welcome back everybody uh we're here today with dr rex dunham who's going to talk to us about fish genetics and all the advances made in catfish genetics dr donald's been working in the area of genetics for over 45 years and has been working with collaborators around the world he specializes in a host of genetic techniques including quantitative genetics traditional selective breeding molecular genetics and genomics hybridization transgenesis gene editing xenogenesis and reproduction mostly with catfish he and his research have genetically transformed the catfish industry twice during his career creating a better fish for our farmers and with that rex i'll turn it over to you okay thank you very much it's a pleasure to be speaking to all of you today as you can see our topic is catfish genetic enhancement traditional and bio technological approaches for food production while protecting the environment we'll be primarily focusing on catfish but to complete the story i'll also be presenting a couple of examples with other fish and from other laboratories and also dr klein reminded me to tell you that although this is focusing on catfish uh all of these approaches can be used in uh most all not all but most all aquatic organisms okay so when we build a house we have we use more than one uh type of tool and the same thing with genetic enhancement we have a variety a whole suite of genetic enhancement tools at our disposal ellis prather on the far left and homer swingle on the far right did the first catfish genetics research at auburn university and they didn't even realize it because at that time they were evaluating different species of catfish to see which one was best for aquaculture and species are indeed species because of genetic differences so when we evaluate different species we're actually doing a genetic evaluation once we've identified the best species the first step in a genetic improvement program is to identify the best performing domestic strains strains and families within strains affect the success of all genetic enhancement programs once we've finished that step then we can begin a focused genetic enhancement program we have long-term options and short-term programs selection for body weight's been very successful across many different species although this is a semi-slow process we can double body weight through about eight to ten generations of selection by 2003 because of the research done at auburn either through direct releases or farmers learning how to do their own selection and also the creation of some catfish companies based on our research about 70 percent of the industry was using uh selectively bred channel catfish now when any genetic enhancement program if we're trying to help the the farmer or the industry we have to be aware of any uh you might say side effects that the program may have on other commercially important traits in the case of selection that's correlated responses to selection and if we select to increase body weight there's some positive benefits the fecundities increase the carcass yield increases disease resistance is a little bit better but unfortunately the tolerance of low oxygen decreases but on the other hand we can overcome that problem through mechanical aeration to make sure that there's adequate oxygen in the water [Music] this illustrates the increase in the kilos per hectare produced by catfish farmers in 2003 versus 1980. during this time period they were able to increase production by threefold so this is a positive impact on the environment and takes pressure off of natural resources so we're producing three times of food on the same uh the same footprint now this is due to farmer innovation we have to give them credit as well as increase farmer skill but also due to impact from research and so there's probably a significant credit here due to improved aeration techniques as well as improvement in genetics it corresponds with increased use of selectively bred catfish in 1974 roger young and r.o smitherman demonstrated that in commercial densities and ponds that a channel female catfish crossed with blue catfish males had improved growth carcass yield and and some other traits over time we learned that this particular hybrid has increased growth rate lower feed conversion improved disease resistance better survival better tolerance of low dissolved oxygen higher harvest stability improved processing yield and so overall the phenotype and production overall value is greatly improved by making this single cross about 20 years ago or or more there were field trials in alabama that showed that under farm conditions these hybrids grew uh produced twice as much as channel catfish and had much better feed conversion efficiency nowadays we have the event the advent of much more intensive catfish production uh increased horsepower for aeration just a few farmers using in pond raceways and several farmers using split ponds the hybrid is an essential component for these systems to work as they have much better survival under these conditions compared to the parent species so when we consider this whole suite of traits that are improved uh the u.s hybrid catfish is probably the best example of genetic improvement in aquaculture that there's ever been now we can have a fish with the best genes in the world but we're not going to be able to impact the farmer unless we're able to produce adequate number of fingerlings for commercial scale use so in 1966 john judisee and the u.s fish and wildlife service already knew that this fish had aquaculture potential but the reproductive isolating mechanisms between those two species prevented commercial scale aquaculture so it was almost 30 years later that we finally had small scale