 και είναι ότι είναι ο εργαλός που βλέπουμε. Παρακολουθούμε, όλοι και ευχαριστούμε πολύ για την συμμειλώνα του Σασαικού Σεμίναρ. Είναι ο Σασαικός του Σεμίναρ, και είναι ο ίδιος κοιμήρς του Σασαικού Σεμίναρς. Είμαι ο Γεωργή Καφετζής, ένας φορματοσυστικής κοιμήρς, σε το Μασσολαιρήσωλετ's λόγο. Και τώρα ήρθε σε ένας θεούής με τον Μπαδεμ. Και as your host for today, I would like once again to begin by thanking Tim Vogels για να ξεκινήσουμε να ευχαριστούμε τον Τύμ Βόγγελς και τον Πάνος Βοζέλος για να προσπαθήσουμε αυτό το very initiative στους the greener and much more accessible seminar world. Όχι όμως, αλλαόμαστε να πάμε back to the reason we all gathered here for today and introduce our guest from the University of Maryland, Prof. Karen Carleton. With undergraduate studies in chemical engineering from Yale University, she went on to University of Colorado and acquired her PhD in physical chemistry in 1987. Following postdoctoral years in Berkeley with Bradley Moore, Karen started her own lab in 1989 in Andover and physical sciences incorporated. In 1995, she then joined University of New Hampshire as a member of the Hubbard Center for Genome Studies and the Department of Zoology before moving about a decade later to University of Maryland, where she has been located ever since and nowadays holds the title of professor in the Department of Biology. With research interest revolving around photoreceptors, color and visual communication in coral reefs, it is with great pleasure that we are having current today here with us and we will be hearing about cyclids in her talk entitled Using Obscene Genes to See Through the Eyes of a Fish. So without any further ado from my side, please all welcome Prof. Carleton. Karen, this stage is officially all yours. Thanks very much, George. I really appreciate the invitation and the opportunity to speak with everybody. And I wanted to thank Worldwide Neuro for this initiative and especially the Sussex Group for keeping the vision community going during these tough times. So I really appreciate that and it's the vision community I think is a really wonderful group to work with. I've enjoyed collaborating and so a quick shout out to Team Opsen out there. So as George said, I have sort of a little bit of an unusual background and with my physical science training, I think about the visual system primarily by following the path of the photon. And so this helps break the visual system down into some key steps that we need to think about as biologists. And so we have an illuminant that's going to send photons out through the environment. They're going to reflect off some object that is then going to be detected by a receiver. And the light will go through the cornea, be focused by the lens and form an image on the retina. And in the retina, there are these photoreceptors that are going to detect that light. Now today we're going to ignore the rods for the most part and focus mostly on the cones and there are multiple types of cones that are useful for bright to daytime vision. And these cones differ because they have different ops and proteins within them that produce visual sensitivities in different parts of the spectral range. And so these different signals can go from the retina into the brain, where the organism can then make decisions about what sort of behaviors to follow, whether to avoid something or whether move towards it. And so vision is an incredibly important part of animals behavior. So vision is really fascinating because there's a very clear link between the genotype and the phenotype. So the genotype is the sequence of the ops and proteins that surround this 11-cis retinal molecule that absorbs the light. And it's the amino acids close to that retinal that shape the absorption wavelengths of that visual pigment. So the sequence or the genotype of the ops and shapes the phenotype or the visual sensitivity of the organism's eye. And this has important functional implications for that organism. Likely the visual system is under strong selection based on whether the animal is foraging or mating or trying to avoid predators. And there can even be differential selection for organisms that live in different environments or maybe are performing different visual tasks. And those that selection might vary even throughout the day, across the seasons, through an animal's lifetime, say between larvae and adults or across evolutionary millennia. So there's a lot of opportunity for the visual system to evolve and for us to understand how and why the visual system works the way it does. I'm going to talk about visual systems today and fish are a wonderful system because there's so much diversity across fishes. So fish make up half of all vertebrates and they've been around for many hundreds of millions of years. Even our model zebrafish is several hundred million years divergent from many of the fish we're going to talk about today. So I'm going to talk about cichlids and I'm going to spend a little bit of time trying to convince you about how cool cichlids are. And for those of you who maybe don't think you know too much about cichlids, if you've ever gone to a restaurant and ordered tilapia, you have eaten a riverine cichlid. And these cichlids come from the river Nile in Africa, so they're riverine generalists, but they're now raised around the world in aquaculture facilities and they produce about 5% of the world's aquaculture output. So tilapia are farmed everywhere in an important source of protein for the world. But in addition to the tilapia, there are something like 2000 species of cichlids that are distributed around the globe. And that includes cichlids in the new world, cichlids in Africa, Madagascar and even a few in India. But if you look at the species distributions across these four different groups, the vast majority of the diversity occurs in Africa. And so we'll spend most of our time today talking about African cichlids. Now, much of the diversity in Africa is within the Great Lakes, although there are cichlids all in the riverine systems throughout Africa. And as these lakes were forming relatively recently, several of them are in the Rift Valley that's forming as the plates of Africa are spreading apart. So as these lakes are forming, the riverine species invaded one or a few times to seed the radiations in each of these lakes. And so in these newly forming habitats, there was sort of a blank slate and cichlids rapidly diversified to fill all the available ecological niches. Now this diversity has arisen quite rapidly. Lake Victoria is less than a half a million years old and it has hundreds of species of cichlids within it. Depends how many species depend on whether you're a splitter or a joiner. But there are literally hundreds of