Wolf spider's reaction towards caterpillars fed a nicotine-rich (top) and nicotine-free (bottom diet). Courtesy of Sagar Pandit.
Next, the researchers decided to discover how the hornworms were utilizing the nicotine. In a previous study, they found that a gene that's normally expressed in the caterpillar's midgut, CYP6B46, isn't as active in hornworms that are fed plants with suppressed nicotine production. This seemed a good place to start, so they created N. attenuata plants that are able to silence the caterpillar's CYP6B46 gene. Sure enough, caterpillars with the silenced gene became very attractive to spiders.
The Important Role of the CYP6B46 Gene
Further work showed that the gene is able to take some of the nicotine in the caterpillar's midgut and transport it to the hemolymph (its blood). Nicotine, Baldwin explained, works by attacking the acetylcholine receptor, which "drives the neuromuscular function of all animals on the planet." What's more, there are no acetylcholine receptors that are resistant to nicotine, meaning that caterpillars aren't necessarily able to eat tobacco because their receptors are resistant to
nicotine. "The way the caterpillar handles the nicotine is that it poops it out," he said. The work with the CYP6B46 gene shows that the hornworm doesn't poop out all of the nicotine — it transports 0.65 percent of the nicotine it ingests into its blood.
But how does blood laced with nicotine help the caterpillar? The researchers figured that M. sexta must exhale the nicotine through their spiracles, which are small respiratory holes they breathe through — the nicotine warns the spiders that the caterpillar is toxic. To see if this was true, "we glued little nicotine trapping devices on the spiracles and then took them off after the spider caught the caterpillar," Baldwin said. The devices showed that the tobacco hornworms were puffing out nicotine from their spiracles when the spiders attacked. "Normally, it would work as a type of toxic halitosis, or bad breath."
Interestingly, this defense doesn't work for all predators. The scientists discovered that the big-eyed bug Geocoris pallens, another major predator in the N. attenuata field, wasn't deterred by the caterpillar's nicotine-rich breath. Baldwin thinks this may be the case because the bugs only feed on the hornworms' hemolymph, which has very low levels of nicotine. The spiders, on the other hand, eat the caterpillar's nicotine-loaded gut. Still, the study is the first documented case of a "toxic bad breath" and an animal being able to use its exhalation as a signal that it has eaten something poisonous, he said.
The research also highlights how important field studies are for determining what individual genes do. "We've discovered the function of the gene CYP6B46 in the caterpillar because of a spider in the field," Baldwin said. "This shows that nature is an important laboratory for discovering gene function."
Check out the full study in the journal
PNAS.
Top image via Danny Kessler. Inset images via Pavan Kumar and Danny Kessler, respectively.
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