Like the males of many animal species, male Drosophila melanogaster fruit flies, a commonly studied lab animal, are aggressive toward one another and even fight when competing for resources such as food and females. Researchers in the lab of David Anderson have been studying aggression in these insects for decades, elucidating the neural basis for heightened aggression in males, among other discoveries.
More recently, Anderson's lab turned its attention to other species of fruit flies to see if these behaviors persist across the genus. Generally, they do-except in the African fruit fly D. santomea. These insects have evolved another tactic: courtship.
"All the conditions we've used to study aggression in other fruit fly species were the same, so we were expecting fighting, but these males behaved sexually toward one another," says Youcef (Joe) Ouadah, a senior postdoctoral scholar research associate in biology and biological engineering in the Anderson lab. "They courted one another all the livelong day, even though they were set up in the lab by us to fight."
In a new study, Ouadah, Anderson, and their colleagues examined the underlying biological mechanisms for such male same-sex sexual behavior (SSB) in D. santomea and traced it to evolutionary changes in pheromonal signaling pathways in both males and females. Although male-male courtship behavior in other insects is often attributed to an inability of males to distinguish other males from females, that does not appear to be the case in this instance.
"It's important to note that despite observing strong male-male courtship behaviors, the D. santomea males can distinguish males from females and reproduce at typical rates," says Ouadah, lead author of the paper. "Things still work out for mating, but the males don't have to fight each other."
The findings are described in a paper published March 20 in the journal Current Biology.
Testing for Differences in Aggression
Ouadah first saw D. santomea's unique tactics when screening several different fruit fly species for aggressive behaviors. Male flies were isolated for a week to prime them for social interactions, and then two random flies of the same species were put together in a chamber with a floor made of food substrate: Tropicana apple juice. The food, like exposure to a female fly, is a stimulating resource that typically causes male fruit flies to fight.
"Our idea was that once we found the biggest, most interesting evolutionary difference in fighting in a different species, we would try to figure out what changed from D. melanogaster," Ouadah explains.
Video footage and audio recordings of the newly interacting male flies quickly revealed a pattern that looked like the opposite of fighting. One of the two flies would raise a wing to a 90-degree angle and then vibrate it to make a species-specific song-two classic fruit fly courtship behaviors usually performed by males toward females of the species. But why?
Previous studies showed that certain pheromones-chemicals that are secreted or excreted to trigger social responses-play strong roles in controlling fruit fly aggression and courtship. To explore this possibility in the wooing male flies, Ouadah reached out to Caltech's Joseph Parker , professor of biology and biological engineering, who studies how organisms interact across species boundaries, with a focus on rove beetles and ants.
"Joe's lab does pheromone profiling through a technique called gas chromatography-mass spectrometry, and they were willing to load fly samples onto their machine in addition to the many beetle samples they already had going," Ouadah says.
With the help of former graduate student Tom Naragon (PhD '25) and current graduate student Hayley Smihula in the Parker lab-who, along with Parker, are also coauthors of the paper-Ouadah found three pheromonal differences between D. melanogaster and D. santomea that seem to be driven by the need for female D. santomea flies to choose suitable mates.
An Evolutionary Explanation
"These changes appear to have evolved from selection pressure on females to maintain reproductive isolation of their species from a closely related and geographically overlapping species; the two species can mate but produce sterile offspring," says Anderson, who is Caltech's Seymour Benzer Professor of Biology, director and leadership chair of the Tianqiao and Chrissy Chen Institute for Neuroscience at Caltech , a Howard Hughes Medical Institute Investigator, and the corresponding author of the paper.
D. santomea are endemic to an island off the coast of Africa, São Tomé, and are still in the evolutionary process of speciation, or establishing a foothold as a species distinct from its ancestors. Because speciation is not fully complete, the insects can still somewhat successfully mate with other genetically similar species-another closely related fruit fly, D. yakuba, that also lives on the island-although the resulting male hybrid offspring are completely sterile, like a mule, the offspring of a horse and a donkey.
Females of the species have come up with their own tactics to guard against this. "We think D. santomea females evolved a sexual aversion to a pheromone produced at high levels by males of the 'wrong' species, D. yakuba, which in turn forced males of the 'right' species to respond by reducing production of that same pheromone," Ouadah explains. "This pheromone also ordinarily prevents courtship among males, so without it, high levels of male SSB appear."
The effects of the pheromone difference are so strong that other types of male fruit flies also appear to be attracted to D. santomea.
"If you put two D. melanogaster males together, they'll fight, and if you put two male D. simulans (another fruit fly species found across the globe) together, they'll fight. But if I put my D. santomea males in front of those other males, they all start courting my guys," Ouadah says. "So, this pheromonal mechanism reaches across species boundaries."
Although the direct reproductive benefits that SSB confers on D. santomea aren't entirely clear, the researchers note that there are possible positive outcomes. For example, in repetitions of the original experiment, Ouadah saw that only one fly out of each pair typically displayed courtship behavior and established dominance over the other-although not through fighting.
"It's in the interests of both members of an interacting pair of males, evolutionarily, to avoid fighting and instead make a dominance relationship transparent and known to both flies through noninjurious means," Ouadah notes.
Application to Other Species
The team has also identified similar social and pheromonal evolution with spontaneous male-male courtship in a second, distantly related fruit fly species, D. persimilis. In fact, the need for species to maintain reproductive isolation from still-speciating neighbor populations is quite common in the animal kingdom.
"Our closest living relatives, chimpanzees and bonobos, show similar patterns of historical cross-species mating," Ouadah points out. "It is tempting to imagine that in many other similar cases, reproductive isolation was the driving force behind the emergence of high SSB levels that persist into the present, as seen in the case of bonobos."
However, the researchers note, the "emergence" of SSB and its "perdurance"-the continued presence of a behavior-are not the same thing.
"We believe our results provide a compelling model for the evolutionary emergence of SSB in many animals, but whether the trait is maintained over evolutionary time after it emerges in any given species will depend on myriad other factors, notably including whether or not it provides a benefit to the individual males that express it," Anderson says.
Ouadah also stresses that the team's findings are solely about the behavior they can rigorously measure and quantify, and do not reflect anything about sexual identity or even sexual intent.
"Even though what we can view as courtship looks identical regardless of the sex of the target, that doesn't guarantee that the male-directed form has the same intent as the female-directed form," Ouadah says. For example, the intent could be to intimidate another male rather than to try to mate with him.
Ouadah is now exploring the genetic underpinnings of the pheromonal changes in D. santomea and, in a return to his original studies of aggression, plans to search for aggression neurons in the brains of D. santomea to see if fighting circuits have changed in the species or if other mechanisms are at play.
The Current Biology paper is titled "Evolutionary basis of male same-sex sexual behavior by multiple pheromone switches in Drosophila." Additional coauthors are Emily L. Behrman of Dartmouth College, Mohammed A. Khallaf of the Max Delbrück Center for Molecular Medicine, Yun Ding of the University of Pennsylvania, and David L. Stern of the Stowers Institute for Medical Research and Howard Hughes Medical Institute. Funding was provided by the National Institutes of Health, the Howard Hughes Medical Institute, the Jane Coffin Childs Memorial Fund for Medical Research, and the Caltech Center for Evolutionary Science.