species and this makes Lake Victorian cichlids some of the fastest radiating vertebrates that we know of. Lake Malawi cichlids are a close second to that. Now, this image here is a satellite picture that's color coded to show water clarity and the blue or the coloration, the smaller the attenuation coefficient, so the clearer the water. And so you can see that both Tanganika and Malawi are quite clear lakes. There's some of the clearest freshwater lakes in the world, whereas Lake Victoria is quite a bit more murky. And so this allows us to kind of do an evolutionary experiment of each of these lakes has an endemic radiation of cichlids. So we get to run the evolutionary experiment multiple times and see what happens when you take cichlids and add large bodies of water. And because these vary in their environment, we can see sort of what the impacts are of different environments. Now, in addition to habitat differences, cichlids have huge variations in behavior. Some cichlids will pair bond and in that case males and females look quite similar to each other. Sometimes males hold harems and so there will be a large male defending a whole bunch of smaller females. In this species, these females live in these individual houses of these little shells. And then oftentimes, cichlids will be female polyandrus and that means that females control mating and they choose multiple males to breed with. And this typically occurs on a large lec. And so here is a rocky lec from Lake Malawi. And you can see several males here all holding territories, all relatively close to each other. And females will then come swim to this lec when they want to breed. They have to find the male of their species and then choose which of the males they're actually going to breed with. And so males tend to try and be quite flashy and try and entice the females to breed with them. Now you might notice there's some other fish that are quite muted in coloration and females tend to be very muted. They want to be cryptic in this environment for reasons I'll tell you in about in just a second. So in addition to mating behavior changes, cichlids also can vary in parental care. Some cichlids will actually lay eggs on a substrate, other cichlids will actually mouth brood their young. And you can see the little eyes peeking out of some of the babies that are being held in the mouth of this female. And so because these females are sort of mobile nests carrying their eggs around with them and they do this for several weeks of time, they want to be quite cryptic so that other cichlids don't notice that they're there because those cichlids will try and ram them and have them spit out their eggs so that they can be eaten. So females tend to be quite cryptic and males want to be quite conspicuous. Now, most of the cichlids I'll talk about today, including the tilapias and the cichlids from Malawi in Victoria, are these female mouth brooders that are polyandrus feeding with breeding with multiple males. So the females will visit a male lec to choose their mates. This requires species recognition, then mate choice. And this leads to large sexual selection with males trying to get all the breeding that they can and large sexual dimorphism. Again, males are very colorful and females are not. Now within Lake Malawi, which I'll talk about quite a bit, there are two major habitats, sand habitats and rocky habitats. And over the sand, you get different cichlid species where males will build these breeding sites or bowers, as we call them. And each species will have a different bower shape. Sometimes they're castles, sometimes they're pits with mounds around them. And again, each species has its male build a different shaped bower carrying huge volumes of sand that they have to then maintain. Over the rocks, the fish have it a little bit easier. The males just have to defend various rocky regions, cracks in some cases, caves and other cases. And they will then try and entice the females to breed. They will rise up and either use kind of vibratory motions or auditory signals or chemical signals, hoping to entice the female to go back to his breeding site where she will lay eggs, he will fertilize them and she'll pick them up in her mouth and then go off and decide whether to mate with additional males. So that's a little bit about cichlid behaviors. So again within Malawi, we have these sand and rock dwellers and from the time of the riverine colonizer, there was this first split between sand and rock that was then followed by massive diversification. Again, 2000 species that have all kinds of different body shapes and colors. And if we look at some of these species, we start to put them in piles and we start to first have piles that are genera and genera typically differ trophically based on what they feed on. So we have predatory cichlids that have these fusiform shapes with large jaws. We have snail eaters that have strong jaws for crushing snail shells. We have fish that delicately pick invertebrates out of the algae or plunge into the sand to capture invertebrates. We have fish that rip scales off of other cichlids. We have zooplanktevores up in the water column sucking in a little zooplankton. And then we have the cows of the lake scraping algae off the rocks. And all of these diverse genera arose from this omnivorous riverine ancestor. Now, if we look closely at a given genus, the thing that differs between species is male color. And so all of these cichlids here are in the same genus and they all feed generally in the same way and they have similar behaviors, but they differ by the flashy male color pattern that they use to try and attract females to breed. And so female cichlids like cichlid biologists use male color to kind of distinguish species. So going to Malawi is great for doing field work. We've been fortunate to go for a number of field seasons. We work at the University of Malawi's field station, which has a compressor for scuba diving. I often go with my husband and some of our students and we collect and dissect lots of fish. We can go out on this boat for several weeks going up and down the lake. It's a huge lake kind of the size of Lake Michigan. And it's a really interesting and fascinating place to be. We've been fortunate to entice some visual scientists to join us. So Justin Marshall and Tom Cronin came for one field season and this is Justin kind of gathering some spectral reflectance data of substrates. And the graduate students are generally really happy to be there enjoying the field work, looking at this huge diversity. This might be, you know, 50 different species all swimming next to each other. And you literally can't decide what to look at first. So people have been studying cichlids and cichlid vision