris brassicae caterpillars (n = 10); Pb-Cg, P. brassicae caterpillars in the presence of C. glomerata female parasitoid wasps (n = 14); Pb-Fr, P. brassicae caterpillar frass (n = 10); and Pb-S, P. brassicae spit (n = 12), based on their volatile blend composition. (B), hierarchical clustering heatmap showing the abundance of each identified volatile compound of each treatment. Clustering chemicals in the heatmap indicate higher correlation. (C), schematic drawing of the custom-designed multi-channel arena. (D) wildtype (WT) caterpillar behavioral preference to the tested 15 odorants. (E), Orco knockout (KO) caterpillar behavioral preference to the tested 15 chemicals. Unfilled bars represent tests that exhibit no difference between paraffin oil and the odorant of interest. Blue bars indicate the cumulative duration ratio that caterpillars stayed in the paraffin oil zone, dark orange bars indicate the cumulative duration ratio that caterpillars stayed in the odorant zone. Relative time spent in odorant zone = Cumulative duration in a specific zone / Cumulative duration in the arena. Error bars indicate standard errors. In (D) and (E), differences were tested between the two zones using a Wilcoxon rank-sum test, P values are presented above bars (n = 31-50). For each comparison, left bar represents paraffin oil and right bar represents chemical compound.