A female mosquito only gets one shot to get reproduction right: She mates just a single time in her entire life. With the stakes so high, it would make sense for these insects to be quite choosey when it comes to selecting a mate. And yet a long-standing assumption in the field was that males controlled the process, and females were simply passive recipients of sperm.
"There's an inherent contradiction in this assumption," says Rockefeller University and Howard Hughes Medical Institute mosquito expert Leslie Vosshall . "If females have no say, then multiple males should be able to mate with them all the time. So how can a female mosquito both be a helpless creature but also the decision maker?"
Puzzled by the paradox, Vosshall and her team in the Laboratory of Neurogenetics and Behavior dove into the moment-by-moment, nuts-and-bolts of mosquito mating. The resulting study, recently published in Current Biology, uncovered the first evidence that scientists had it backwards: What makes mating possible is a subtle behavior of the female—a physical movement of her genitalia. Moreover, no subsequent physical pairings trigger this behavior again, regardless of how many males try, or how often they try—and they try a lot.
"It's a very fast, very subtle change, but it entirely dictates whether mating occurs," says lead author Leah Houri-Zeevi, a postdoctoral scientist in the lab. "If she makes this movement, it happens. If she doesn't, it doesn't matter what the male does—no successful mating will occur."
Obscure mechanics
Depending on whether she's living a short and dangerous life in the wild or a long and cushy one in the lab, a single female mosquito can produce up to 1,000 eggs in a single lifetime.
Following her lone mating, she stores the male's sperm in internal reservoirs. Every 3–4 days, she feeds on the blood of a host, and once sated, draws from these sperm reservoirs to inseminate and lay her eggs in fresh water.
Despite studies on mosquito mating going back to the 1950s, the role of the female in the process remained obscure. The speed of the process—the interactions that lead to mating take 1–2 seconds—makes it challenging to capture, and might have been combined with hidden biases for what the female role in mating could be.
"There's a long history in biology of assuming male agency and female passivity," says Vosshall. "This study is a reminder that those assumptions can get in the way of seeing what's actually happening, even in something as well-studied as mosquito mating."
For the current study, the researchers investigated the mating practices of two of the most invasive mosquito species in the world: Aedes aegypti and Aedes albopictus, better known as the yellow fever mosquito and Asian tiger mosquito, respectively. Collectively, they can spread dozens of viruses to humans, including yellow fever, dengue, Zika, and Chikungunya.
They analyzed the step-by-step interactions of different mating pairs both within and between species, including female mosquitoes that had never previously mated and those that had.
A three-step process
Using high-speed, high-resolution cameras, deep learning, and transgenic mosquitoes with fluorescent sperm, Houri-Zeevi and her colleagues discovered that the same three-step process leading to a successful mating between a virgin female and a male occurred in both species. First, the male contacts the female genitalia with his genital tip. In response, the female chooses whether to elongate her tip to about twice its resting length. This behavior is critical for mating. If she doesn't elongate her tip, mating cannot take place. If she does, the male's internal genitalia interlocks with the female's tip, and sperm transfers from one to the other.
The researchers found that the "key" to unlocking the critical female response in Aedes aegypti is rapidly evolving male structures, called gonostyli, that are inserted into the female genital tip and vibrate rapidly when the male attempts copulation.
Houri-Zeevi and the team also observed what occurred when a previously mated female and male attempted to interlock: Step two didn't occur. That apparently prevents step three—successful insemination.
"After one successful mating, she will never elongate that tip again," Houri-Zeevi notes.
Interspecies matings
They found this tip elongation mechanism in both species, demonstrating that female control over mating is shared in mosquitoes that diverged about 35 million years ago. However, they also noted differences between the two species, suggesting that within each species, there is a specific female lock and a specific male key.
That idea is bolstered by the fact that Asian tiger mosquitoes and yellow fever mosquitoes diverged from a common ancestor so long ago that they cannot produce viable offspring, so mating between these species is a genetic dead end for the female. However, that doesn't stop the males from trying to mate with females of the other species. Houri-Zeevi and the team discovered that male Asian tiger mosquitoes—which have far larger gonostyli than their yellow fever mosquito counterparts—used their gonostyli to override the female mating control of yellow fever females, and mate with them without the female genital tip elongation behavior. This "lock picking" could only be done across species, and the Asian tiger mosquitoes could never override the female mating control of their own females.
That finding may help explain a striking pattern that entomologists in southern regions of the U.S. have observed: When Asian tiger mosquitoes move into an area, the population of yellow fever mosquitoes drops or vanishes.
It may also help improve methods of mosquito population control, some of which rely on ill-fated pairings between intentionally sterilized males and wild females. "It's really important for people who work in an area to understand how the biology of females of a local wild population is going to interact with males from a genetically modified population," notes Vosshall.
Going forward, the researchers will explore the finer details of the lock-and-key mating mechanism for each species. "We want to understand the neuronal code the female is using to sense male stimulation and then make her decision," Vosshall says. "The question it comes down to is, how does she choose between different suitors given that it's a once-in-a-lifetime choice?"