since Russ Fernald first started this work back in the late 70s. And all the cichlids that were looked at early on proved to be trichromats. So just like humans, cichlids have three different cone types. The short wavelength cone is in a, the short wavelength visual pigment is in a single cone. And the longer wavelength pigments are in these joined double cones, which we think neurally are independent of each other. These form a highly organized retinal mosaic and they are sort of evenly distributed across the retina. So we expected because they had three different cone types. And to produce three different visual pigments, you need three different ops and genes that they would indeed have three ops and genes one for each cone type. And it's the ops and surrounding that retinal that causes the option shift, shifting the absorption spectrum of the retinal out to different wavelengths that produces these different spectral sensitivities and the different types of cones. So we were surprised as we started doing more micro spectrophotometry with help from people like Ellis Low and Farron Sarosi, that we started to find multiple types of visual pigment combinations. Some species were UV blue green and green sensitive, others switched off the UV to become violet sensitive, and some cichlids even had a sort of more human like blue green and red sensitivity. So I thought this was going to be really exciting, we would go in there and sequence our three ops and genes and we find these major sequence differences and we would totally understand cyclic vision by looking at ops and gene sequence variation. And I couldn't have been more wrong. So we started sequencing ops and genes and we found that there were actually seven different cone ops and genes. And we were able to work with David Hunt and Jim Bowmaker and Phyllis Robinson. You can actually take each of these genes, express them in a protein expression system, sort of add 11 cis retinal and stir, and you can measure that we have a UV sensitive visual pigment from this gene, a violet, a blue, a blue green, two very similar green pigments and a red pigment. And these are all because each of these ops and genes produces a visual pigment in a different part of the spectrum. So, cyclic therefore have sort of a whole range of visual pigments. And it seems that the way they build their visual system is to take one of these short wavelength sensitive genes and express it in the single cones, and two of these longer wavelength genes and express them in the double cones. So, it became rather than a story about gene sequence, it was a story about gene expression changing which set of these available genes were expressed. So, I'm going to talk a little bit about the molecular mechanisms that cyclics can use to tune their visual sensitivities. Just first of all, we know that ops and sequence can have big impacts on visual sensitivity, changing individual amino acids that are close to this retinal molecule can produce measurable shifts in the visual pigment absorption. So, the absorption will peak at a different wavelength depending on the sequence. But when we went to sequence the ops and genes across cyclics, what we found was most of these ops and were the sequences were quite similar across many different species. And it was only the shortest and the longest wavelength genes that actually had significant sequence variation to produce small spectral shifts. And so this confirmed that it was not ops and sequence differences that could explain the large visual pigment differences we were observing that it had to be something else. So again, the shortest and the longest genes could vary visual pigments by a small amount, but most of the genes in the middle were pretty stable, they didn't vary much. The big way that cyclics change their visual sensitivities is by expressing instead of say an SWS1 gene and SWS2 gene. And so by changing totally the amount of which gene is expressed, they can produce big shifts in their visual sensitivities. And this then explained these large differences we had observed with microspectrophotometry. When we went to do gene expression using quantitative PCR, we found that this species was UV blue, green and green sensitive because it expressed the UV blue, green and green genes. This one switched off the UV gene and turned on the violet sensitive gene, and then this species expressed the blue, green and red genes. And I'll just say here we've got seven different cone ops and genes, but two of them are so similar that we can't genetically distinguish them always and so we often group them together here. So we have six different genes that are varying or just six different classes that are varying between species. So now if we look across 50 different species we can see, so we call these the short combination, the medium combination and the long combination, and we can compare how frequently cyclics can switch between these different combinations. And we found when we looked at a bunch of sand dwellers and a bunch of rock dwellers that all lived within the same bay, that we could identify genera, where one sister taxa was say, medium sensitive or one was long sensitive, or one was short and one was medium, or one was even short and long. And so sister taxa are differing by which of the ops and gene combinations they're expressing, and that suggests it must be a very labile process easily changed between species, which was sort of totally surprising. So cyclics have some of the most variable visual systems between species that we know of. Well, we wanted to see if this occurred in the other lakes as well and so, working in Lake Victoria with only sea housing and in Lake Tanganyika with Hans Hoffman we collected a bunch of species. And just to give you a hint, here's some of Justin's downwelling light data in Malawi to compare with only sea houses data in Lake Victoria. Lake Victoria being more murky has essentially no ultraviolet violet or even very little blue light present. And so these are very different light environments. And in agreement with that, we found that the Lake Victorian cyclics don't express all of the options. They primarily only express this long pallet combination, being longer wavelength sensitive because there's no UV or violet light to detect. That differs from again Lake Malawi where we have multiple combinations, both the long, the medium and the short. And then we found that in Lake Tanganyika, which is just as clear as Lake Malawi, we also get species that have all three expression combinations. And so both of these lakes are clear and have the species that differ in which ops and combinations they're expressing. We were then able to use this phylogeny to kind of reconstruct what we thought the ancestral