production commercial production of hybrids and this was based on uh uh common uh car pituitary extract technology developed at auburn and there was only one farm gold kiss that was trying this technology so for about an eight year period we're fluctuating between a million to five million hybrid fry produced in the industry then about 2000 we developed a new technology based on lhrha induced spawning and that coupled with some other factors allowed us to double and triple hybrid embryo production allowing commercial scale application finally of this great hybrid key components included bag spawning better better hatchery and fertilization techniques and another key factor in 2005 auburn partnered with eagle aquaculture to commercialize the hybrid technology and um that gave an example that convinced other farmers that they should they should try this now eagles very organized had a factory type assembly line to produce hybrid embryos so when you look at this graph about 2005 you see a huge jump in the production of hybrid embryos and since that time a steady increase in hybrid embryo production based on our lab's technology last year they estimate that 350 million hybrid catfish fry were produced so uh again now we revisit this change in farmer efficiency and in 2020 if we look at the production records catfish farmers are producing eight and a half times more catfish per hectare than what they were in 1980 so they continue to improve you've got producing more and more food on a smaller footprint theoretically that also is more efficient and makes a smaller carbon imprint uh takes pressure off of natural resources so aquaculture actually uh becoming more and more environmentally friendly and also if you look at that curve look at about 2005 and we start to see a steep incline that corresponds with the increased adoption of hybrid catfish so we have other factors including the intensive systems that are working hand in hand uh with the hybrids to improve production but we can do better uh the strain of channel and blue parent has a strong effect on the production of the f1 embryos here's an example where we compared au1 and au7 over uh five uh six consecutive years and au1 always produce two to three times more hybrid fry uh and that was of course that's due to the females but uh unfortunately all males aren't created equal either as well so we have an impact due to the strain of blue used also then we can take those strain differences and we can improve uh hybrid embryo production further by selection for females that are highly productive we can also continue to improve the performance of the hybrids in regards to growth disease resistance carcass yield so different genetic types of hybrids are improved but there's still differences there so strain apparent affects hybrid performance both of these hybrids here uh are improved compared to the parents we're using two different types of males and the one with the rio grande grows 40 percent faster than hybrids produce with the other strain of males one thing we're working on now is using combining abilities evaluating that to determine what's the best way to continue to make a better and better channel blue hybrid in this example the key is if you look at the pi diagram on the left the blue shaded area is a general combining ability due to the dams or mothers and on the the red shaded area is the combining ability due to the blue catfish sires or males and what this tells us is that if we select for faster growing channels and faster growing blues and then hybridize those that will also increase the the hybrid growth rate we have a little bit different result when we look at dress out percentage in this case on the left you see that the green pie is the major genetic component this is the specific combining ability what that means is what we have to do is evaluate different pairs or sets and select for pairs of fish that produce the better performing progeny and that's how we would improve hybrid performance for this trade for many years when we write grants we complain we're using a wild fish we really need money so we can make a genetically superior growing fish for most major aquaculture species we can't spin that story anymore because if we look take wild catfish then we domesticated them then we selected them and then we used hybridization and other programs if we compare the production of the wild fish to what we have now it's a 10 to 20 fold difference but we can continue to do better and of course new aquaculture species have not gone through this story yet we're trying to making the hybrid progeny is uh very laborious and tedious so we're trying to look at things like xenogenesis to make uh embryo production more efficient xenogenesis is a method of reproduction in which successive generations differ from each other it's analogous to human surrogacy in that case we can have one woman carrying the embryo that has the genetic material from another mother i mean from an uh from another female so uh the baby is not genetically related to the uh birth mother at all with xenogenesis we have so that's what we mean by successive generations are are different in the case of xenogenesis it's the same concept but the host is carrying the gonads and the gametes from the donor uh so to do that we sterilize a the host generally through triploidy triploids are sterile the gonad development is atrophied the gametes are not viable and then we isolate stem cells from the blue catfish in this example and we introduce those to the sterilized host and in this case what we're trying to do we have a xenogenic channel catfish male everything about him is channeled catfish except for he produces blue catfish sperm therefore the channel