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Fruit flies lose their virginity lightly – and then become choosy

29 June 2021

Mate choice is important for females, who often invest much more energy in offspring than males. However, being too selective is a bad idea, as they might end up not mating at all. Biologists have wondered for a long time how females optimize their chances. Scientists at the University of Groningen have performed experiments with fruit flies that reveal the explanation: mating induces a behavioural change in female flies that makes them more choosy than when they are virgins. The results were published on 21 June in Nature Ecology and Evolution.

The fourteenth-century French philosopher Jean Buridan described a donkey who starved to death because he couldn’t choose between two bales of hay. Evolutionary biologists have their own version of this decision-making problem: in many species, females invest more energy in reproduction than males. Thus, males try to mate with as many females as possible, while females are choosy as they have to select the best males to father their offspring. But, like with the philosopher’s donkey, being too choosy may result in no offspring at all.

On the right Jean-Christophe Billetter, left first author Philip Kohlmeier | Photo University of Groningen
On the right Jean-Christophe Billetter, left first author Philip Kohlmeier | Photo University of Groningen


In 2005, theoretical biologists suggested an explanation: what if female species’ mate preference changes over time? This would be especially beneficial for organisms like flies, who store sperm for later use and mate multiple times. ‘The sperm from the last mating is favoured for fertilization, so a first mating with an inferior male is of little consequence but it does ensure that the flies can reproduce’, explains Jean-Christophe Billeter, associate professor of Neurogenetics of Social and Sexual Behaviour at the University of Groningen.

The explanation sounds plausible, but is it actually true? It turned out to be difficult to prove this in a controlled experiment. Billeter: ‘It is very difficult to distinguish between learned behaviour and a programmed change in mate preference.’ However, Billeter and his group have ample experience in studying mating behaviour in fruit flies. And he had noted changes in the way that the flies process cues after a first mating. ‘For example, food acts as an aphrodisiac – but only in females who have mated before, not in virgins.’ This means that virgin and mated females pay attention to different cues. Other scientists also described differences between virgin and mated flies.


As there are lots of lines of fruit flies with specific mutations, this model organism would allow a careful study of how mating preference is governed. Billeter and his colleagues performed a series of experiments to study mate choice. The basic set up was to observe one female with two males, each from a different strain. These were NL (a Dutch strain) and Tai (originating from West Africa). As fruit flies mate for about twenty minutes, all it takes is to wait to see which of the two males will mount the female. ‘We observed thousands of petri dishes, often overnight, each with two male flies and one female, to get our results.’

Fruit flies | Photo Billeter lab
Fruit flies | Photo Billeter lab


In a first experiment, they found a very clear result: virgin flies showed no preference for NL or Tai males. But mated females became choosy; they subsequently mated with Tai males in nearly 100 percent of the experiments. ‘It was a bit of luck to find such a huge difference between the strains’, says Billeter. Another series of experiments was then performed to find out how this preference came to be. A male pheromone in the sperm fluid was already known to be responsible for mating-induced changes in female behaviour. And when virgin flies mated with male mutants without pheromones, they did not become choosy. This indicated that the females were not learning to become more choosy, but, in fact, underwent a programmed physiological change leading them to become more selective.

As the pheromone induces the production of a substance called ‘juvenile hormone’ in the females, the next step was to confirm that this hormone plays a role in developing selectiveness. And indeed, virgin females that were fed this hormone developed a preference for Tai males. But how are Tai and NL males distinguished? ‘We figured pheromones were involved, so we then tested females without a sense of smell. And indeed, they did not develop a preference for Tai over NL males.’

Mating fruit flies | Photo Billeter lab
Mating fruit flies | Photo Billeter lab


At this stage of the study, Billeter visited a conference where Chih-Ying Su from the University of California in San Diego gave a talk on pheromone receptors in fruit flies. They started a collaboration and, using electrical recordings, the Su lab showed that the Or47b Odorant receptor neurons become less sensitive to pheromones after mating, as a result of juvenile hormone signalling. ‘So, we figured that the juvenile hormone reduces the sensitivity of the flies’ olfactory cells to this pheromone.’ The final piece of the puzzle was finding the pheromone that activates Or47b. ‘This was identified, and we predicted that Tai flies would have higher levels of this aphrodisiac pheromone than NL flies.’ The hypothesis was confirmed: levels in Tai males were twice as high as in NL flies. And dousing NL flies with the pheromone made them as equally attractive as the Tai males.

This leads to a clear picture: to virgins, all males smell equally lovely. But mating reduces their sensitivity for an aphrodisiac pheromone, and thus induces preference for males with higher levels of this perfume. ‘We could only discover this sequence of events because fruit flies are such a powerful model system, thanks to the availability of off-the-shelf tools to manipulate neurons and behaviour’, acknowledges Billeter. ‘But, now that we have confirmed, in this model, that mating actually flips a switch that induces mating preferences, we expect that researchers will be able to find this in other species as well.’


And there are wider implications. ‘We have now discovered a trigger that can change the behaviour of an individual by changing how it responds to a cue. The first author of the paper, postdoc Philip Kohlmeier, is an expert in social insects, such as ants and bees. He is now eager to find out if similar triggers are involved in how social insects specialize in different classes, such as foragers and nurses.’

Reference: Philip Kohlmeier, Ye Zhang, Jenke A. Gorter, Chih-Ying Su and Jean-Christophe Billeter: Mating increases Drosophila melanogaster females’ choosiness by reducing olfactory sensitivity to a male pheromone. Nature Ecology & Evolution, first online 21 June 2021.

Last modified:01 July 2021 09.50 a.m.
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