Articles, Blog

Eleanor Bath (Oxford): Understanding Female Aggression in Fruit Flies

August 16, 2019

Why do females fight? For 150 years, since Darwin published his theory of sexual selection, scientists have thought that females don’t fight. Because… they shouldn’t. If you think of aggression, you’ll probably think of males being aggressive. That’s ’cause, across the entire animal kingdom, from humans to birds, even to fruit flies, which I work on, males tend to be more aggressive than females, towards each other and even towards inanimate objects, for, seemingly, very trivial reasons. It’s really hard to find videos of females doing the same sorts of questionable behavior. So, why is this? It’s kind of a… a biological theory behind it, actually, which is that the sexes choose really different paths to reproduction. Which is that females have to be choosy, and that’s because females invest a lot in each offspring. So, you go from the gametes stage, where females have to invest in these really large eggs, whereas males invest in tiny sperm. And then it goes on beyond that gamete stage as well. You can think of females having to go through pregnancy, lactation, even beyond like this long-tailed tit having to feel… feed 12 chicks by itself. And, because females have to be choosy, because they invest so much in each offspring, it means that, you know, they don’t want to mate with every male around. They want to mate with the best male. Males, on the other hand, don’t have the same sort of restrictions. For them, the best option to have the most offspring is to mate with as many females as possible. But there’s a bit of a problem here — if females don’t want to mate with every male, but males want to mate with every female, it means that males have to compete for access to those females. But, females, because there should be in theory lots of available males, don’t have to compete for access to those males. So, females shouldn’t fight. But if you actually look at the animal kingdom, you find that lots of females fight. You find it in mammals — so, female hyenas are actually more aggressive than male hyenas. They’ll fight for social dominance inside the pack, and more dominant females have a better chance of raising their offspring. You find it in birds, and females compete for access to nesting sites, so these are hollows in tree trunks, and the better your nesting site the better your chance of raising offspring and the better their chance of producing their own. You even find it in insects. So, here, this burying beetle female has had to compete with other females to get access to food to feed her larvae, down here. So, females do fight. But they don’t fight like males. Females tend to fight for resources for their offspring. So, there’s this really interesting link between reproduction and female aggression that you don’t see in males. So, it’s something that’s really specific to females, and I want to check out more what’s happening, what this specific link is between reproduction and female aggression in fruit flies. And fruit flies are a great system for this because we know females are aggressive. They’ll fight over access to food. We also know that males are aggressive, so they’ll fight over access to females, they’ll fight over… over access to food — apple juice. Males will even fight over access to headless females. We also know that sex and reproduction has a really big influence on females. So, sex completely changes female fruit flies. It turns them into completely different animals. So, before mating, females don’t produce many eggs. After mating, they start pumping out hundreds over their lifetime. And these eggs are actually really costly to produce. You require a lot of protein to build an egg. The problem is that, for females in the wild, they live on rotting fruit. There’s not a lot of protein available, so there are kind of small patches where fermenting yeast produces this protein, which females need to produce these eggs. Mating also changes females’ receptivity to remating. Before they mate, females are generally pretty keen; after mating… after mating females are not receptive to any other males. Sex also changes a whole lot of other aspects of female fly behavior. It changes their sleeping patterns — mated females sleep a lot less than virgins. It also changes things like their immune system, gene expression, their feeding behavior. So, it seems like mating is a really good place to start looking to test the link between reproduction and female aggression in flies. So, how do you make female flies fight? So, the first day, what you can do is… basically, with fruit flies, you can collect a whole bunch of eggs, then ten days later these eggs will emerge into fully formed flies. So, you’ve got a sort of a seven-hour window from when they emerge as flies until they start mating. So, in that seven-hour window, you can be pretty sure they’re virgins. So, I collected a whole bunch of virgin females and I kept them in these individual vials. So, here, they’ve got some regular fly food, down there, which doesn’t have a lot of protein in it. Then, after I’ve let them recover for a day or so, I paint the flies so that I can identify them later. So, I’ll give them a nice little red or… or yellow spot. The next day, I will either mate them or I’ll keep them as virgins and not mate them, so that I can control their mating status. And then, the final day, I’ll fight them. So, on the day of fighting, I’ll start by putting them, in the morning, into what I call a starvation vial. So, essentially, what it is is a vial that just has some damp cotton wool in it. So, they don’t have access to food, but they do have access to water. I leave them in there for two hours, so it’s not too traumatic for them, and then it just makes them a little bit more motivated, a little bit more hungry. And then I put them into a contest arena, which is actually just a small plastic cap that has sort of the regular fly food in it, that they’ve been living on for their lives, and the small red dot, here, which is made up of live yeast and is really high in protein. So, then, I’ll pop the flies in there and then I’ll record them for 30 minutes. So, for those of you who’ve always wanted to know what a fly fight looks like, here we have an actual video. So, this orange stuff is the regular fly food. Here, you can see one of them is painted yellow, one of them is painted red, and they’re eating a little yeast paste in there, which you can’t really see on this video. So, here we have the flies starting to headbutt each other and starting also have a go with their legs as