 or that's the idea, I appreciate it. So I'm gonna talk now about insects. We are switching over from the mouse side over to these guys that you see on the picture here. I'll go through a number of projects. Some of them form more like a background, so they're already published. Some of you have read about them. Some of you have heard me talking about them, but they sort of form the background to what I'm coming to towards the end. First half will be about Rosophila. Second half will be about Manduka. Okay, so today we basically think that there is about six million species of insects. We also think that there is about 20, 25,000 species of vertebrates on earth. So already this number gives you the sort of why they are so fascinating. They have invaded everywhere. They are the most successful organism on earth. We should learn from these guys how to optimize. I just saw a TV program the other day where a German architect studied the wings of the Colorado beetle to build light and very sturdy constructions. And this is now standing outside Albert and Victoria Museum in London, a huge new roof all built on the structure of the Colorado beetle. And in different ways we can study the diversity and the functionality of insects, I think, to learn and to do things in different ways. I chose to do this about olfaction and about how insects are run by different kinds of olfactory inputs. This is the head of Rosophila. Some of you are not insect people. Some of you might not even be olfactionists. So I just do some basic, this is the main nose of Rosophila and down here is the second nose. This is the antenna. This is the pout. This is the proboscis where they more or less taste mainly. I will mainly talk about the antenna today. We look closer on the antenna. The antenna has about 1200 sencilla. Sencilla are in insects, I would say 1200 little noses because each one has its own confined environment. You know, as Stuart showed before, in your nose all neurons lie and float around in the same snot, right? So your nose, there is no separation, but in an insect about two, three, four neurons are separated into each one of these little noses and can have its own chemical environment when it comes to the snot which is called the sencilla lymph in insects. So you can have different odor and binding proteins for instance in all these different sencilla, which there is. So if we look at what we can do there, so we can record from these single hairs, we can record from each neuron present in those hairs which is a huge advantage of insects. One thing that we have specialized in is also is in combining biology and chemistry as Stuart talked about before. So we let the insects tell us what is interesting in an odor. If you collect the molecules of a banana, you might get 100, 200, 300 molecules depending on how good you are in catching the molecules. But of course, not all those molecules are interesting to the insect. And it's a huge natural chemistry, natural product chemistry effort to identify each one of those. So what we do is that we hook it up and we take half of this effluent to the mass spec and we take half, let it flow over this sencilla and tell us what is interesting to this specific neuron on the fly antenna. And we can do this in a high throughput way and test thousands of compounds on all the neurons on the insect antenna these days. High throughput GC-MS, electrophysiology. Okay, if we then look at the brain, this next level, of course, then you have the antenna loads here. We have the antenna and the maxorelpal with olfactory sensory neurons that we talked about already. They target single glomeruli inside the antenna lobe. From the antenna lobe, the green here, from the 50 glomeruli, we have projection neurons heading up in the brain. Up in the brain, we have the mushroom bodies, we have the lateral horn. To compare to what we talked about before, mushroom body is a little bit like cortex, lateral horn is a little bit like amygdala. Mushroom body is a lot involved in memory, plasticity. The lateral horn is more involved in innate behavior. So what happens at these different stages? We have encoding, of course, out on the antenna. That's where everything has to be detected. Just like Stuart said before, if it's not detected, it cannot be processed. And we have some pre-processing going on, much thanks also to these 200 local neurons shuffling messages around the glomeruli in the antenna lobe. And then up here we have categorizing of basically what is good and what is bad. And that's what I wanna talk a lot about today. When we do all stuff, we always try to think about behavior. So we are basically not interested in things that are not vital to insect behavior. We always wanna think about what does this mean to the insect? What does this odor mean in the end? So this is the choice that a fly is put in front of, right? In all of these apples lying there, some are much too green to be good for a fly. Some are overwrought them to be good for a fly. But a few of those apples are just right. And that's what the female has to find to put her eggs because she's choosing