taxa might have been. And so these pie charts show the probability that the ancestors of each of these groups have had one of these three different pallets. And you can see that the ancestor of all of these groups was most like the riverine species, again the tilapia, which also has the long wavelength pallet. So this suggests that both within Tanganyika and within Malawi, they have separately re-evolved this ability to have species that differ in which of the ops and genes are expressed. And so not only is it labile within lakes, but it has re-evolved in both of these lakes independently. We wondered why all of these species would have all of these genes if they weren't being expressed. And so we decided to look through development and we chose to look at this riverine outgroup species to begin with. And so the tilapia is easy to raise in the lab and it produces lots of young. And it allowed us to look at how much opsum was expressed in species or individuals as a function of their age. And so we took a couple of different brews and we would just sample a couple of individuals every couple of days. And what we found was that the very young fry expressed mostly the UV gene. That then turned off by about two months and was replaced by the violet gene, which was then turned off and replaced by the blue gene so that the adults have blue expression in their single cones. The double cones do something somewhat similar. So they have this blue-green gene on early, but it gets turned off. And the red gene really takes over just like this blue gene does in the tilapias. And we've got a little bit of green ops and expression as well. So we have a lot of variation through development with all seven of the cone ops and genes, including the two very similar ones that we have distinguished here being expressed. So we started to wonder then what was happening in the lake species if this riverine species was showing so much variation. And so I'm going to code this this way. So here again is the tilapia results for larvae, juvenile and adults in the single cones and in the double cones. And what you see when you compare what's happening in the riverine tilapia with other species, what you find is that in the lake species, they turn on the larval genes in some species and then just keep those on. Some species go partway to the juvenile stage of the ancestral type and become medium wavelength sensitive. And then some species just turn on this adult expression combination. And so we have highly modified the developmental patterning seen in the riverine outgroup in the lake species. And so this might be because the lakes are more stable environments. And they've modified sort of that developmental progression to produce the three different visual combinations that we see varying between species. We even see one species here which actually has changes in the single cones but no longer changes its double cone. So it seems that single and double cones are decoupled in this developmental programming. So as we were doing these studies we were collecting fish in the wild. And then when we're starting to bring some of them back into the lab. And we noticed that fish that in the wild had very clean ops and expression combinations. When we brought them back to the lab would start to express additional genes. We wondered were they now going from being trachromatic to pentachromatic. So were these being uniquely expressed in individual cone types or was there some sort of co expression going on. And it turned out to be the latter. And so we started to do in situ. So Brian Dalton was actually a graduate student with Tom Cronin who came and worked in the lab. So you can see the retinal mosaics that he's taken a picture of with the single cone in the middle and the double cones around. And here he's just labeling with one gene. You can see the alternate double cones that are getting labeled as you move down a row. When he labeled with two different genes you can see that some of them are always in opposite members of a double cone. But other genes are only co expressed in double cones. And so what turned out was that these genes, the extra genes that are appearing in the lab are being co expressed and are not in their own cell type. This gene expression turned out to be even more complicated because it turned out that, well, so sorry. So producing two different visual pigments in the same cell essentially is like finger painting. It allows you to mix and match visual pigments to produce a pigment that's intermediate. And this is a way then by tuning the ratio of these two genes to infinitely tune the visual pigments that are present in the retina. And so cyclics now have sort of an infinitely tunable set of visual pigment sensitivities. Well, this co expression that we are observing turned out to be quite a bit more complicated because it turned out it also varied spatially across the retina. And so for this set of genes here, this gene was present in every single double cone across the retina. And that's shown in the regions where it's blue. So where there's pure RH2A expression, that's in blue. But there's co expression of this LWS that increases as you go dorsal ventrally. And so where it turns red, that means 100% of the cones are co expressing both genes. So we have some dorsal ventral variation with this pair of genes. When we looked at these two genes, which are co expressed, they vary temporal nasally with pure expression of this blue gene in the nasal region and the temporal region and then co expression in the nasal region. And then for the single cones, we had this region right here in the middle, which we call the Aryan centralis, it has high ganglion cell densities. And so in the center here, it's pure UV expression, but in the periphery, we have pure co expression. And so these different variations suggested that there's sort of a region in the middle here, this area centralis where co expression is quite low, but in different parts of the periphery we're getting different amounts of co expression. So crazy so much variation every time we started to look at a different factor we found more variation. Well, this co expression turned out to be responsive to light, and it turned out to show some environmental plasticity. And so Pratima in the lab was able to take some wild caught fish that we had shipped, and we started sampling the first day they arrived from the airport, and then she sampled them several days later, having them live in the fluorescent lighting of our lab. To compliment those experiments Miranda took some fish that were raised in the lab and moved them either to bright lights, or to more solar like illumination which you get out of these metal halides. And she also sampled over