An experiment in editing the gene suspected to be the primary odorant receptor for the sex pheromone in codling moths (Cydia pomonella) took a surprising turn when mating the gene-edited males and females resulted in no viable eggs produced—a sign of the gene’s possible role in reproduction, as well. (Photo credit: Gyorgy Csoka, Hungary Forest Research Institute, Bugwood.org)

A funny thing happened on the way to testing the CpomOR1 odorant receptor.

Researchers at the USDA-Agricultural Research Service conducted an experiment on codling moth (Cydia pomonella) using the CRISPR/Cas9 gene-editing method to study the role played by a specific gene known as CpomOR1. The gene is highly expressed, or “switched on,” in the antennae of male codling moth, which has led scientists to hypothesize that it is the primary odorant receptor for codlemone, the main codling moth sex pheromone.

To test that hypothesis, a team led by Steve Garczynski, a research geneticist at the USDA-ARS Temperate Tree Fruit and Vegetable Research Unit in Wapato, Washington, edited CpomOR1 in codling moth eggs to produce adult moths with the gene knocked-out. They then sought to test if edited males would show a reduced response to codlemone. The result of testing on the males was inconclusive, so the researchers mated the edited male and female adult moths, hoping to test the response in their progeny. That’s when things took an unexpected turn.

The female moths with the knocked-out CpomOR1 gene produced significantly fewer eggs than unedited females (similar, in fact, to the number of eggs produced by virgin female moths), and none of the eggs produced viable offspring. Evidently, CpomOR1 plays a role in codling moth reproduction, as well.

The results of the study are detailed in a report published Thursday in the Journal of Economic Entomology.

Despite the surprise, the discovery none the less advances entomologists’ understanding of the CpomOR1 gene in codling moth. “Unexpected results are a good thing,” says Garczynski.

In female codling moth, the CpomOR1 gene is also known to be expressed in the tip of the abdomen. Garczynski says the initial hypothesis was that the gene’s presence there was part of “some sort of feedback loop to let females know that they are producing codlemone.” Now, after this study, he and his colleagues posit that CpomOR1 activation in the female abdomen sends a signal back to the egg to prepare it for fertilization.

New research is underway to edit other pheromone receptor genes in the female abdomen tip via CRISPR/Cas9 to study their function, and Garczynski says he’d also like to pursue further studies to clarify CpomOR1’s role in egg production and viability.

In the long run, research on gene editing in codling moth is aimed at finding possible avenues for improved control of the species, which is a global pest of apple and pear orchards, but such goals remain far off, for now. “How that would happen, I have ideas, but none that are practical at this time,” Garczynski says. “A research goal at the lab is to use CRISPR/Cas9 genome editing to identify new protein targets that can be exploited in the future for codling moth control. How this happens will depend on the maturation of CRISPR/Cas9 delivery techniques or development of specific compounds targeting critical proteins.”