the environment for her offspring. And in this way, how many genes will be coming into the next generation. And that is done a lot by all factions. So to choose the right apple out there, that is basically what we're interested in. So if we often talk about all factions, we always say this that, wow, we have 1000 receptors and therefore we can smell an enumerate number of molecules or millions of whichever camp you belong to, but a lot more than the receptors we have, right? And that's because we have this cross fiber. We're playing different keys on the olfactory piano. So that's sort of how we normally think about all factions. But what we see surprisingly in several cases now, and these are the ones I will mention in the beginning is this, that there is only one key being hit and you can predict what will happen if you hit this key. So basically we have red lines going through the frame saying good or bad, saying go or stop. And this has been a little bit surprising to us because we always thought that outside sex, which we knew from sex pheromones in moths for instance, where you can totally predict the behavior that we will get from the male. Beyond that, we thought it was always a lot of cross fiber coding going on. So I go through a few examples fast. You have heard of several of them I'm sure, but this was the first really strong example that we found back in 2012, geosmin in Drosophila. It turned out to be a really cool system and it's really, if you hit this button, if you activate the geosmin receptor on the Drosophila antenna, you will have stopped. And this is the key that will activate it. An overripe, moldy fruit lying in your kitchen, you can smell it when you come home because your nose is also very sensitive to geosmin. But this is what happens. And what we saw there was the most amazing specificity we ever found in any receptor in Drosophila. Only out of all of those, these are all the synthetics, but then we went on with the GC, as I talked about in the beginning, tested thousands of compounds and we never found anything except geosmin that activated this. Therefore some labs are now picking this up as a new, not optogenetic, but geosminonetic, whatever you would call it because you can really put this one in, into other neurons and use geosmin as a specific activator of neurons. But we only found one ligand that fits here. We did the calcium imaging in the brain, only one single glomerulus activated at any concentration, yes? Yes, one receptor, over 56. Then we did the same at the next level. We looked at projection neurons with the patch clamping in the brain of Drosophila. We had the same, we only got activation by one single odor, geosmin, and we expressed the calcium indicator in the projection neuron level. Again, only one glomerulus activated in the whole antenna load. So there is really one red line going over at least the two first levels of the olfactory chain in Drosophila, only telling about geosmin. The question is why? Well, if you check where geosmin comes from, it comes from microorganisms like penicillium or streptomyces, and when you let the fly experience these, they die very fast. The larvae die, the adults die, and so on. So this is bad stuff for the fly. Here we also got a very nice thing, way to do the reverse, because we had microbiologist colleagues at the institute and they had streptomyces that had more totally normal, except that they didn't have the geosmin enzyme. So they were normal microorganisms, but they didn't smell of the geosmin anymore, and then the flies didn't avoid it at all, and they died like flies. So this geosmin is the totally necessary order to keep the flies away, and it only activates this single channel. One single receptor on the antenna, it only responds to geosmin. If you put in up the genetics there, you activate it, you get the full stop, and so on. So it's really a red line going through the brain. So basically, what it does is that it tells the fly about very bad toxic food sources. It's like when you open your fridge and there is some old dinner in there, that smells bad, you go really high. Disgusting. Because it is food, and they end up in position site. Stay away. Because they would eat that. They eat it until, because in the fruit is yeast and they like yeast, but the second succession will be these microorganisms after the yeast, and then it becomes toxic. So we call it an ecologically dedicated line, yes. Sorry, I have to speak louder. It's a really risky system, you've got one neuron. But it's very sensitive and specific. I mean, you have one pheromone receptor to find your female. Smallest, same, right? But that's what we found. There is no other systems responding to geosmin on the antenna. Is this one? Sorry? Yes, it's in all other flies that we have looked at, except in one that lives in a flower all its life and is never exposed to this kind of stuff. So it's evolutionarily very conserved all over the Drosophila flies. So it's one ecologically dedicated line going through all these first levels of the olfactory system. Some other examples that now we got hooked on