catfish female on the left she recognizes him as normal mates and the product is 100 uh hybrid uh hybrid embryos uh so that would be an improvement on our current technique we can transfer those stem cells at different life stages blastula fry even sub-adults but the data to date indicates it probably works better with fry sometime shortly after hatching now the japanese and others have produced xenogenic salmon that when mated together produce pure rainbow trout and there are xenogenic zebrafish males that depending upon the stem cells introduced we're able to produce sperm from silver danio goldfish and even mud loach so we did have success uh here on the left these are hybrid fingerlings that were produced by mating a xenogenic channel catfish male with a channel catfish female so you have two channel catfish parents but all the offspring are hybrids on the right to show that i'm not lying those are the control channel catfish and you can see the morphological difference now the problem was we were having more and more success but we weren't getting the fertility and the fecundity that would allow commercialization so we're trying to overcome that problem you have to have high enough transformation rate colonization of the cells and proliferation one aspect we've been looking at is the correct uh time and development that will give you uh the highest colonization and proliferation of those donor cells this is actually a graph from xenogenic white catfish but we have almost an identical uh illustration with channel catfish so we can uh mark these cells with fluorescence and then later 45-90 days later measure the rate of proliferation of those cells and what we see here is if we injected the stem cells anywhere from 0 to 12 days after hatch when you examine these slides you can see individual slides fluores cells fluorescing or sometimes they stick together in clusters so we're looking at the data two different ways but in both cases you can see between four and six days post hatch we get the greatest number of stem cells that take hold and start growing which theoretically will result in more fertile morphic and xenogenic fish okay the next step that we want to look at is molecular genetics the latest technology revolves around snp single nucleotide polymorphisms where we're looking at the genetic variation at each individual base this technology allows us to do what's called genome-wide studies where we can find in the genome which chromosomes chromosome areas have the primary genes for a certain trait in this case we're looking at resistance to eduardo ictaluri and we see that the it appears that the main genes that affect this trait are on chromosome 1 12 and 15 of channel catfish similar experiment but for columnaris and we find there's a different set of genes that have the greatest importance for survival located on chromosomes 7 12 and 14. so the next step that we're working on is actually doing marker assisted selection and genomic selection where we select for these dna markers and in some cases not all cases that will actually allow you to make better and faster genetic improvement we've done a lot of genetic engineering with fish and it looks quite promising the most common type of research is where in the past is where growth hormone genes have been transferred with catfish we can increase growth 50 percent uh maybe even double and triple uh growth rates with this technique but with a wide variety of fish um you know 20 but in some cases as much as 10 to 30 30-fold increase in growth has been accomplished but of course the latter is not a very common result in the case of catfish and salmon that growth hormone increased growth horm affects muscle structure so these fish have increased number of muscle fibers glycogen globules mitochondria but a reduced number of fat globules just like selection we have to be aware of what other traits are affected when our goal is to transform one trait in this case you can have plyotropic effects where one gene affects more than one trait and the growth hormone channel catfish have uh better survival at cold temperatures growth hormone has a role in osmoregulation the transgenic catfish also have better resistance to high salinity another promising area is the transfer of anti-microbial peptide genes if we when we transferred sucropen to channel catfish we were able to increase bacterial disease resistance in some cases two to four fold uh recently uh we have looked at we took different types of anti-microbial peptides and uh compared them in in vitro uh sucropen catholic sidon pluricidin and an ampicillin antibiotic and the most promising one was alligator catholicidin uh we've been able to produce transgenic fish with the cathode side and gene and uh in this case this data comes from a challenge uh with uh where they're being challenged with columnaris and the catholic side and transgenics have four to 4.5 times