relatively short time scales. And what we found in both of these cases was there was a lot of plasticity and response when we changed the fish from a wildlife to a lab environment. So in the wild the fish have pure expression, and when they start being raised in the lab, even after three days they're co expressing these additional genes in these cells. Similarly fish that were raised in the lab have co expression but when we move them into a more solar like environment with a lot more UV content. Now they go back to being more pure expression. And this happens over just a few days. And so this gene expression shift is very rapid. And amazingly so and might help cyclids adapt to environmental shifts as they're moving about in their local environment. So we have then a whole bunch of different mechanisms by which cyclids can tune their visual systems. So we have shifts in gene expression, major shifts turning genes on and off, and then subtler shifts that are due to co expression that are in response to changes in the environmental lighting. Well, we wondered then were any of these this variation functional, did it. Sorry, matter at all I'm going to leave the head. And so, move back. So we have these many different mechanisms that cyclids are using. They change sequence they change gene expression. I'm running out of time so I'm not going to show you that they change in lens transmission, they change in chroma for expression, and they even have some gene losses, particularly in the central and south American species. And in doing sort of a survey across a whole bunch of species. We found that many fish now use these same mechanisms for tuning their visual systems. And so, cyclids are just the tip of the iceberg, as it were, with fishes ability to tune their visual systems to adapt over both evolutionary and then shorter term time scales to adapt to different light environments and likely also ecological foraging and and behavioral traits. So I encourage any of you who are interested in different groups to keep looking because I think we'll continue to find a lot of variation across different groups of fishes. So again, we wondered whether any of this variation really might matter. Would this is maybe this variation is just random noise, or maybe there's just a lot of individual variation. And so I'm going to tell you three quick examples of why we think this variation is correlated and important for both behavior and ecology. So I'm going to start with an example of coding sequence variation, which I probably convinced you was not important but which actually turns out to be important in some instances. So, working with only Seahouse and again on the Lake Victorian cyclids, we found that there were two species that varied in the sequence of their ops and genes having three amino acids that differ. And these cause functional shifts in the visual pigment sensitivities, both based on MSP done in Jim Bowmaker's lab and doing protein expression that work that was done in the Japanese lab of Norio Cata. So these functional differences might be important. Again, remember that the Victorian cyclids only ever expressed the blue, green and red opsons and so this red opson might have some impact. And so only was able to go to the field and measure the light environment at the surface one meter down and two meters down. And what you can see is that the lights environment shifts quite rapidly with depth in murky Lake Victoria. Well correlated with that they found that the species that had the longer wavelength LWS version occurred at depth and fish that had the shorter wavelength LWS version occurred shallowly. Coincident with that, they also found that the fish that lived shallow were blue in color and the fish that lived deeper were red in color. And so this suggested that this environmental gradient produced a visual gradient and evolution of the visual system, which then fed back on female mate preference, such that male colors evolved to produce red males at depth and blue males in the shallows. And these two groups are actually different sister taxa their species and so vision and visual sensitivity differences have actually driven speciation in this case. So just a small difference in sequence tuning can actually have a big impact. We haven't found anything that spectacular in Lake Malawi, but there we focused more on differences in gene expression. And so one example of the variation that we found in Malawi is that we wanted to explore how gene expression might relate to foraging types. And so we took 50 different species that come from different foraging guilds, and we broke them up into whether they were breeding in the water column on algae or down in the mud or feeding on other fish. And we then compared those groups as to how much of the UV ops and they expressed. What we found was that fish that were up in the water column had higher UV ops and expression fish that were down and feeding in the mud or feeding on other fish had lower UV ops and expression, suggesting that UV vision is important for foraging, and that shouldn't surprise anybody. Even these algal feeders often will opportunistically feed on zooplankton when they are available. So because UV vision is so important for contrast and detecting these little zooplankton, it makes sense that these fish would maintain their UV sensitivity. The last example I want to talk about for functionality has to do with thinking about the co-expression. Again, this co-expression is fairly complicated because again we have one pair of genes that increase in co-expression dorsal ventrally and another pair that increases co-expression from temporal to the nasal region. And we think that these gene expression differences are the result of the fact that different parts of the eye are looking at different backgrounds when they view their world. And so because the ventral retina is looking up against the downwelling light, that downwelling light is quite broad and it might be advantageous to actually co-express genes because they can better collect the light, the broader light. And this variation might also be beneficial for looking at the space light. And so you can see now in black here's the downwelling light and in black here is the space welling light and you can see the co-expressing gene is broader and actually will detect more of the background light, giving the fish an ability to better detect contrast. And we did some calculations to kind of confirm that there was an enhanced ability to detect contrast with this co-expressing combination. Now, not only as the co-expression spatially variable, but it's not totally random which genes are being co-expressed. So if we look at how the genes are laid out in the genome, these three genes are next