Mutagenesis of odorant coreceptor Orco fully disrupts foraging but not oviposition behaviors in the hawkmoth Manduca sexta

Abstract

The hawkmoth Manduca sexta and one of its preferred hosts in the North American Southwest, Datura wrightii, share a model insect-plant relationship based on mutualistic and antagonistic life-history traits. D. wrightii is the innately preferred nectar source and oviposition host for M. sexta Hence, the hawkmoth is an important pollinator while the M. sexta larvae are specialized herbivores of the plant. Olfactory detection of plant volatiles plays a crucial role in the behavior of the hawkmoth. In vivo, the odorant receptor coreceptor (Orco) is an obligatory component for the function of odorant receptors (ORs), a major receptor family involved in insect olfaction. We used CRISPR-Cas9 targeted mutagenesis to knock out (KO) the MsexOrco gene to test the consequences of a loss of OR-mediated olfaction in an insect-plant relationship. Neurophysiological characterization revealed severely reduced antennal and antennal lobe responses to representative odorants emitted by D. wrightii In a wind-tunnel setting with a flowering plant, Orco KO hawkmoths showed disrupted flight orientation and an ablated proboscis extension response to the natural stimulus. The Orco KO gravid female displayed reduced attraction toward a nonflowering plant. However, more than half of hawkmoths were able to use characteristic odor-directed flight orientation and oviposit on the host plant. Overall, OR-mediated olfaction is essential for foraging and pollination behaviors, but plant-seeking and oviposition behaviors are sustained through additional OR-independent sensory cues.

Keywords: CRISPR-Cas9; Manduca sexta; Orco; insect olfaction; insect–plant interactions.

Fig. 1.

CRISPR-Cas9–directed mutagenesis of M. sexta Orco. (A) The M. sexta Orco protein has 7 transmembrane domains. The first transmembrane domain was mutated generating a predicted frameshift (yellow) and translational stop signals (red) (B, Top). The M. sexta 10-exon Orco gene (18.85 kb) was accessed through the annotated official gene set (OGS) (gene ID Msex2.12779), with scaffold and base pair coordinate location on the positive strand (12,214 to 31,059 bp) within scaffold JH668978.1. A proximal region to the start of exon 2 was targeted for CRISPR single-guide RNA (sgRNA) design. Two sgRNAs (target1 and target2) on opposite DNA strands were multiplexed and injected. Target 1 was designed with a mismatch nucleotide, indicated in red to raise in vitro transcription efficiency. PAM sites indicated in purple, Cas9 cleavage sites 3 nucleotides from the PAM site indicated by arrows. (B, Bottom) Three germ-line insertion deletion (indel) mutations were recovered and sequenced (mutations 1, 2, and 3). All indels were generated by sgRNA target 1, insertion indicated in red, deletions indicated as dash lines. (C, Top) Mutation 1 was used for downstream functional analysis. Sanger sequencing chromatogram of wild-type (WT) individual and mutation 1 allele shows insertion event, indicated by box. (C, Bottom) Indel generated a frameshift mutation introducing 2 stop codons downstream, giving rise to a truncated 72-residue protein, indicated through the predicted amino acid sequence.

Fig. 2.

Genotyped M. sexta Orco KOs do not express Orco or respond to conspecific female sex pheromone. (A, Top) Genotyping primers designed to amplify 201 bp of the targeted exon 2 of MsexOrco. Orco KO allele produces a 212-bp amplicon, characterized by the (−1, +11) indel, which introduces a Bcl1 restriction enzyme cutting site not found in the WT allele. Insertion indicated in red, restriction site underlined, arrow points to enzymatic cut site. (A, Bottom) PCR product amplified from individual caterpillar horn tissue samples loaded on a 2.0% agarose gel. Lanes 1 and 2, PCR product with no restriction enzyme added. Lanes 3 through 8, PCR product cut with restriction enzyme. Lane 3, no cut bands from a WT caterpillar. Lanes 4 through 6, ∼200-bp band of PCR product is still dark, with lighter bottom bands at ∼150 and 50 bp, indicative of Orco HET individuals. Lanes 7and 8, ∼200 bp is mostly digested and breaks down to a darker ∼150-bp band and ∼50-bp band, indicative of the homozygous Orco KO individual. (B) Expression of the M. sexta Orco protein in the olfactory sensory neurons (OSNs) was characterized in the WT (Left) and KO (Right) on paraffin-embedded male antennae sections. Nickel-DAB staining with HRP conjugated secondary antibody detected Orco-positive OSNs only in the WT but not in Orco KO hawkmoths. Top, transversal; Bottom, sagittal sections. (Scale bars, 10 μm.) (C) Single sensillum recordings (SSR) from long trichoid sensilla of adult male antennae stimulated with 10⁻² (vol/vol) bombykal in mineral oil for 0.5 ms. (Top) Representative SSR traces, odor stimulation indicated by the gray bar. (Bottom) Mean net responses from 3 sensilla per hawkmoth (± SD); circles represent results from individual hawkmoths. Total number of hawkmoths tested n = 5 per genotype (***P ≤ 0.001; not significant, P ≥ 0.05, 1-sample t test versus zero). (D) EAGs from clipped male antennae stimulated with bombykal; same concentration as in C. (Top) Representative EAG recordings. (Bottom) Box plots show the net response, corrected against solvent (*P ≤ 0.05, ***P ≤ 0.001, Wilcoxon rank sum test versus zero). Box plots show the median net EAG amplitude (horizontal line in the box), the 25th and 75th percentiles (lower and upper margins of the box) together with the 1.5× interquartile range (whiskers), and individual data points (circles). Genotyped individuals were all confirmed with EAG recordings, matching genotype to a physiological phenotype.