greater survival than the non-transgenic controls so uh this these techniques can increase production efficiency and profits these days we're always talking about animal welfare but are we really serious about it so this is a technique where we can make healthier fitter animals that have better animal welfare for aquaculture but as you know we're slow to accept it in society so by definition genetically modified organisms are not organic but if they reduce or eliminate chemical use and antibiotic use is that not beneficial is what we call truly organic things to think about now the goals of consumers can be are different often than producers rather than production consumers are interested in healthier more nutritious and tastier food so one thing we're examining is omega-3 fatty acid levels and as uh as you know uh omega-3 fatty acids have many important human a long list of uh human health uh benefits uh catfish freshwater catfish have very low levels of omega-3 fatty acids compared to salmon anchovy and tuna so we've been transferring the saturation elongated genes in an attempt to increase omega-3 fatty acids in channel catfish and uh we've had success we want to do better and continue to research in that area we've been able to increase the omega-3 fatty acid level 30 to 100 percent another important aspect of this is the ratio of the omega fatty acids that you consume also have health effects so this also alters the omega-3 to omega-6 ratio in a uh in an improved manner a more healthy manner we're not doing this type of research but another interesting example the canadians transferred the anti-freeze protein gene from winter flounder into atlantic salmon now in the arctic in the antarctic the water is actually below zero c because of the salt therefore the blood fish would die in those environments but as you know there are species that survive there and that's because of the anti-freeze protein genes the canadians goals i consider risky transgenic research because in this case what their goal was was to make a salmon that could be cultured closer to the arctic circle by doing that you're expanding the geographic range and essentially making an exotic species and if we're worried about environmental risk exotic species are probably the most risky example in the fish world that we have but they were able to produce salmon that made this anti-freeze protein but it didn't affect cold tolerance they also experimented with goldfish it also did not lower the lethal temperature they could survive but it did increase their survival at the low end of their their natural temperature tolerance 20 years ago the first commercialization of transgenic fish is ornamental zebrafish and since that time several other species in this case fluorescent protein genes from jellyfish were transferred and you create these new colors and black light at night you have neon fish swimming around glowing in your aquarium so now excuse me the latest development is gene editing where we have targeted genetic mutations so we're trying to disable genes instead of inserting genes and we have crispr technology and others that allows us to take scissors to dna and delete and disable these genes myostatin is a muscle regulating protein that prevents us from continuing to grow and grow and grow during a mammal's lifetime and also it slows down the growth of fish there are natural mutations of this gene where it doesn't function properly or or at all leading to and these mutations have been found in cattle and dogs where you have this double muscling arnold schwarzenegger phenomenon and there's actually been a handful of humans that have uh mutated and the myostatin was disabled so the goal here is well what happens if we delete the or disable the myostatin gene from channeled catfish are we going to end up with a meteor uh larger channel catfish so our lab members have accomplished that and uh during the first uh 30 days of growth we obtain about a 30 percent increase in growth rate and if you look at the two uh histology slides in the center the one on the left is a control the one on the right is a myostatin mutant and it's obvious to the naked eye that the myostatin mutant has about 30 to 40 percent more muscle fibers now we've developed f1 fish with this mutation if you look at the yellow highlighted area i pinch myself and wonder because the mutants at low density in ponds are growing three times faster than the controls so we're repeating that with a higher density and growing on to food fish another growth regulator that we're looking at is mc4r which has a role in fat metabolism as well as growth and we've uh learned and others have learned that it actually has a critical role in fish reproduction so if we knock out this gene uh the fish uh become become sterile and uh the the data that we and uh one other lab has indicates this is it can actually be a major uh regulator in the hpg axis additionally by knocking out this gene [Music] about 50 to 70 percent increase in growth can be achieved as well as 50 percent better feed conversion efficiency and uh and uh this in the in regards to growth uh this mutation functions in a recessive manner if you look at the yellow highlighted area again the homozygous individuals that group is the one growing the fastest the second one down is a heterozygous and they grow more no differently than the than the controls uh so a result that surprised us is the mc4 knockouts uh on le on the left if you look at epa that omega-3 fatty acid by knocking out mc4r we double the the epa level which if we insert the elongated gene from salmon we get the same doubling of that uh fatty acid if we look at dha we by knocking out mc4r we get a significant increase and uh but it's not quite as effective as knocking in the elongates so we have these uh excellent results for genetic engineering and gene editing but are we going to impact aquaculture are we going to impact food production one concern is environmental risk in order for there to be a risk these fish would have to be more fit in regards to reproduction foraging ability predator avoidance swimming ability in the natural environment the data today strongly suggests that they are less fit and would likely be outcompeted in the wild but with today's societal uh caution and attitude that's not going to be enough in order to use these fish we're going to have to control or confine these so how do we do that not only for transgenics but other controversial genetic types used in aquaculture so physical confinement in my opinion