to each other, these three genes are next to each other and this gene is on its own chromosome. And the genes that are co-expressed are actually fairly far apart. So these two genes are co-expressed, these are co-expressed and these are co-expressed. Sorry. And so it's the spectral nearest neighbors that are being co-expressed, suggesting that has evolved to produce the visual pigments that would best tune the collected light when they're co-expressed in the same cell. So I hope that gives you some confidence that what we're seeing is not totally random and that it is perhaps beneficial for the fish. Well, at this point, we had done as much as we could to try and explore the variation in ecology, and we still couldn't explain a lot of the variation that we're seeing. And I didn't know at that point if we were just not very good ecologists or whether there were some ecological events that maybe happened some long time ago that we no longer could observe. And so we decided at this point to kind of switch gears and start to try and focus on genetically how these visual systems were shifting from one to the next. And again, remember that they're modifying the developmental program of the ancestor, which goes from short to medium to long as they grow from larvae to adults. So cichlids are amazing for another reason and that is they can actually be crossed between genera. So we started making crosses between different genera that had different expression combinations. And that allowed us to use classical genetics to try and identify the genomic regions that were important for turning on these different ops and genes. And so here's a cross that we made between this species that expresses the blue and red gene, and this species which does not. And what you can do is you can genotype a whole bunch of markers. So each of these little black tick marks is a different marker on each of the chromosomes, so chromosome one, two, three and four. And at each of these markers on the different chromosomes we could test whether those markers were or were not correlated with ops and expression in the F2 of this cross. And so using those F2 it kind of mixes up the whole genome and allows you to determine whether individual parts of the genome are important for a particular trait in this case that trait being ops and expression. Now the opsons are on this chromosome here and this chromosome here. And we found some pretty significant factors that were important say for expressing the blue ops in here, and for expressing the red ops in here. And so we wanted to see whether we could actually figure out what genes were doing this and genetically what was the mechanism for impacting this. And because these genes are far away from the opsons, the genetic change is not some cis regulatory change of the ops and promoter it has to be some kind of gene that's going to act on the opsons. So probably it's going to be some transcription factor. So we narrowed down this region here by adding lots more markers and adding more individuals, and this was work done by Jane and the lab. And we were able to narrow it down to about a megabase region, but it's still contained 180 genes. And we started then to use transcriptomics and some other methods to try and identify which gene in this region was going to be a transcription factor important for opsies. The idea is that there will be some gene in this group where when it's up regulated turns on the ops and and when it's down regulated down regulates the ops. So it should be correlated with ops and expression across many different species. And what we found was a gene called retinal homeobox one, which was indeed highly correlated with the blue ops. And when Jane started sequencing this gene, she found that some species had an intact regulatory region upstream of where the gene was, and others had a big deletion there, about a 400 base pair deletion. So we then decided to look across the diversity of the African species, the lake Malawi species that I've shown you a couple of times now. And across both the sand and the rock dwellers, we found that there were species that had this region intact and others that had it deleted. And when that region is intact, you can see if an individual has either both its copies, good or even just one of its copies having that intact region. The blue ops and is highly expressed, but when that region is deleted, shown by these white boxes, the blue ops and is quite weak. And so across the whole Malawi flock, we can sort of explain a large amount of the blue ops and expression by the genotype or the presence and absence of this regulatory region upstream of this gene. And so we can actually explain about 60% of the variation with this individual locus, which genetically is a huge effect. Well, we went on to try and explain and find the genes that were in some of these other regions. So, then was a postdoc in the lab and he was able to show that tbx2 tbox2 was a transcription factor important for turning on the red gene, and actually turning off some of the green genes. And he did this work in collaboration with Laura in a Roops lab at the NIH. And then Pratima was able to show that MITF is the gene that explains this contribution to the blue ops and expression. And again, it works in combination with this RX1 gene to control blue ops. Now you'll notice MITF has a smaller effect than this gene, but we wanted to test whether these genes actually do indeed have the effects that we think they do. And so we've started to work now with Scott Junty, who recently came to the University of Maryland and has the capabilities to make these CRISPR-Cas9 mutants. And his lab was making a mutant of the MITF gene and it happened to be the exact one that we were interested in. So we've been raising families to try and test the effect of having the coding sequence of this gene mutated. And what we find is that if we have homozygous mutants, heterozygous mutants and homozygous wild type, they do actually differ by the amount of blue ops that is expressed. And so by actually knocking out the MITF coding sequence, we actually get more blue ops and expression than we see in the wild type. Now the effect is a little bit weak, and so we're trying to repeat this in some more families. But we think that this supports the idea that MITF is controlling blue ops and it gives us hope that we can knock out other genes to try and explore their effect on how ops and are expressed. Now, for all of these locus or loci that we've discovered now, we started looking to see why it was that these genes had an impact. And we found that each of these three genes actually had some large, either insertions or deletions. And so for this RX1 gene, we found again, some of the species have