well, which you can see. Red’s getting a bit angry, here. And Yellow is having a little bit of a go at Red as well. Now, there are a couple of different types of female fly fighting behavior which are really specific to females. So, one is this kind of fiddling around with their legs, which we call fencing. The one that’s sort of the highest intensity of female aggression is called headbutting. So, here we’re going to have a look in a bit more detail at flies headbutting. So, we have Yellow chasing Red off and, just to slow it down a bit for those who haven’t spent hours watching fly videos… Yellow coming around, having a go and headbutting Red off the food cap. So, that’s what fly aggression looks like. So, the first question we want to ask was essentially, do mated females fight more than virgin? Does mating increase female aggression in fruit flies? So, for these graphs, basically what’s going to be happening for all of the graphs is… on the y-axis, we’re going to have the total fight duration, so, the total amount of time female spend fighting in an observation period. And in this particular experiment we took two virgin females, down here in blue, and fought them against each other. We took one mated female and one virgin female, and fought them against each other. And then we had two mated females fighting against each other. So, if you look at the two virgins first, you find that they don’t spend a lot of time fighting. They’re not really very aggressive. If you change it and put in one mated female, all of a sudden you get this increase in female aggression. What happens if you have two mated females? You get a further increase on that mated-and-virgin pair. What’s really interesting is that, between the red and the orange, there’s not actually a significant difference. But between those two and the blue, there is a big difference. So, mating makes females a lot more aggressive, even if there’s just one of them in there. So, that’s pretty cool, that you see this direct link between mating and increased aggression. But what is it about mating that makes females more aggressive? So, the first sort of logical question was, is it because of eggs? As soon as females have mated, they start producing a whole lot of eggs. And you can imagine, if you’re eating for 2 or 200, that you have to get a lot more food, that your body tells you to collect more nutrients. But, just because you need more food doesn’t mean that there’s more available in the environment. You know, there could very well be other females who are in exactly the same situation. So, to get that food, you need to get aggressive. So, what we did was we tested females that couldn’t produce any eggs, so these are completely sterile females, and we said, okay, if it’s sort of these costs of egg production that are driving female aggression, these sterile females shouldn’t show that increase in aggression after mating, because they don’t have these costs. So, our prediction was that you’ll see a bigger difference in terms of levels of aggression between mated fertile flies and virgin fertile flies than between mated and virgin sterile females. So, here we have… we have our two sterile treatments down here, whoo… and our two fertile treat… treatments down here, and we had two pairs. So, we had… in this case, we had females fight against someone from their own mating status, so you have two mated females or you have two virgin females fighting against each other. So, if we have a look at the virgins, first, for both the sterile and the fertile females, there wasn’t a lot of fighting going on. And there’s no difference between these two, which we wouldn’t expect. Then, if we look at fertile mated females, we see that same dramatic increase in female aggression that we saw in the last graph. So, mating increases female aggression there. But, what happens with mated sterile females? So, here, you still see an increase in female aggression after mating with sterile females. But what we’re really interested in is, is the difference between these two bars smaller than the difference between these two bars? And the answer is that there is no difference. So, egg production doesn’t actually change how much more aggressive you get after mating. So, it’s not eggs. It’s not the costs of egg production driving this increase in female aggression, which kind of blew us away a little bit because it seemed like such an easy, logical answer. So, we thought, okay, let’s take a few steps back, you know? What happens before egg production? What else happens during mating? The answer is that males transfer a whole lot of stuff to females. So, we said, alright, let’s take a look at that stuff. Let’s take a look at that male ejaculate. And the male ejaculate, at least in fruit flies, it’s a bit of a cocktail. So, you get sperm. You also get the seminal fluid, which contains a whole lot of proteins that can actually induce changes in female behavior. You also have what I like to call the zest of life. So, we decided that the first obvious thing to check was sperm. You know, it’s crucial for fertilization, at least in this species, and it’s needed for a lot of those post mating behavioral responses I talked about earlier, such as increasing egg production, reducing receptivity, immune changes, that sort of thing. And sperm’s really interesting because it doesn’t just act on its own. It actually interacts with these proteins that are involved in the seminal fluid. So, it seems like a good candidate, both sort of by itself but also as sort of a vehicle for other proteins to induce these changes in females. And this picture here is actually the female reproductive tract, these tubes, and the green and red dots are sperm from two different males. So, it kind of shows… I like it because it illustrates that a lot can happen in females after mating, particularly related to the male ejaculate. So, we predicted that sperm would be necessary for increased female aggression after mating. So, in this situation, we have our blue is our virgin, which were, again, not very aggressive, then we have our orange, which was our control males… our control females, who mated to males that gave them everything in terms of an ejaculate, who transferred sperm. So, here, again, there was that same sort of increase in aggression after mating. But then, for our experimental treatment, we had females who were mated to males that didn’t produce sperm. So, they produced the rest of the ejaculate, they just didn’t produce sperm. So, we predicted that, if sperm is involved, you should see a bar that’s pretty similar to virgins, which is exactly what we found. We