is not enough because we all learn from jurassic park that even though physically confined some idiot could steal the eggs and spread them try to spread them throughout the world so we want a technique where we have total reproductive control genetics is [Music] maybe the best route to accomplish that so the fish can only make when the hatchery manager intervenes one approach is actually transgenic in this case what we're doing is inserting a gene usually a short hairpin rnai a gene that knocks out or prevents the expression of primordial germ cells which are critical they're the precursor for gametes so if you destroy those the fish cannot produce eggs or sperm this is obviously the top fish this is normal sexual development of a channel catfish male bottom is obviously a female here we have some fish with the transgenic knockout females either the ovaries do not exist or there's no ova developing inside here are a couple males one of them has no testes at all and the other one a greatly atrophied test testes so we can't have a farm if all of our fish are sterile so we have to be able to reverse that sterility in a sample of embryos so that we can grow some brood stock so these constructs these transgenes are designed so that we can add a compound to the hatching water that will turn that sterilizing gene off and allow normal development and the top fish in this case we're using a gene that we can turn off with application of copper sulfate so the top fish is a transgenic fish hatched without copper sulfate low levels and the bottom fish is a a transgenic fish with a sterilizing gene but that gene's been disabled by hatching and copper and we see the fantastic ovarian development one problem though these transgenic fish without gonads apparently they're important for growth so we saw a 25 decrease in growth and survival this may have been due to the fact that they were in head-to-head competition with more aggressive controls and we have preliminary data that indicates we might be able to correct this problem through selection another option is gene editing again but in this case targeting reproductive genes followed by hormone therapy to restore fertility again we use crispr technology to target and mutate those reproductive genes such as lh fsh and gene rh so then we can take normal spawning hormones like hcg lhrh and spawn the fish they can only reproduce through the intervention of the hatchery manager again some of these fish can produce can ovulate and spermate but the gametes are not viable so on the left we have eggs from a sterilized female and they were non-viable so the fungus is attacking and destroying them on the right we have a sister who's been hormone-spawn and now the eggs are fertile and developing normally again we need to be concerned uh that we're affecting other important commercial traits on the left we have different hpg axis knockouts and they are growing as depending upon the knockout they're growing as well or better than controls for added redundancy and security we tried making some double triple and quadruple knockouts in this case some of the fish actually are growing slower at the same rate or some triple knockouts are growing faster than the controls of course uh so we can uh we're very close to having total control of these fish um but the next major hurdle is public acceptance the issues there are politics government regulation environmental risk education and food safety so there's different terms transgenic gmo genetically engineered bioengineered and then of course gene edited this technology can benefit the farmer the processor and also the consumer but the controversy is is it safe so the uh when we apply one one concern is well if i eat a growth hormone fish is it the same as taking steroids well we apply insulin and growth hormone to correct medical defects we do it by injection or nasal sprays instead of oral because the digestive process is going to destroy the dna otherwise if we ate a carrot or a pig we'd become a pig man or a carrot man and obviously that doesn't occur now these major scientific organizations uh u.s national academy of science royal society of london fao who and even the european food safety authority which bans all genetically engineered food all of these organizations have done analysis and concluded that in all almost all cases transgenic there's no logical reason why there would be a safety issue with transgenic meat but there is one thing that we do need we need to treat each be responsible and treat these on a case-by-case basis uh the main safety consideration is allergenicity so if we took a peanut corn or shrimp gene that produced a protein that was responsible for the allergic uh response and people who have that problem then we could theoretically produce a transgenic food that would be a health risk for those particular people another pro hurdle we have to overcome is the anti-gmo movement and this is a career this is a job if you go online these organizations are soliciting donations even if they do not want the public to accept this technology some of them have 30-year careers if the technology was approved and accepted they no longer have a job now another problem we overcome is human nature is to be leery of radically new technologies this is a painting from 1802 that's propaganda material used from the 1802 anti-vaccine society most of us would probably agree that vaccine technology has had a very positive effect on humanity but the message they're sending here we have a line of people from 220 years ago they're getting taking vaccine and within minutes they start growing horns and they have cows growing out of different parts of their their body and obviously that's ridiculous even the