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.

The olfactory co-receptor IR8a governs larval-frass mediated competition avoidance in a hawkmoth

“…Unfortunately, the oviposition rates of Orco −/− moths were too low to draw any conclusions regarding the involvement of Orco in the oviposition avoidance. One explanation for the conflicting result with our previews study 30 in terms of oviposition in Orco −/− moths is that Fandino et al (2019) used whole and large D. wrightii plants which probably provided a very strong visual stimulation. Interestingly, however, mutation of Ir25a did not affect the oviposition behavior (Fig.…”

“…A recently published functional analysis of the moths’ antennal lobe 45 revealed three glomeruli that strongly responded to acids. In another study 30 , activation of two of these glomeruli was not affected by knocking out the OR-coreceptor Orco, supporting that these two glomeruli become innervated by IR-expressing OSNs. When performing calcium imaging experiments with moths that either lacked a functional IR25a or IR8a, the responses to acids in Ir25a mutants were unaffected compared to control animals (Fig.…”

“…To determine which olfactory pathway is governing the detection of 3-methylpentanoic acid and hexanoic acid, we performed electroantennography (EAG) measurements on wild type (WT) moths, and on odorant co-receptor heterozygous ( Orco +/−) and homozygous ( Orco −/−) moths that were recently generated in our lab using CRISPR/Cas9 genome editing 30 . While WT moths and Orco +/− moths exhibited robust EAG responses to the acids, Orco −/− moths showed reduced responses (Supplementary Fig.…”

“…To determine which co-receptor is involved in the acid detection and acid-driven oviposition avoidance, we used olfactory receptor co-receptor (Orco) mutant moths 30 , and generated Ionotropic receptor 8a (Ir8a) and Ir25a two mutant lines. The M. sexta genome v.1.0 (Mansexv1.0) fasta file and the GFF3 file were submitted to the CHOPCHOP (http://chopchop.cbu.uib.no) database for CRISPR/ Cas9 target selection sites.…”

“…The tobacco hawkmoth Manduca sexta (Lepidoptera: Sphingidae) is an established model for insect olfaction 13 and odor-guided behavior 29 . The recent identification of 73 OR genes and 21 olfactory IR genes and their expression patterns in male and female moths 13 and the establishment of the Crispr/Cas 9 technique in M. sexta 30 has made the species to an even more powerful model for olfactory neuroethology. The larvae of these moths feed on various plants of the family Solanaceae, including coyote tobacco ( Nicotiana attenuata ) and jimson weed ( Datura wrightii ).…”