In female flies, sex is more complex than yes or no

When it comes to mating, male fruit flies (Drosophila melanogaster) put on quite the display. Coming across a likely female, a male will follow her around, exude pheromones, play a song on one wing and lick and tap at her genitalia. The female fly will then decide whether to mate. The neural circuits that underlie male mating behavior have been studied for many years. But female fly mating circuits have remained more mysterious, in part because female behavior during mating seemed more passive.

Now, three new papers have begun to fill in the blanks about female fly mating behavior. The new work shows that female flies have a much more complex set of mating behaviors than thought. Scientists have also outlined groups of neurons in the fly brain and nerve cord that control a female’s mating response, revealing the biological intricacies behind what looks relatively simple.

Females have been flying under the radar in many biological studies. In the case of fruit flies, scientists were hampered by behaviors difficult to observe. “Female behavior has taken a backseat to male behavior because male behavior is so obvious,” says Bruce Baker, a geneticist at the Howard Hughes Medical Institute in Ashburn, Va., and an author of one of the new studies. “The female behaviors are difficult to see. These papers make the female a much richer model to do behavioral and neurobiological studies on.”

When faced with a suitor, the female fly must first assess whether the male is a good bet for a mate. But her considerations do not end there. Many other factors affect whether a female fly will be receptive to a male’s entreaties. “She needs to look at her internal state, whether she’s a virgin or not, whether she has mated recently, is she sleepy, hungry and so forth,” Baker says. (Virgins are more likely to mate.)

A female fly integrates all these cues when a male comes a’courting. If receptive, she will slow down and wait, opening her vaginal plates to allow the male to copulate. If she is not receptive, Baker says, a female fly gets violent. She might fly or run away, kick and even thrust her ovipositor (the organ used to lay eggs) in a hapless suitor’s face, “which discourages him substantially.”

Jennifer Bussell, a neurogeneticist at the Rockefeller University in New York and colleagues took a closer look at receptive female fly mating behavior. Using cameras placed under a receptive fly, the researchers showed that the female doesn’t slow down. Instead, her behavior is more subtle: She moves at the same rate, but performs a series of pauses near the male fly. “She stands still near the male,” Bussell says, “so she can listen to the courtship song, take in his pheromones and see if he’s a really good mate.” Writing in the July 2 Current Biology, Bussell and colleagues show that a group of neurons, located at the base of the fly nerve cord and rich in a certain protein (transcription factor Abdominal-B), controls this pausing behavior. When Bussell silenced these neurons, pausing behavior stopped and females no longer mated. “They pause much less, they don’t give the male a chance,” she says.

But pausing is just one part of the female response to a potential mate. Another study led by Kai Feng and Mark Palfreyman at the Research Institute of Molecular Pathology in Vienna and published July 2 in Neuron, looks at how information from the reproductive tract of a female fly makes its way to higher processing centers and influences behavior. Previous studies have shown that during mating, males produce a protein called sex peptide with their sperm. This sex peptide binds to cells inside the female’s reproductive tract and changes her behavior, making her unreceptive to other male advances for seven to 10 days.

Feng, Palfreyman and colleagues were able to show that the cells inside the reproductive tract connect with just two neurons, called sex peptide abdominal ganglion or SAG neurons. These cells, located at the tip of the nerve cord, project toward the brain. If sex peptide is present, it binds to reproductive tract cells and conveys the signal to the SAG neurons, which reduce their activity in response. In the end, the female fly becomes unreceptive to male mating efforts. 

What’s still unclear is exactly how neurons link up in the female fly brain to process external cues like mating songs and internal cues and to produce a yes-or-no mating response. A third paper, from Chuan Zhou, Baker and colleagues at HHMI and published July 2 in Neuron, looks at specific neurons within the brain to try to tackle this issue. The authors show that neurons in two brain regions, called pCd and pC1, respond to male courtship songs and to the scent of male pheromones. Silencing the activity of the neurons in these regions made virgin flies slow to mate. Increasing activity in the same areas resulted in a speedy mating response.

All three papers provide new insights into how female flies make the decision to mate. But each offers a very different piece of the puzzle. The pCd and pC1 neurons in the brain may be important in the decision to mate or run away. The SAG neurons project up toward the brain, and may connect with other brain cells and to pCd and pC1 neurons, sending information about sex peptide to the brain. The Abdominal-B neurons are at the output end, controlling the behavioral result of all the previously processed information.

But the three types of neurons do not yet link up to each other. Future studies may help put the pieces together into a common neural circuit. There are probably many other brain cells involved, processing pheromones, hunger, movement and other information. In addition, some of the neurons these papers describe may not end up truly involved at all. It could be that out of a large cell population, only one or two really control a specific response or information highway. “We have puzzle pieces that go in the female mating puzzle,” Palfreyman explains, “and we also have pieces in here that may really belong to another jigsaw puzzle. We need to find the rest of our pieces, but we also need to figure out which pieces don’t belong.”

The recent progress suggests the female’s time has finally come in fly studies. “When I started my project, I was surprised to learn that despite people studying fly courtship for many years, almost all of it has been done from the point of view of the male,” says Bussell. “Now we have three papers all at once identifying specific neurons! It’s an exciting time.”