automobile was met with resistance there were laws or proposed laws when they first came out saying you needed to have someone walk in front of the car waving a red flag some places had two to four mile per hour speed limits there were laws proposed that said if you saw a horse coming you didn't want to make the horse afraid so you needed to hide the car take it apart wait till the horse passed and then reassemble the car and continue your journey obviously there's no reason to have cars if you have those laws and but perhaps in retrospect maybe we should have been more concerned about the automobile in some cases we have political agendas to overcome uh the alaskan congressmen openly admit they're trying to protect their clientele their anti-aquaculture and of course therefore they're also anti-gmo because they're trying to eliminate the competition for the commercial alaskan fishermen but a huge obstacle we're all involved in is public education the the vast majority of people really don't understand where their food comes from they don't understand biology and they have even more trouble with genetics the two ladies on the right are two of my daughters the one on the far right is amy when i uh wrote my first book she was 16 i gave a copy of the book to all of my children about two months later i asked her what she'd thought about it now she ended her education with an associate's degree and she was a she was a straight a student her entire academic career so she's an intelligent educated person she also has a very wry sense of humor and uh must have had a bad experience with freshman biology she told when i asked her if she how she liked my book she said well dad i opened the book the first word that i saw was allele so i closed the book so this is the education that a large part of the public has been bombarded with through the years another interesting example is the jimmy kimmel show had an episode where they interviewed west coast uh grocery shoppers they asked them what they thought about gmos and of course they all said oh man that's so horrible i would never eat such a thing so then they asked him well what is a gmo nobody could define what gmo stood for thomas hoban in north carolina did a very interesting study regarding genetically engineered food in 1994 now he knew because of this propaganda he needed another way to evaluate the the data why people were answering the way they were so he asked two additional questions he asked them if you've ever have you ever eaten a hybrid fruit or vegetable and sixty percent responded no then he asked is it ethical to eat a hybrid fruit or vegetable about 60 percent of north carolinians and now this is a rural basically a rural state said no of course we have all eaten hybrid fruits and vegetables that's quite common he also did an international study and he asked a true false question ordinary tomatoes do not contain genes while genetically modified ones do if you just guess 50 of the people could get it right the canadians were the only ones that hit that 50 just by guessing united states was only 45 percent but austria france germany italy 32 to 35 of the people could answer that question correctly obviously they could have done better with a random gas or drawing marbles out of a hat so we do have a transgenic meat on the market now the first one as most of you are aware our growth hormone transgenic fish aqua advantage produced by aqua bounty so fish was the first one to reach the market fda approved these fish for consumption in 2015 but they were not marketed for several years because of labeling problems and laws that were activated to prevent their import about the same time the canadians also approve them and i don't have an exact figure but uh approximately 20 metric tons or so have been uh sold in canada aquabani eventually got approval to grow these in the united states and their first u.s grown uh transgenic salmon are uh on the market in united states have been on the market i believe for a few months now now in order for that to happen the u.s had to enact labeling laws uh the anti-gmo people are really upset with those laws because there's a variety of ways that you're able to label it and this particular technique instead of having a red label that says genetically engineered it's a green label that says bioengineered and obviously they disagree with that approach so now we have gene editing and some countries uh tightly regulate uh this new technology where we're instead of putting dna in we're removing dna we're disabling dna so some countries have fairly lenient laws others very strict there's two lines of thought sometimes regulation is triggered by the process and sometimes by the product in america the first thing that triggers regulation is process so for example you could have a fish with a natural mutation and if you were to recreate that very same mutation with gene editing and the two fish were identical even though they are totally identical the artificially produced one is going to be regulated the other one is not because of the process gene-edited fish have been commercialized for the first time uh aqua bounty again is there uh it's probably myostatin edited nile tilapia that are being marketed in argentina the so we have a suite of genetic tools that we can use my hypothesis prediction is in the future all of our aquacultured organisms will be developed by multiple uh sets of of genetic tools so instead of having so just selected fish or just triploid fish you may have selected triploid hybrids sex reverse transgenics etc to build the best house we'll need to use as many tools as possible to address different traits thank you very much it's not the end it's only the beginning
2021-12-06