found that, if you don’t get sperm, you’re essentially fighting like a virgin. So, sperm is really crucial for this increase in female aggression after mating. But, as I discussed before, that might not be the end of the story. Because sperm also interacts with all of these other proteins and has a lot going on, we didn’t want to just stop here and say, okay, it’s just sperm. So we said, let’s have a look at some of those proteins. Because what about the rest of the ejaculate? The rest of that cocktail is still there. So, the one that we really wanted to look at is this nasty little protein called sex peptide. So, this is it here, in this beautifully colored picture, and it’s 36 amino acids long, so it’s pretty tiny, but it’s really powerful. So, all of those responses I’ve told you about before — increasing egg production, reducing receptivity, immune changes, gene expression, even reducing female lifespan — those are all caused by sex peptide. And not only does sex peptide have dramatic effects on females, it also comes into the females bound to sperm. If you don’t have sperm, a lot of the effects of sex peptide are actually really diluted, because… but they work together. So, we thought, well, it seems pretty likely that sex peptide could be involved in increasing female aggression after mating. So, again, we have our virgin females in blue. Again, not very fighty. Then, we have our control females, who were mated to males that gave them the entire ejaculate. Again, showing that same increase in female aggression. But this time, instead of removing sperm, we mated females to males that did not produce sex peptide. They produced the entire ejaculate except for sex peptide. So, here we see that, actually, it’s kind of an intermediate response. So, you see that females that mated but didn’t get sex peptide, they don’t show that full increase in mating that we see when you get sex peptide, but they’re not quite like virgins either. So, this tiny little peptide, this 36-amino-acid-long protein, is at least partially responsible for increasing female aggression after mating. So, sex peptide is also pretty important. So, these results show that males can actually alter female behavior towards other females through their ejaculate, which I find pretty creepy. But, I mean, that raises a whole lot of other questions, like, why would males do this? So, the first option is that females actually use it as a cue. So, females mate, they get a whole lot of sperm and they say, okay, now I’ve got this sperm, I have this sex peptide, that’s a sign that actually I’m gonna really soon be producing a whole lot of eggs. That means that I’m gonna need to get more food, so I need to get aggressive. So, maybe it’s in the females’ benefit. You know, maybe they’re able to detect that all of a sudden they’ve got this huge amount of sperm that they can use to change their behavior. The other slightly more disturbing possibility is that maybe it’s actually a form of male manipulation. Maybe it’s not in the female’s best interest. So, this is a possibility because you could imagine, you know, a female mates to a male, has some eggs with his sperm, they both then go their separate ways, and she goes on to mate with another male. And, sort of, how much she wants to invest, in terms of time, in terms of energy, could be dependent on the male she’s mating with. So, you can imagine if you have, you know, a bit of a dud male, you don’t want to invest a lot of your time and energy in producing his offspring, whereas if you had a really good male it’s actually really beneficial for you to invest energy in fighting for his offspring. But that first male is not gonna see it like that. The first male wants the female to invest as much as possible in his offspring. So, if he can make her more aggressive, it elevates her energy level, but it could have flow-on effects for her. Maybe she has less energy later on in life. Maybe aggression results in injury. Maybe it shortens her lifespan. So, it could be that, actually, males manipulating females into fighting is actually a form of sexual conflict. So, as well as sort of these, you know, broad questions about what the evolutionary purpose is, there are also a whole lot of questions that this kind of research leads to, like, how do sperm and sex peptide do it? How do they transmit these complicated messages to… to result in female behavior? Do females just detect that their sperm or storage organs are full? Are their receptors involved to pass neu… to pass messages up to the brain? What about other seminal fluid proteins? There are another 150 that trans… get transferred in the ejaculate and we’ve seen that sex peptide isn’t fully responsible, so maybe some other proteins are involved. You also have the question, sort of, are more aggressive females actually more successful? If you increase your aggression, maybe you succeed initially, but you can imagine that… that comes with a high cost. If you’re spending your entire time fighting off other females, maybe you have less time to eat, maybe you have less time to mate, less time to lay eggs. So, is there actually a benefit to being aggressive? I’m trying to answer that question about, who is… who is this benefiting? And also some really tricky questions like, does it depend who you mate with? Does this effect change? Do different males induce different levels of aggression in females? And, then, the flow-on effects from that. If you’re a female, you not only have to worry about how many resources are available in the environment, or, you know, your mating status, you also have to think about the mating status of other females in the environment and who they’ve mated to. So, it has some serious evolutionary and ecological consequences. And then, of course, there’s the question about, could this happen in other species? So, in a lot of insects, there are really similar proteins, really similar reproductive systems, that suggest that maybe you could see the same increase in aggression after mating in females. Potentially, you could even see it in a broader range of animals, so from birds, reptiles, mammals… all of these have sperm and seminal fluid proteins, as well as instances of female aggression. So, could there be a link there? These are obviously very different life history strategies, so it’s a big jump, but, could this be a potential mechanism to link reproduction and female aggression across species? And the million-dollar question, of course, is, what about one other species? Could it happen in humans? And, with that, I’d like to thank my co-authors, and my funders, and iBiology. Thank you.


