Fertilization competition discovered in mating fruit flies

Researchers at the University have discovered two mechanisms male fruit flies use to ensure that more of their genes get passed on to the next generation: displacement and incapacitation of a previous male’s sperm.

In most insect species, the second male to copulate with a female sires most of her offspring. Scientists have long puzzled over this strange phenomenon, also seen in birds and some arachnids.

Some species have evolved infinitely more elaborate means of ousting sperm from the female than the fruit fly. In rove beetles, the male deposits a sperm packet that expands inside the female like a balloon, literally pushing the first-male sperm out. The female then uses a specialized tooth to pop the packet and release the new sperm. In crickets, any remaining first-male sperm gets eaten before the female is inseminated again.

“Second-male precedence is so common that we’re really interested to learn that there are multiple complex mechanisms behind it, even within a single species,” said Catherine Price (Ph.D. ‘99), first author of a paper published July 29 in the journal Nature. “By studying the balance between cooperation and antagonism between males and females, we may gain a greater understanding of what happens in a fertilized female.”

Female fruit flies mate with multiple males, storing the sperm in three specialized storage organs (the long, tubular seminal receptacle and two mushroom-shaped spermatheace) and using it as needed to fertilize eggs. However, the odds of becoming a father are not equal for all the males: the last fruit fly to mate with the female tends to sire the most offspring.

“Not only are male flies in competition with each other to mate with a female, but their sperm are in competition to fertilize the eggs once inside the female,” said Price.

“This leads us to believe that the males have evolved mechanisms for encouraging females to use their sperm, and females may have evolved means of mediating competition between sperm from different males.”

Price and Jerry Coyne, Professor in Ecology & Evolution and co-author of the paper, examined the mechanisms male fruit flies use to ensure paternity, focusing on the apparent displacement and incapacitation of stored sperm by the ejaculate of later-mating males.

The researchers obtained male fruit flies with sperm that had been labeled with green fluorescent protein, enabling them to distinguish between first- and second-male sperm in a female’s reproductive tract. When the researchers mated females with males that had GFP-labeled sperm and then with males that did not have the label, they counted far less fluorescent sperm in the seminal receptacle.

“The first males’ sperm seems to have been physically displaced here, but where it goes remains somewhat of a mystery,” said Coyne. Displacement occurred shortly after mating, but only if the second male had viable sperm. Seminal fluid alone could not cause displacement of stored sperm.

Coyne and Price also noticed that the number of stored first-male sperm used to fertilize eggs dropped considerably after the female was remated. This “sperm incapacitation” effect became much stronger as more time passed between the first and second copulation.

“As the sperm sit in the females’ storage organs, they must undergo some change that makes them more susceptible to damage by something in the second males’ seminal fluid,” Coyne said.

“Previous research has shown that a fly can incapacitate and displace his own sperm if he mates with the same female twice, so we know that second-male sperm precedence does not rest on a genetic difference between the sperm of the first and second male,” said Coyne.

The evolutionary underpinnings of second-male sperm precedence are still unclear, especially since the reproductive interests of the male and female are different. “It may be in the female’s best interest to get rid of old sperm because it could get damaged if it’s stored for too long,” said Price. But for the male, the truth is in the numbers: it is to his evolutionary advantage to fertilize as many eggs as possible and to prevent the fertilization of as many eggs as possible by his competitors. “The mystery is why the second male nearly always defeats the first male,” Price said.