this. We saw, ah, there are some red lines. Let's look for some other ones, right? So we started looking and the next one we found is involved in orbit position. So the female has one receptor on its antenna. When you activate it, the female will drop her eggs. It's very cool. Sometimes you get this unasked for control. One day a student came and said, ask, couldn't I please, do I have to test limonene in this bioassay? Because every day I have to clean the tubes of eggs. And it was another experiment. But the females were all dropping their eggs when it smelled of limonene. So there is one receptor that detects limonene. Limonene is the component, as you know, of orange or lime or lemon peel. And we started looking at this because females really go for these places to put their eggs. They really like orange peel for some reason. Then you start thinking, hmm, flies, Africa, oranges, China, how is this connected? Why would Rosofila Melano-Gastor go for oranges or lemons to put their eggs? So we had to go back to Africa and have our colleagues from all over the place start sending us crates of any fruit that they could find. We got fruit, I've never seen them. I never heard of them. The basketball-sized fruit hanging from trees and a very strange one. In the end, we got this one, African squirrel nutmeg. You would probably say that that's an orange, right? Looks like an orange, smells like an orange, but it's not even related to an orange. But it's definitely one of the candidates where Rosofila Melano-Gastor could be pre-adapted to now in your kitchen at home, go for the orange or the lemon. So this one smells very strongly of limonene. Why? We find now that the main enemy of Rosofila hates this. So the parasite toy that goes for 80% of the larvae in nature hates the smell of limonene. So it really protects the larva from the main enemy out there. Again, here, if we put in optogenetics, we activate this red line, the flies will drop their eggs. That one, we don't know yet. The next line we found is on the pout. This one goes for good stuff. It attracts the flies heavily. It detects ethyl phogliacal. Ethyl phogliacal is a product of one of our best wine yeasts. We use it to brew wine, the brethanomyces. It's also used for some kind of beers. And this gives a yeast that is very rich in antioxidants. And what we could show, you know, there was something called paraquat that we used for quite some time to kill off insects. It's totally forbidden these days. But it's one of the strongest antioxidants or oxidants that we know. And when we fed the insects this kind of yeast, they lived much longer. They died quicker still with paraquat, but this seemed to protect them. It seemed to be very healthy stuff. And there seemed to be also to be a red line going through the system for this. Last one I'm gonna mention, back to the parasitoids. This one we found just a couple of years back. Here, the fly has again one specific receptor, only detecting the pheromone of their parasitoid. This again, the guy that will kill off 80% of their larvae. So both the adult and the larvae has developed a receptor that detects the pheromone of these guys. And it's a very strong repellent also for the female fly or for the larvae. So they really try to run away from this. And again, we could activate it with optogenetics and so on, and it has exactly this effect. So again, the red line, I told you about four red lines now, going through the system. So we find this, that means that we can also start looking at the nervous system, both peripherally and centrally and see how is that built to take care of this information. And I will just mention this briefly, as we're also in a neuro audience here. So what we looked at recently was that we, for the first time we built an imbibu atlas of the antenna lobe. So, you know, before we took the brain out, you cut the antenna nerve and everything goes boop, and then you fix it and it goes boop. And then you try to image in the live animal and there is really no correspondence between the fixed brain reconstruction and the live antenna lobe or olfactory lobe. So what we did is that we expressed NC82 in the live animal and then we did a full reconstruction of the antenna lobe which indeed was very different from the one we had before from fixed material. And this allowed us also to look at all the input. We use different methodologies to look at input, to look at output. This came out in cell reports a couple of years back as well. This is wrong, it should be 16, was last year, two years ago. But one of the things we found there was that in these that I talked about, the red lines that have this sparse coding, very specific lines that seem to take care of specific odors, ecologically relevant, we found many projection neurons. Before the conventional wisdom was that each glomerulus has about the same number of projection neurons, usually one, maybe two. Here we found that this could have between eight and 12, from the josmin one, that's the most innovative one beyond sex pheromones. So from each one of these really important odors, there seemed to be a lot