Drosophila has been a workhorse of genetics for roughly a century. But as a solitary insect, it hasn’t allowed researchers to investigate the genetic basis of complex social behavior in ants and bees.
Now, a pair of studies published today in Cell, here and here, and previously posted here and here on BioRxiv, use CRISPR to manipulate odor receptors in ants for the first time. The findings reveal that without pheromones to mediate their social behavior, the ants behave like solitary insects. The researchers also discovered, to their surprise, that the brain region that processes olfactory information is atrophied in ants that are missing functional odorant receptors. This suggests that ant brains need sensory input to develop properly, something long thought to be a feature of the more complex mammalian brain. The Cell studies illustrate how CRISPR “expands the toolkits” for researchers eager to study phenomena in non-traditional genetic models, says Yehuda Ben-Shahar, who studies the evolution of behavior in flies and bees at Washington University in St. Louis.
Ants, which have evolved to communicate via biochemical cues, have almost 400 chemosensory genes called odorant receptors compared with only about 50 in fruit flies. All 400 of these genes require the same co-receptor, called orco, to form a functional odorant receptor. So Daniel Kronauer’s group at Rockefeller University and Claude Desplan’s group at New York University decided to target orco to eliminate the function of the entire gene family.
It took about two years for Kronauer and his graduate student Buck Trible to develop a protocol for CRISPR gene editing in Ooceraea biroi, the clonal raider ant. It entails collecting and incubating eggs, injecting the CRISPR reagents, hatching and raising the larvae, and then integrating the larvae back into a colony to be fed by worker ants.
Desplan’s group conducted a similar experiment in Harpegnathos saltator, the Indian jumping ant. In these ants, when the queen of the colony dies, some workers can become pseudoqueens to replace her and lay eggs. The researchers introduced a mutation by injecting CRISPR into eggs, and then raised some of the workers to be pseudoqueens to make the desired crosses.
Each ant species has its advantages. In the clonal raider ants, once a mutation is introduced, it is easy to propagate clonally. The Indian jumping ants can be easily crossed to combine mutations, a technique that is frequently used in Drosophila genetics, explains Clement Kent, a social insect biologist at University of York in Canada.
When the researchers in both groups disrupted the orco gene, they found very similar effects. As expected, the ants had olfactory deficiencies—for example, they were no longer repelled by the scent of a Sharpie pen (a simple assay Kronauer’s lab designed). And when, in the case of Desplan’s work, the mutant ants were presented with odorants known as alarm, attractant, or repellent pheromones, they failed to retract their antennae in the way that the normal, wild type ants did.
But they were also oblivious to other ants’ pheromone trails, which function as social cues, and failed to exhibit normal mating behavior. Instead of clustering together like normal ants, the mutant ants just walked around by themselves. “You never see an ant that doesn’t join the colony,” says Kronauer. “That’s where we got very excited.”
To the researchers’ surprise, in both species, the brain regions that process olfactory information were underdeveloped in the orco mutants compared to normal ants, suggesting that olfactory input is required for normal brain development. Further experiments in both labs showed that the knockout ants not only have atrophied antennal lobes in the brain, they also lose the peripheral sensory neurons in the antennae. It isn’t yet clear if the sensory neurons don’t form in the first place, or if they form and then die off at some later point during development.
“This is completely different from anything that has been reported in insects,” Kronauer says. The assumption had been that the insect brain is pretty hardwired, developing independently of sensory input. That is not true of vertebrate brains, in which early life sensory deprivation may lead to atrophy of the corresponding processing areas in the brain, a process thought to allow for more context-specific learning.
Ben-Shahar says the studies demonstrate the feasibility of applying CRISPR to non-model organisms, and hence elucidating how genes affect behavior in ants at the molecular level. “The strength of these papers is that they are a proof of principle that this is now feasible,” he says.
Desplan next plans to use CRISPR to knock out the olfactory receptor in Nasonia, a small, solitary parasitic wasp that is closely related to ants. The researchers hope to decipher orco’s role in this closely related asocial species, which also has approximately 400 olfactory receptors. Comparing brain development in the two species could elucidate how chemosensory input broadly regulates brain development, Desplan says.
“Now it’s becoming very clear that there are things that go beyond social behavior that we can study in ants,” Kronauer says. “There are fundamental questions in neurobiology that we can ask that you couldn’t answer in flies.”