A dysfunctional odorant co-receptor renders hawkmoths incapable of foraging, but not ovipositing

July 18, 2019    No. 8/2019 (203)

CRISPR-Cas9 provides a new tool for a comprehensive analysis of moth olfaction for studying interactions between insects and plants

Scientists from the Max Planck Institute for Chemical Ecology have used CRISPR-Cas9 gene editing to disable the olfactory co-receptor Orco in tobacco hawkmoths. Behavioral studies demonstrated that foraging is visibly disturbed in these moths. Although the insects were able to navigate to Western Jimsonweed flowers, they abruptly landed on the plant without even trying to drink nectar with their proboscis. Despite overall difficulties, some hawkmoths were able to successfully locate the plant and lay their eggs. This research approach expands our understanding of how moths detect their host plants (Proceedings of the National Academy of Sciences of the United States of America, July 2019, DOI: 10.1073/pnas.1902089116).

CRISPR-Cas9 gene editing allows for a targeted inactivation of genes. This is achieved in two parts: The Cas9 enzyme acts as molecular scissors for DNA and a single stranded guide RNA binds to the Cas9 enzyme and directs it to a specific location in the region of interest, in this case the Orco gene. Richard Fandino developed a technique for quick injection of the molecular components – dissolved in a buffer solution – with a quartz glass capillary into the sphingid egg, without damaging the egg and the developing tobacco hawkmoth embryo. The solution is dyed red to visualize the injection. Photo: Richard Fandino, Max Planck Institute for Chemical Ecology

The study of ecological interactions between plants and insects which are mediated by chemical signals is a topic of central interest in the field of chemical ecology. How important is the insects’ sense of smell as the recipient of plant signals in such interactions? A team of scientists led by Richard Fandino from the Department of Evolutionary Neuroethology has now explored this question experimentally. The researchers applied CRISPR-Cas9 in the tobacco hawkmoth Manduca sexta for a targeted gene modification.

Manduca sexta presents an excellent research organism with a very well-described ecology that may be important in uncovering some of the mysteries of insect-plant interactions from the perspective of the insect”, explains Richard Fandino.

The target of the CRISPR-Cas9 molecular scissors was the gene coding for the co-recptor Orco (Odorant Receptor Co-Receptor). This protein plays a crucial role in insect olfaction. Together with the so-called olfactory receptors it forms a complex: The olfactory receptor defines the molecules that are detected when smelling an odor. Together with Orco, the olfactory receptor transmits the stimulus, which elicits a neural response. CRISPR-Cas9 moths had a mutated Orco gene which rendered the receptor dysfunctional. Comparative studies between moths carrying this mutation and moths whose sense of smell is intact provide invaluable information about the importance of olfactory receptors.

Unusual behavior of moths without Orco

The researchers observed that foraging moths without a functional co-receptor Orco landed abruptly on the flower of a Western Jimsonweed (Datura wrightii), without hovering in front of the flower and extending their proboscis. “This was a completely novel behavior. We had in fact expected that the loss of olfactory function would affect the animals more, and were therefore surprised that most moths can still find flowers and plants. However, when foraging, the moths land on the flower instead of drinking nectar. This behavior indicates a so far unknown function of olfaction which we have to further explore,” says Ewald Grosse-Wilde, one of the main authors of the study. “A likely explanation is that moths usually test the smell of a flower while hovering. Moths without olfactory co-receptor may not be able to find the smell that tells them that the nectar in the flower is a good food source.” (see press release “Scent guides hawk moths to the best-fitting flowers”, May 19, 2016).

Experiments with mated females in order to test ovipositing on Datura plants, however, showed that differences between wild-type moths and moths without Orco were not as significant as were differences observed in foraging. After all, more than half of the gravid moths exhibited a characteristic olfactory directed flight towards the plant and laid their eggs.

Insect nose versus ancient nose

Insect olfaction is based on different receptor types. Apart from olfactory receptors, there are also the evolutionary much older ionotropic receptors, which are also involved in detecting many odors. The ionotropic receptors could be considered the “ancient nose” of all arthropods. The highly sensitive olfactory receptors have evolved much later in the course of evolution, possibly when insects learned to fly (see press release “The first insects were not yet able to smell well”, March 27, 2014). The olfactory receptors likely evolved from one single Orco gene.  The olfactory system based on these receptors could be described as the “insect nose”. The two “noses”, which developed independently from each other, could explain why moths without Orco are not completely anosmic.

“The hawkmoth’ capacity to flexibly adapt its behavior towards plants possibly indicates that the ancient nose has evolved independently to detect chemosensory plant cues and that these are sufficient for localizing a host. The insect nose, on the other hand, detects olfactory cues that provide more qualitative information about its host. Therefore, floral foraging and pollination behaviors rely heavily on the insect nose. Our study shows that olfaction is integrated differently with other sensory cues in foraging and oviposition behaviors”, says Richard Fandino.  

The CRISPR-Cas9 revolution in ecological research

CRISPR-Cas9 opens up new opportunities in ecological research. ”To be able to genetically modify animals like the tobacco sphingid moth has long been a dream for us. In this long-term teamwork we managed to use the CRIPSR-Cas9 technique in order to render a gene critical to olfaction non-functional, both as a proof of concept and to show the importance of olfaction for behavior,” co-author Bill Hansson, who is the director of the Department of Evolutionary Neuroethology, emphasizes.

The goal of this research is to better understand insect olfaction and its role in the interaction with the processing of further sensory signals, such as visual cues of flowers and leaves of a plant, but also humidity or CO2.The research approach could help to find out which plant cues provide crucial information to the moths regarding the decision of where to lay eggs. Such insights are important in order to ecologically reconcile the protection of beneficial insects, such as pollinators, with the control of plant herbivores [AO/KG].

The video shows a foraging hawkmoth with a functional Orco (first part) and a hawkmoth without a functional Orco exhibiting a disrupted foraging behavior (second part): The latter is landing abruptly on the flower without extending its proboscis and trying to drink nectar. Video: Richard Fandino, Max Planck Institute for Chemical Ecology.


Original Publication:
Fandino, R. A., Haverkamp, A., Bisch-Knaden, S., Zhang, J., Bucks, S., Nguyen Thi, T. A., Werckenthin, A., Rybak, J., Stengl, M., Knaden, M., Hansson, B. S., Grosse-Wilde, E. (2019). Mutagenesis of odorant co-receptor Orco fully disrupts foraging but not oviposition behaviors in the hawkmoth Manduca sexta. Proceedings of the National Academy of Sciences of the United States of America, DOI: 10.1073/pnas.1902089116

Further Information:

Dr. Richard A. Fandino, Max Planck Institute for Chemical Ecology, Hans-Knöll-Straße 8, 07745 Jena, Germany, phone +49 (0)3641 57 1703, e-mail rfandino [at] ice.mpg.de  

Dr. Ewald Grosse-Wilde, Faculty of Forestry and Wood Sciences, Czech University of Life Sciences Prague, Czech Republic,  e-mail ewald.grosse.wilde [at] gmail.com

Download of high resolution images via http://www.ice.mpg.de/ext/downloads2019.html