of redundancy in the lines that go from the glomerulus up to the next level. And it's totally correlated to the lifetime sparseness of the tuning of the receptor out there. If we have more broadly tuned receptors taking part in the piano playing that I showed you in the beginning, then we usually have only one projection neuron. So there is quite a strong correlation between this. Then we go up, we look at the next level as well, we look at the projection neurons, we have different tracks, you see we have one main track, this is the GH146 line for those who are in drosophila, this is the MZ699 line, these are all excitatory neurons, these are inhibitory gavergic neurons. And we have looked at both of these now with different techniques, we have cut them with two photon laser to see what the effect is when you take them out. We have done a lot of imaging up in the lateral horn because we see that in the lateral horn we can see different parts that seem to be correlated to all these different things that I showed you in the beginning with being good or being bad. And basically the take home message is that we find all these different areas depending on if something is attractive, if something is repellent, some of them seem to be concentration coding, but the main interesting thing for us is that we have different areas coding for attraction and the version up in the lateral horn. And now we're working onwards on this, but we have several publications out by now. So this, read more about this in the literature, I will not go deeper into neuroscience today. I wanna go back to Neuroethology and go into sex a little bit. So Neuroecology of Rosophila pheromones. So some years ago, several different labs looked at virgin flies and they could see that there was something in the order of virgin flies that activated a neuron on the male antenna that could be expected to be involved in sex behavior because it expressed the fruitless gene. The fruitless gene is always involved in male sexual behavior, but no one could really find what it was. Just that it was activating this neuron, right? So we went in with some new chemical methods, thermal desorption GCMS, really cool method. You don't need any solvents or anything. You put in the whole fly, the machine heats it up to the temperature you specify, it takes it into the machine, it cools it down to on liquid nitrogen, collects everything that comes out and then into the machine. So no solvent involved or anything like that. In this way, you can really pick up minute amounts of odors that is emitted by anything. You can put a piece of banana, you can put an insect or whatever you like in there, but you can pick up more or less all odors. So we did that and we could identify 85 compounds coming out of a fly. A lot of new ones that no one had seen before, but among these, we found three that really activated the male antenna. Simple compounds, methyl chloride, methyl meridate, methyl palmitate. Yes, straight chain compounds more or less. Very small amounts, but very active. So what we basically found to make this long story short was that one receptor, and that is the one expressed in this fruitless neuron that I talked about before, increases the copulation success of the male greatly. And this is the fruitless positive one. Then we find that it's co-located with another neuron, 88A, not fruitless expressing. And this one attracts both sexes together. So these compounds seem to have a dual function, both increase the copulation success of the male and bring the sexes together. But these are new drosophila pheromones that have not been identified before. You know of cis vaccine elastate, which is the main that we know of since long back. This came out two years ago as well. So then we started looking more. And during these studies, we had this hunch that they seemed really fond of pieces, these flies. They are weird guys. But they seem to be attracted to each other's dropping. So we started looking at this a bit as well. If you look at a blueberry, drosophila really like blueberry. So without a fly near, it looks like this. When a fly has been there, it looks like this. So they really constantly drop things after themselves. As they are eating or something, they will drop feces behind them. So we started looking at this a bit. So we looked at how attractive is blueberries on their own or where the fly has been. And it's a big difference. It's really a strong preference for the one where there has been a lot of droppings on it. And then we started looking at the frass, at the feces. And what we saw is that the methyl chloride, the methylmeristate and the methylpalmitate occur quite a lot in the feces of the fly. So these pheromones that we identified from the fly before, they also constantly drop behind themselves on the substrate where they're eating. So the whole substrate becomes sort of an attractive source for the flies to gather and mate on the substrate. So we started collecting large amount of shit from the flies. And yeah. So I thought that you got this. Now that was the beginning. That one is done. I've gone into