a deletion, but actually some of the species actually have an insertion. Similarly for the TBX2 gene, the causative effect is this deleted region, but we found that some species have this insertion. And for the MITF region, the effect is actually caused by an insertion in this locus. Now these insertions are not just random sequence, it turns out that they are transposable elements. And we think that then what's actually happening in these cycloads is you get a transposable element pasting itself in, and then it goes to cut itself out and it creates a gap. And so by having these cut and paste transposable elements, it's creating regulatory variation that is explaining ops and spectral tuning. So here's a little cartoon that shows this network, this genetic network. And we've got our RX1 and MITF gene that are both acting on the blue opson. We have the TBX2 gene that's acting on the red opson. And also we think on a regulatory region for the RH2 opsons, the green opsons. And so we think what's been happening over relatively recent times is that there's transposable elements kind of hopping around the genome that are cutting, being pasted in and then cut out. And that then creates these mutations that cause this whole network to be modified. And so this seems to be something that might happen and explain sort of the rapid diversification that we're seeing. So before I end, I want to convince you that we're not just genetic nerds that spend all our time in the lab. We also like to think, we do go to the field, but we also like to think about behavior. And so Danny is a student who recently graduated and he really wanted to think about cycloid behavior. I kept telling him I don't do cycloid behavior, but he forged ahead anyway. And he was able to train cycloids to recognize blue targets and to tap them and to get rewarded for tapping them. And he then started asking these fish, can you tell the blue target apart from say gray targets of different brightness or even other colors. And so here are some of his experiments to show you kind of how the fish behave. Again, the fish will tap the target and then get rewarded. You can make it a little bit harder. Sometimes the fish has to think about it then. And he came and asked the fish to look at and choose between different colors. And so this was a way that Danny was able to prove that cycloids do indeed have color vision. They aren't just using all these visual pigments for brightness, they actually can use them for color. And working with Karen Cheney and Justin Marshall, he was able to actually quantify cycloids' ability to discriminate, say blues from reds going through purple. And he could figure out when cycloids got confused and use that to quantify how good their color vision was. And it turns out cyclic color vision is about eight times worse than human color vision, but still quite sufficient for discriminating the colors that are so important for their many activities in the wild. So with that, I can perhaps answer some of these or discuss some of these unanswered questions that I think would be very interesting to ponder more about some of the things we don't know about cycloids. There's still so much to learn. I want to end by just thanking all of the people who did the work. Several of the folks working on genetics. Miranda and Melissa are now trying to make some more crisper knockouts. We've had some great bioinformatic help. Zika is a new student working on some more behavior in the lab. Again, a huge shout out to the wonderful vision community who've been greatly helpful in our thinking about the cyclic system, but have also involved me in thinking about coral reef and even deep sea fishes as well. And so thanks to all of those folks and also thanks very much for all the folks who have funded our efforts. And so thanks for listening. Thank you very much Karen for this fascinating talk and thank you for stopping screen sharing already. I have a couple of questions of mine but at this point I would like to remind to the audience that they can go ahead and post their questions in the chat or join us in the very Zoom room that we are currently sitting in and I have provided the link in the YouTube chat. So the first question is from Tom Badden as cyclics very obscene expression etc do spectrally dependent behaviors and up shifting accordingly. I think it was the last word. What what came before accordingly. Sifting. Oh sifting. But I do not know what he means by up maybe he will join us soon and he will clarify that. He says end up and up accordingly. So, I mean there, the opposite expression variation does correlate to some degree with behavioral differences. And some of that has to do with foraging but there's many other behaviors that all of the cyclics do. So, you know, I can explain the fact that they've got UV sensitive cones or not, but all of the other variation, I have no idea why it's occurring. It's not correlated with anything that we've come up with it's not correlated with cyclic color it's not correlated. I mean there's some correlation with light environment. Beyond that, there's really, you know, in Malawi, all of those types occur in Tanganika all of those types occur. Why, I don't know. Yeah, it's really interesting. What one question I have is, so when you take them from the wild and you put them in different light conditions in the lab. Do you observe that this change is also age dependent, in a sense that does plasticity go away. The older the animal is. So I should say, so the species that have the short wavelength combination, they remain plastic through their whole life. We can move adults back and forth and they're still plastic. They will be more plastic if they've gone through different environments through development, but they still remain plastic. However, the tilapias. Well, we don't actually know about tilapia plasticity early in development. I should ask Miranda that question, but the adults do not seem to be plastic. And so it seems that some species are more plastic and others and the ones that kind of retain the larval combination of option expression are the most plastic, which may make sense because if plasticity is something that arises early in development, if you keep those early genes on, you're going to retain that plasticity. Yeah, that is right. I see where you're taking this. As there are not any questions right now in the chat. I would like to remind people, I mean, Tom is already here. I would like to remind people that they can be joining us in the zoom room for a postdoc informal get together. The live transmission may be in 10 minutes from now. I would like to remind to the audience that this was the last but certainly not least talk of the season of the Sussex Vision Seminar