Fig. 3.

M. sexta Orco KOs show disrupted or reduced olfactory responses to plant headspace and to ecologically relevant single odors. (A) D. wrightii headspace collection was used to stimulate male and female antennae (for gas chromatogram see SI Appendix, Fig. S4). (Top) Presumed neural activity patterns from Ca²⁺ response in male and female antennal lobes (ALs) after stimulation with D. wrightii headspace in solvent. Pattern of responses varies based on sexual dimorphic morphology of the M. sexta AL (Top; ref. 119). False color-coded images are representative Ca²⁺ activity responses from right ALs; entrance of the antennal nerve is in the upper left. Images were generated by subtracting the frame before stimulus onset from the frame with the maximum response. Calcium activity was normalized for each image and color-coded (see color bar). (B) Heat-map representation of median EAG response values (millivolts) corrected for the response to the solvent from male and female antennae (***P ≤ 0.001, **P ≤ 0.01, *P ≤ 0.05; n.s., not significantly different from 0; Wilcoxon rank sum test versus zero). Box-plot representation and corrected median values with individual P values are shown in SI Appendix, Figs. S5 and S6 and Tables S2 and S3. Sample size of all 3 genotypes for headspace and single odorants male and female in parentheses (n). (C) Neural activity in the AL of males and females in response to stimulation with benzaldehyde (floral odor) and hexanoic acid (vegetative odor) as these elicit the strongest EAG responses in the Orco KO hawkmoth.

Fig. 4.

M. sexta Orco KO were slower to reach a flowering plant and showed disrupted foraging. (A) The percentage of male hawkmoths that flew toward a flowering D. wrightii plant (stimulus source) in a 250-cm-long wind tunnel did not differ between genotypes. Total number of hawkmoths tested (n) in parentheses. (χ² = 2.1344; degrees of freedom = 2; test of equal proportions). (B) Orco KO hawkmoths took longer to contact the plant. The time to contact was calculated from take-off until males hovered in front of the flower or contacted the plant in a 5-min free-flight experiment (Holm corrected Wilcoxon rank sum test). The box plots show the median net contact time (horizontal line in the box), the 25th and 75th percentiles (lower and upper margins of the box) together with the 1.5× interquartile range (whiskers), and individual data points (circles). (C) Three-dimensional flight tracks in response to the stimulus source, hawkmoth (not to scale) depicts start of flight toward nonflowering plant (not to scale) in a wind tunnel. Flight tracks were chosen based on time to contact median values. (D) Two-dimensional heat map representation of the cumulative relative distribution of all tracked hawkmoths from start to stimulus source; sample size (n). Single pixels represent points of relative transit of the hawkmoth color-coded from maximum transit (red) to no transit (dark blue) (see color bar). (E) The relative frequency of track-angle (flight direction relative to the wind) toward the flowering plant is shown as the percentages of track angles toward stimulus source (0°). (F) Percentage of free-flight males that foraged was significantly reduced in the Orco KO males compared with WT and Orco HET. Foraging counted when males unfurled the proboscis and contacted the flower with the proboscis (χ² = 41.118; degrees of freedom = 2; P < 0.001; Holm corrected test of equal proportions). (G) Images captured from an infrared filter camera above the flower in wind tunnel flight experiments. Orco HET male with proboscis extended preparing to forage (Top) and Orco KO male immediately after contacting the flower, without proboscis extended (Bottom).

Fig. 5.

Orco KO gravid hawkmoths have reduced attraction toward nonflowering D. wrightii; however, females that flew to the plant used olfactory-directed flight behaviors. (A) The percentage of mated M. sexta females that flew and contacted a nonflowering D. wrightii plant (stimulus source) was significantly reduced in KOs compared with WT and HET females (χ² = 11.261; degrees of freedom = 2; P = 0.004; Holm corrected test of equal proportions). (B) Time to contact was measured from take-off to physical contact on the plant. Orco KO female time to the stimulus source was not significantly different compared with WT and Orco HET (Kruskal–Wallis H test). Box plots show the median net contact time (horizontal line in the box), the 25th and 75th percentiles (lower and upper margins of the box) together with the 1.5× interquartile range (whiskers), and individual data points (circles). (C) Three-dimensional flight tracks in response to the plant stimulus, hawkmoth (not to scale) depicts start of flight toward nonflowering plant (not to scale). Flight tracks were chosen based on time to contact median values. (D) Two-dimensional heat map representation of the cumulative relative distribution of all tracked hawkmoths data from start to source; sample size (n). Single pixels represent points of relative transit of the hawkmoth color-coded from maximum transit (red) to no transit (dark blue) (see color bar). (E) The relative frequency of track angle (flight direction relative to the wind) toward the nonflowering plant is shown as the percentages of track angles toward stimulus source (0°). (F) The percentage of total females that elicited oviposition behaviors (contact on leaf and abdomen curl) on whole D. wrightii plant were significantly reduced in the KOs compared with HET or WT females (χ² = 13.735; degrees of freedom = 2; P = 0.001; Holm corrected test of equal proportions). (G) Fifty-five percent of total Orco KO females laid a similar number of eggs on D. wrightii plants compared with WT and Orco HET. Bars and error bars represent mean ± SD, with no significant differences between genotypes (not significant; one-way ANOVA).