the frass now. So I finished, that was where I finished telling you about the 47B and the 88A. And that was the first story. And I jumped over to frass because the first story made us interested in the frass. So that's now I'm in there. So we collected a lot of this to really analyze what it is. And we could do all the experiments. We could knock out the receptors and show that the attraction to this source is all built on activation of the pheromone receptors. When you take them out or when you take out orco, you lose all the attraction to the frass. OK, we also did it in our fly walks. This is an experiment that we built out up during the last 10 years where we have total control. We can, more or less, watch each step of the fly. Yes? I'm going to the distance itself. And we should have come out of the pheromone. Yeah. What is it going to act as a factor? Because they add the pheromone. In the intestinal tract, they add the three pheromone compound. And if we knock out the pheromone receptors, there is no attraction anymore, which for us means that it's these compounds that are the active ones. We also did it in our fly walk. And there, we saw that frass is actually the most active compound we ever saw. So the green curve here is how much attractive a fly gets when it gets the odor of something. And this is the frass odor. So it's really attractive to these guys. We checked different species, and then we found that the odor of frass from each species is more or less specific. So it, again, adds to this, that you add this to the fruit where you are. And that will sort of be a message that here on this fruit has been at Rosofila Milano-Gaster. I'm sitting here. I'm smelling very nicely. And you should come here and mate with me. This is a good substrate. Might also be a good substrate for offspring. So far, frass is constantly deposited, highly attractive. The attraction is based on pheromones. And it seemed to be somewhat species-specific, at least. So all of these label lines, if you think about it, someone said before, having one line, that's gambling. And what does it mean? If you have label lines that will trigger specific behavior, that means that you open up quite heavily for exploitation of your system. And that's what we found. Earlier, of course, it has been found in deceptive flowers. These are flowers that smells like sex, like the left one. An offspring's orchid. This orchid will attract males. And the males will try to copulate with this flower because it smells like a female. It looks like a female. And the male will fly from. We published this a long time ago. The other one smells like an obi-position substrate. It triggers a red line in the female fly line saying, you have to go here because it smells of rotten meat. And you should go there and drop your eggs. And in this way, it also takes part in the pollination without getting any reward. These are not producing any nectar. So they're just fooling the flies by smelling good. And they do it by activating these red lines through the brain. So what we looked at now was another aspect of this. Or we didn't even know that we would look at it. Because this is one of those where your hypothesis is turned totally around in the middle of the project. So I'm always trying to think like a fly. So if I were sitting beside Anna, and Anna was co- or sneezing, and then I would maybe move to this chair, because I think, she might carry some bad cold disease. And you should stay away from your fellow beings. That's the way we react sometimes. And that's what we thought here as well. If you were a fly and you were smelling, hopefully, that one of your friends had a cold or was sick in some way, it would be a good idea to stay away from your friend. So that's why we started looking at this. And the hypothesis was that flies should stay away from conspecifics infected by bad bacteria. So we started poking flies at this little red suture here with different kinds of needles infected by different bacteria. So we added some good bacteria that they carry in them all the time anyway. Some ones that are good for us, like to bacillus. You know you eat it in your yogurt and so on. And then we took three that are very bad. One of them is bad, but they can recover from it. Two of them are lethal. So we started seeing, what will this? Can they smell it? And will they move over to the other chair if they smell it? So how is fly behavior affected by this infection? So we started looking first at the odor of the bacteria on their own. There was no real effect. The flies didn't choose at all. But then when we added flies or feces from flies that were infected, totally the opposite happened. The flies went very nicely towards those that were infected, either to the flies themselves or to their feces. It became super attractive instead of the opposite. So instead they got closer and started hugging their friends more or less instead of moving over to the other chair, right? And we started looking at other things. Fly walks, same results. But we looked at feeding. We had a cafe system where the flies can feed. And then we had