  • Reply Joe Tyndall October 4, 2017 at 3:23 pm

    Human female aggression is COVERT.

  • Reply Tyler_ Lalonde- October 17, 2017 at 4:30 am

    No it’s not hard to find.

  • Reply pramitbanerjee January 24, 2018 at 2:19 pm

    so cool. This is the kind of presentation I want to give someday.

  • Reply Andrew Raphael Lukasik April 27, 2018 at 7:54 am

    I find female aggression fascinating. Precisely that human females, as opposed to fruit flies females, are exposed to one another set of DNA-like instructions – culture . Culture that can, for example, tell them that they're anti-thesis of aggression (illustrated at 22:42 here). As a result, often seems to have rather hard time mentally processing instances of their aggressive capabilities or behaviors.

  • Reply Benjamin Trias August 7, 2019 at 2:00 pm

    I don’t have a degree in biology so please excuse me if my comments seem inadequate. But I don’t really see the point of benefits for females or males. Wouldn’t any either contribute to the species? Aren’t the manipulators not the genes in the end? The part addressing the benefit seems to imply some intentional behavior on the part of the flies. Isn’t the aggression resulting from the contact with the sex peptide and sperm not simply mechanical? Do you answer the question as to whether more aggressive females are more successful, isn’t the fact that this behavior is generalized the outcome of successful reproduction? It seems to me that even this aggression being an incidental result of the contact between sperm, peptide and the female, this aggression will lead to females to secure more food and so to ensure they’re laying more eggs? Isn’t this the obvious conclusion? Or am I missing something?

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