Series and we will be continuing beginning of September with a talk from John Dowling. And yeah, like Tom if you want to continue with what you were saying earlier. But if not, I would like to ask a really naive question like do we know anything about bipolar cells and RGCs in secrets apart from like gradients of topographic distributions and so on. No, nothing. Okay, very interesting. Yeah, so I would love to know right what's happening developmentally as these options are shifting on and off. Presumably, well, Russ from Fernald had shown that the cones, they're not dying so the cones are remaining stable, presumably the rest of the neural pathway is also stable but I have no data to support that. And you did mention that when we have like these changes, they are also retina region dependent right, or did I get this wrong. Yes, you're correct. So we can. If we change the room lights right the the ventral region of the retina will change. We can actually we did one experiment Brian did this, where you can actually use fluorescent lighting from below. And then we could induce a small change in the dorsal retina as well so it really depends on the light which direction it's going and where it's falling in the retina. Very, very interesting. Yeah, so maybe I can just chime in. So, yeah, that was really interesting. So I just sort of follow up for my question. I mean, I think you're sort of answering it in. I guess the answer is we don't know but let me see if it really is so in fish or let's say in lower vertebrates whatever that means. So this this idea that keeps popping up that certain behaviors are linked to certain cone systems at the circuit level right so I think an extreme example would be maybe the UV dependence of break up trade take out the UV cones that goes. Right. So you could argue that, therefore those cones are responsible for that behavior and if then the UV cones were to shift spectrally or otherwise, the entire behavioral program would shift automatically. So, in taking that then in the reverse, maybe using these fairly stereotype behaviors, you could get at the circuits that might be existing in the fish. Right. So, so for example, the fish after source of writing reflex right which happens to be spectrally approximately like the red cones at least in supernates. Right. So, if you now have a sicklets that is not expressing the red option is those are writing behavior like what spectral sensitivity with that then follow with it. It doesn't go away. Does it use a different cone because that would then need a different circuit. Yeah. So I'll tell you a different behavior that we did a little bit of work on we've done some op the motor response work. And for that work we took fish and we split them between the lab fluorescent lighting and these fluorescent metal halide lights. So we had fish with more LWS expression and fish with less LWS expression. And we were able to show that the optomotor sensitivity tracked the amount of LWS expression. And so, and that has to be an only one cone type. So, it didn't matter what how much green ops and was expressed it wasn't sensitive to the single cone options. It only depends on how much of the LWS expression, how sensitive they were in the in to that optomotor so. But the cell types are still there. And so it seems to me that it can't be just one ops and gene that determines a particular behavior it has to be something wired to a cone type, and because we can put multiple options in those cones. So I'm not doing both sides. I realized of the same answer. It seems like it has to be something that's not spectrally dependent only. Yeah. Now I can see what you mean. I mean, this would be a very unhappy fish, but if you take out the red ops and stick UV option and then see if they suddenly track UV stripes but not red ones that might give you a way too difficult an experiment to actually do it but. Well, I think that, you know Fabio Cortesi and Justin Marshall are making knockouts in, you know, anemone fish, and I think are intending to do some of those kinds of experiments. So I've been trying to knock out the UV gene but have not yet succeeded and so perhaps we'll be able to do those experiments at some point. So I will step right in, even though I have waved my moderator rights at this point and I would like to communicate to you a question from Cendric van der Berg that appeared in the chat right now. What do these environmentally induced changes correlate to in terms of delta s indetection or discrimination behavior, both luminance and color, are they greater than one. So I'm going to answer it a slightly different way. When you co express options in a double cone cell that will increase contrast by two to 5%. So if Justin is listening, he will say that's a pretty small impact, but as an evolutionary biologist, I would say a two to 5% increasing contrast is a huge effect. It might allow you to detect a predator, you know, one foot closer or one foot further away than you might have and that would maybe make the difference between whether you're dinner or not. So even small contrast changes could have a big impact. Some of the some of the effects on color can be three, four and five JND. So they're not insignificant impacts. One really nice atom you want to go ahead. I can but you had a question. Really nice one like how deep are these legs like what depth are we actually talking about, even for the deep inhabitants. So the lakes are 700 meters deep. However, being near the equator, they actually go and not sick below 200 meters because the water doesn't turn over. So, so the fish only live in the top 200 meters. Because when we are there and we're scuba diving and there's no anechoic chain or no, you know, pressure chamber anywhere close. So we have not really sampled deeper although there are sicklets that live quite deep and presumably have different visual systems I think there's a group from Hull in that has done some work on the deeper species and they've done some transcriptomics on that on those fish. Thank you very much. I just wanted to say hi real quick. I really enjoyed your talk. Thanks a lot for coming. It's really good to see where the sick would work scone to I haven't had an update in a while now. Great and you're in the UK now. No, I'm back in Sweden again. Okay. Okay, lots of cool stuff going on there. Oh yeah. Only for only for me right now everyone else is on vacation. I'm the only one working. You're the only one without the summer house. So maybe next year I'll have one. I haven't integrated enough yet. So you think you're there for a while then. Yeah I think so I have funding for five years and my wife actually has a job she really likes this time so we're probably going to hang out for a little while.