either a tube with pure feeding stuff. And in the other one, we spiced it up with some frass. So we mixed up some frass in this feeding matter. And again, it became super attractive. So the flies went and they started eating of the frass of their sick friends. Not a good idea, basically. Then the final, we checked, was always position, because that's the final decision of the female. Very important for the offspring. Because these bacteria will also infect the offspring and kill them off in the end. Here they really avoid the bacteria. That had been shown before. So the odor of the bacteria themselves is bad. But if you have the flies, if you let infected flies stay on this medium before, it becomes highly attractive again. So the female flies choose to put their eggs where there were sick flies before. All of these totally against the hypothesis that we had from the beginning. So why? Why should it be like that? It talks against all sort of sense. So we looked at this. Again, we went into our thermal desorption system and we looked at what happens here. And if you look here, these are the pheromone compounds. Here are the healthy flies. Here are the infected flies. So the infected flies has a 20 to 30 fold boost in the amount of pheromones that they produce. So it seems like the system is hijacked, right? So these bacteria, the bad ones, not the good ones, there is no effect of the good ones on the pheromone production. Only the bad ones, they really affect this production. If you look over time, this is the bacterium that they can recover from. So there is a boost and then they recover and it goes down again. These are the ones that are dying more or less. But the pheromone production goes up enormously and then they die and it drops, of course. But I mean, there is this very strong effect of these bacterial infection on the pheromone production of the flies. So we also checked it on the sencilla. Much stronger response in this olfactory neurons when you expose them to this. Yeah, I'm coming to that, yeah, coming to that. So which mechanisms might be involved here? So we started looking, first we looked at the immune system. Well, when we knock out the immune system, we get less increase in the pheromone, right? But we cannot create this boost by artificially activating the immune system. Then we looked at the metabolism and hormonal mutants. Also, we could knock it down but we could not mimic what is going on. So we haven't gotten to the mechanism yet that the bacteria over evolution use to boost the pheromone of the fly. But what we also have to look at, is it any good for the bacteria to do this? So we had to see, can they multiply via the fly? So the red stuff here are live bacteria in the thesis of the fly. So the bacteria really pass through the fly, they come out. We put them onto an agar plate and we could grow very nice new bacterial colonies from the press of the fly. So it's really good for the bacteria. Second question is then, is it bad for the fly? Yes, it is. The flies die pretty fast after they have been infected. So it's good for the bacterium, it's bad for the fly and it really seems like this is a way for the bacterium to hijack the pheromone system of the fly. So it could also be the case that the fly feels sick and he or she says, better mate quickly because I'm dying anyway. So we checked how good copulation is and it's really much, much worse in the sick ones and even worse if you go down and look at what comes out of this. So that is not the case. It's not really worth it copulating anymore because the output is almost zero in the end. So as far as we see it right now, it's the bacterium, here is the summary. Here are healthy flies, right? Normally this is the way it should be. You activate the pheromone line, you find healthy friends and their press, you meet them, you mate and you have healthy offspring. Then we have the other one I talked about, the jostmin and these things that stop you from going to bad places and get harmful microbes. But then we have these devious bacteria that go into the fly, modify its emissions and override the bad channel, sort of attract healthy flies, they get infected, you get infected offspring of someone and overall it spreads these bacteria to many flies around. We haven't gone on to the ecological stage yet to see how it spreads through the population but that is a plan that we have right now. So basically these different points here, this is right now coming out in nature communications in one of the coming weeks. Okay, where are we? Still some time. So sorry, who is speaking? You have to speak loud because my hearing is bad. How it changes the pheromone production? No, we don't know. We're looking at that as well because we don't even know the enzyme because it's so recent that the pheromone was identified that we don't know how it's produced yet. But of course we would like to find the place where these guys go in and crank it up but we don't know yet. So once more, do you know? There's also an area to say, I am resorting, do we have some confidence that the resorting is not going to be gathered