Gene Referees Sex Differences in Fruit Fly Fighting

Study Pins Control of Aggression on Gene Known to Regulate Courtship

In love and war, female and male fruit flies play by different rules, all governed by a single gene that works differently in the brains of each sex, report researchers at HMS and the Institute of Molecular Pathology in Vienna.

Photo by Graham Ramsay

Fighting like a female or like a male is hardwired into fruit fly neurons, and defeated male brawlers tend to “remember” their loser status, adopting losing strategies in subsequent bouts. The findings appear in a pair of papers by (clockwise from left) Oulu (Lulu) Wang, Steven Nilsen, Alo Basu, and Edward Kravitz, with others including researchers in the Vienna lab of Barry Dickson.

Fighting like a girl or fighting like a boy is hardwired into fruit fly neurons, according to the study, which appears in the December Nature Neuroscience. The results confirm the gene is a key factor underlying sexual differences in innate behavior, including aggression. And the findings mark a milestone for an unlikely new animal model to study the biology of aggression and the way nervous systems give rise to behavioral differences.

“Aggression is a very serious problem in society, and it’s a problem that ultimately must have biological and genetic roots,” said co-author Edward Kravitz, the George Packer Berry professor of neurobiology at HMS, who developed the fruit fly fighting model used in the study and whose lab hosted the bouts. “We want to understand how complex behaviors like aggression get wired into nervous systems. I can’t think of a better way to address that question than with fruit flies. And no one gets hurt.”

The gene, dubbed fruitless, is known for its role in male courtship. Large and complex, the gene makes a set of male-specific proteins found exclusively in the nervous system of fruit flies, in about two percent of neurons. The proteins, made by sex-specific splicing of transcripts beginning at the first of four promoters within the gene, are necessary for normal male courting, from the initial recognition of a mate to transfer of sperm. Males missing the proteins do not court females, and they sometimes court males. In females, fruitless makes truncated RNA, whose function is unknown. Females normally do not court, but with a male-specific splicing of the gene transcript, they perform the male courting ritual with other females.

Now, the researchers have shown, the same gene directs another sex-specific behavior—aggression—and they have found sex-specific fighting patterns. Head butts and shoving, for example, largely define female fighting (in contrast to the male soccer player who head-butted another during this year’s World Cup). Males prefer lunges; they rear up on their back legs and snap their forelegs down hard, sometimes nailing an opponent that is slow to retreat.

Photos courtesy of Nature and Eleftheria Vrontou

Battle of the sexes. Fighting female fruit flies tend to head-butt (left) and shove (center) their female opponents, while scuffling males mostly rear up on hind legs and snap down hard in a move researchers call a lunge (right). Sex-specific versions of the fruitless gene appear to control the sex-specific aggression patterns.

The flies undergo a major role reversal when the male and female gene versions are switched. With feminine fruitless splicing, male flies adopt more ladylike tactics, mostly the head butt and some shoving. With the masculine form of the fruitless gene, females instinctively lunge to the exclusion of their usual maneuvers.

Unraveling Sex Roles
The gender-bending fruit flies were first developed to study courtship in the Austrian lab of co-author Barry Dickson, director of the Institute of Molecular Pathology. Dickson created male flies with the female version of the gene and female flies with the male version.

In Dickson’s courtship studies, male fruit flies with the female fruitless gene were not acting like males, but it was not clear that they were acting like females, either. (Ultimately, courtship behavior is constrained by pheromones and anatomy, which do not change.) He contacted Kravitz, hoping that aggression studies would resolve the lingering question of male behavior changes.

Meanwhile, co-author Steven Nilsen, a postdoctoral fellow in Kravitz’s lab, had similar questions and was staging contests between another line of mutant-fruitless flies without such clear brain-switching genetics. So postdoctoral fellow Eleftheria Vrontou, the lead author, packed up the flies from the Dickson lab and took them to the Boston fruit fly fight club.

For the past five years, researchers in Kravitz’s lab have been methodically scoring fruit fly fights to determine the normal aggression patterns with the long-term goal of documenting how genes and molecules change those patterns. They stage male fights on food-filled bottle-cap–sized cups decorated with a headless female (a live female would fly away, leaving males nothing to fight over). Female flies fight over an extra dab of fresh yeast paste—their version of dark chocolate, Kravitz said. The flies are videotaped. The movies are replayed in slow motion to record each move and countermove.

“Ed has systematically developed reproducibly aggressive behavior in flies and paved the way for serious analysis,” said Laurie Tompkins, program director at the National Institute of General Medical Sciences, which funds the work. The fruit fly aggression model is part of a new trend to use fruit flies as models to study complex behaviors, including sleep and responses to painful stimuli, Tompkins said. “Drosophila have marvelous advantages in terms of genetic tricks,” she said, “and flies in many respects behave and respond similarly to humans.”

Foundations of Behavior
Fruit fly fights vary a lot among individuals, but hundreds of encounters have revealed patterns unique to females and to males. In one striking difference between the two sexes, male fly fights establish a pecking order. Males appear to remember when they have won or lost and who beat them up, according to a study in the Nov. 14 Proceedings of the National Academy of Sciences led by Alexandra Yurkovic, a former undergraduate in Kravitz’s lab. After the first fight, a winner wins or draws in most subsequent fights against the same and other flies, and a loser loses most remaining fights.

Quantitative analysis shows that a male fly who lunges first is more likely to win, but it takes about a dozen encounters to reach the turning point of a fight. That’s when researchers see the sequence of five successful lunges that defines a winner, and the five retreats in a row that mark a loser. The winner stays with the food cup and the headless female. The loser takes longer and longer to return to challenge the victor, but will persist in fights that the researchers cut off at five hours. Unlike animals with fangs, sharp teeth, and claws, losing fruit flies appear to be in no danger of mortal wounds. Combined with the appeal of the food and female, a losing fruit fly takes little risk in persevering, the researchers presume.

“Losers can become winners,” Kravitz said. “To do that, the loser has to lunge. We know they’re capable of lunging, because they do it a small percentage of the time when paired with naive flies, but they don’t lunge against a familiar opponent.”

In contrast, females trade victories back and forth during a fight, allowing both to feast on the yeast in turns, according to previous work led by Nilsen and Bun Chan, postdocs in the Kravitz lab.

The male version of fruitless, whether in a male or female, also prescribes this hierarchical male relationship pattern, and the female version dictates the lack of one, according to the new paper from the Dickson and Kravitz labs. Just as the female version of fruitless is necessary for female-style aggression, the researchers found, the male version is necessary and sufficient for male-style.

The findings provide a welcome guidepost to help enable future research to track down the underlying neural circuitry, said Bruce Baker, a biology professor at Stanford, who first linked the fruitless gene to male-specific courtship behavior. “That’s a pretty big thing,” he said. “We can think about understanding in molecular detail how we go from the initial genes and the proteins they encode to the nervous system that causes our body to respond in certain ways.” More generally, he said, such studies form a potential bridge between systems neuroscience studies of behavior and modern molecular neuroscience research into individual neurons and synapses.

—Carol Cruzan Morton

top