before you're off? Yeah, yeah, I see what, yeah. But I mean, these are really bad. So everyone will get them and everyone will die. Toxoplasma is a UNICEF protocol. But this is not a system. I mean, we, no, no, they're only bad. Yeah. So these that we have looked at so far. We have to, we have, we are, I mean, this is the first study. First thing is that it was totally against our expectations. So now we're dissecting onwards in it. So you're right. Okay, so now I want to move over to Manduka. There should be a Manduka there, but I don't know where it is. Let's see if we go back. Did it already fly? Ah, here she come. Here she come. So now I want to talk about Manduka and its interactions with flowers. And specifically this interaction that you see here in the front going on now. It's quite a feat with that long, long proboscis getting the right place, finding the right flower, injecting it at the right site and so on. And that's what we are interested in. So already Darwin said that there must be co-evolution between this length of flowers and the length of the proboscis, right? So we have a whole bunch of tobacco flowers ranging from more or less B pollinated ones that are very flat and more or less only produce pollen up to these extremely long, longiflora that are about this long. And all of these have their specific pollinators, more or less. We work with Manduka sexta and it has this length and you see that that would match Nicutiana a lot about. So the first thing we wanted to see is, is there odor specificity? Can the moth choose this flower in comparison with all the other ones? We used our wind tunnels to do this. We can have a flower outside. We can bring in the odor and we can see what does the moth do in there. So also what we looked at was the energy budget of this. And we found that on Alata, there was quite a lot of nectar present, but we also wanted to know what does it cost for the moth to do this. So the first thing we did that, we could show that the moth really chooses the right flower depending on what it smells. But then we looked at this, how much time or how much energy does it cost for the moth to do this. So we built this machine where the moth had to fly into a funnel like this. And then while it was in there, we could measure the CO2 coming out, which means that we could measure how much energy was spent by the moth while extracting the nectar out of the flower. And in this way, we built the energy budget. And in the end, what we could see was that the only flower that really paid off, when you calculated what it cost for the moth, you're standing like this all the time and then finding the right place with your tongue and then holding the right place. And then with a small one, it started tumbling around, it didn't find the place and so on. And the only one that really paid off was the one that fit to the tongue, so where you could find the right place. So this was the first study that we did there. Yeah, we are right now setting up field experiments in Utah and Arizona to check this in the field as well. These were the first ones. These are pure lab experiments, but I agree. How much do you have to spend flying between them as well? That's great. Okay, so, but you saw here that they really find them out there in the night, right? So our first question, how did I really pinpoint that opening, that little minute opening to find the flower? Luckily, we're in the institute of where Ian Baldwin is also a director and he has made Nicutiana a model system. So he can modify them in any way, more or less. And he has one mutant where he has taken out the main compound of the flower, Benzylacetone. So it doesn't smell of anything anymore. Here's the normal amount, here is the mutant. So the enzyme for producing Benzylacetone is gone. So we started thinking if we could use these to dissect the mock behavior. So we built a big tent in Jena, eight by 24 meters. And in there, we put out flowers, mixed the one, the normal one smelling and the abnormal ones not smelling. And what we saw was that the flower contacts were about the same. So they seemed to look for those white spots in there, even a dusk, they're crepuscular. But what we then saw was that in the one non-smelling flower, more or less no seeds were produced. So no pollination occurred by the moths and no nectar was removed from the flower. So something was failing in that interaction, right? They found them, but they didn't go on. They didn't pollinate, they didn't suck out any nectar. And we did the same thing in the wind tunnel. We could again see that the flowers were contacted at about the same rate, but the time spent was much lower and the nectar remaining again was all of it left, none taken out of the flower. Okay, so what is the interaction here? There must be some sensory input missing in this interaction. We started looking again at the moth and what we found, or I thought back to a long time back when we were looking at all the structures of the moth head, there are actually some sencila sitting out on the very tip of the proboscis. They look weird, but some of them have this porous structure that could indicate something here. We started doing in situ hybridization, we did for Orco, we could find Orco expressed in the proboscis, which that's the olfactory receptor, co-receptor. So there is, that was a sign that there might be olfaction going on out there. We looked for all, we found quite a few of receptors out there actually, both IRs, GRs, ORs, and so on. They fell all over the place, even one falling among the pheromone receptors, pheromone receptor, play down here, but also falling out, and this OR 23 especially, it falls between those that have been characterized as flower odor receptors in other moth species. So we started looking here more in detail, we did in situs again, we could find that Orco is co-located with some sensory neurons in the proboscis, and then we did recordings. And when we did recordings, we found that these, there is a cell here responding extremely much to the benzyl acetone, to that compound produced by the flower. You see the response here. When we checked this, sorry, when we checked the specificity, we found that it is not just one compound, there is a host of compounds, but all of them are flower odors, produced by the flowers that Manduka liked to drink next to her. So here we seem to have some kind of system that likes to find the way into the flower, right? But we had to show this, this is good when it's good to have good students around. My student found out this device here. So the same receptor is expressed on the antenna. Now, the same receptor is expressed on the antenna. So if we couldn't isolate the antenna from the proboscis, we couldn't really say that it was the proboscis that was involved. So what we did is that we did this thing here, clean air, benzyl acetone, and then an exhaust that would take the odor out before it could ever reach the antenna. And this way we could say, if something happens here, if there is a choice, it's definitely the proboscis that does it. Now I have a movie coming up, which is bad, but it will, if we let it run once, it's usually better the next time. How long? So I will go out, cause, let's see. Oh, what happened now? Okay, maybe you can, with some good will, you see something moving to the right here, right back. So here, hold on. So here is the time moving, and I don't know why it turns green on my computer, but there is a little bit to the other one, but you see it's constantly going down this one. So we could do statistics here. How much do you spend with your tongue in right and left to where you wanna go, sort of? So let's see if we can move this one on. Ah, here's the same thing again. Let's move on. There, we could do statistics here, and it's highly significant that they go with their tongue in the direction where the nice smell is. So, yep. Oh, they don't like to fly without antennas. We want as natural behavior as possible. And if you chop off the antenna, they're so unhappy that they don't like to behave at all. Yeah. So here it seems like we pinpointed a new olfactory channel coming from the proboscis of the moth before we knew of the antenna and of the tongue, but here it was, it's now the proboscis, and this is out in Eli last year. So, it sort of connects very nicely to the moth behavior. We find that they choose the right flower and they have a specific detector on the tip of their tongue to find their way into the flower. So, final wrap up, what we have been talking about here. First, I showed you that we have these ecologically dedicated lines in the fly system, detecting geosamine, detecting limonene and so on. The lateral horn seem to take care of this information in different areas, depending on its behavioral significance, if it's good or if it's bad. Then, newly discovered Rosophila pheromone, the methylaurate and these guys that are really novel Rosophila pheromones. Frass contains these pheromones and makes it highly attractive. And finally, I showed you that pathogen infection boosts the pheromone production in ways that make the flies highly attractive. Then we went on and we looked at Manduka and that it chooses the optimal flowers based on odor. And then finally also that the final stages in nectar feeding is based on proboscis smelling, which is a totally new pathway. We have just now filled the neurons coming out of the proboscis and they don't target the antenna lobe. So there must be something else going on in there. They still target the subesophageal ganglion, which is normally the taste place of the insect brain. These are the people involved. These are my group leaders, Marcus Kanadan, Silke Saxe. Marcus does a lot of the behavior, Silke does a lot of the neuro. Honey was a very good postdoc that did a lot of the red line stuff that I showed you in the beginning. Max Planck, strange enough, keeps us giving us very nice funding every year so that we don't have to spend time applying for too much money. Basically also in the end, Honey is now in Yale so that he is doing a career now together with John Colson over there. So I think that's basically it. Thanks for the attention. Any questions? Welcome. Thank you.