cc  >> Cathy Vrentas: Hi, everyone,  welcome to Wednesday Nite @ the  Lab here at the UW-Madison  Biotechnology Center.  Tonight we welcome Terry Barry,  who has a diverse background at  UW-Madison including a  bachelor's in zoology and a PhD  in endocrinology and  reproductive physiologies, all  from here.  And his background is also very  interesting, he's done work in  many countries including a Peace  Corps stint in western Samoa and  work in Japan, Hawaii, Maine and  the Philippines.  And for ten years, up to 2006,  he owned one of Wisconsin's  largest fish hatcheries.  In 2006 he co-founded the  start-up company Aqua Most,  which is involved in  commercializing a specific type  of water purification system.  So please join me in welcoming  him to Wednesday Nite @ the Lab  today.  (applause)  >> Terence Barry: Thank you,  thanks for coming.  This is an overview of my talk,  I'm just gonna start out by a  real quick overview of what  aquaculture is, just to get you  up to snuff.  Then spend a little time talking  about the UW Laboratory of Fish  Endocrinology and Aquaculture,  which is what I call my lab.  And share a little bit about the  facilities, turns out it's one  of the best-kept secrets on  campus, I'm told, when people  see it. 

 And then what I decided to do, I  really mulled over how best to  approach this topic, but I think  what I'll do is give you a quick  overview of a bunch of stuff  we've done in the last four to  five years, versus going into  depth in any one thing.  I decided to do that just to  show you the breadth of the kind  of things that we do in our lab.  And then I'm gonna end up by  talking about our ongoing  projects.  So to start out with,  aquaculture is actually the  rearing of any organism under  controlled conditions, and it  includes plants and things like  mollusks and oysters and so on.  But I'm gonna focus specifically  on fish, which is what I do.  Unlike terrestrial organisms,  there's many, many types of fish  that can be reared in culture.  And aquaculture isn't just  rearing fish for food, it can  also be rearing fish to enhance  the natural stocks, to be  raising bait fish for sport  fishermen, and so on.  Raising fish for biomedical  research purposes and so on.  So all of that encompasses what  we call aquaculture.  There's many different kinds of  fish and the systems that we use  and the problems we have to  solve are actually unique to  each species, which is one of  the joys of actually working in  this field. 

 In my career I think I've worked  on 20 different species of fish  so far.  Some of them are carnivorous,  some are herbivorous, some live  in the marine environment,  others in brackish water, others  in fresh water.  They have fish that are raised  for food, for example, I did a  project in the Philippines where  literally they had to go out  into the mangrove swamps and  catch the seed stock that they  raised out to food size, whereas  other fish are actually, you  inject the fish with hormones  and you get the fry and the eggs  and you raise them yourself  completely in captivity.  Temperature requirements are  highly variable, we've got, just  on the right here, Wisconsin, we  raise fish that are categorized  as either cold water fish, cool  water fish, or warm water fish.  We once had fish in my lab from  the Amazon that we raised at 32  degrees Celsius and side by side  we had a bunch of lake trout  that we were raising at 5  degrees Celsius.  So fish have a lot of needs from  that angle.  In the middle here are two  really important aquaculture  species in North America.  The catfish is a very large  pond-based aquaculture industry  in the South, a little bit under  duress right now because of  foreign imports and the cost of  feed's going up.  The one on the bottom there is  an Atlantic salmon, you've  probably heard a lot about the  salmon aquaculture industry in  Chile, Scotland and so on.  And there's a lot of move to  actually diversify some  off-shore net pen aquaculture in  America as well, that would be  mainly that species. 

 And on the right are a bunch of  marine species, the one on the  lower right is something called  the "scatofagus argus," which  means the "roaming poop-eater"  and that's the fish that I  worked on when I was in the  Philippines.  (laughter)  So besides the diversity of fish  there's also a highly,  there's a lot of  different ways you can raise  fish and these are the four  principal ways to do that.  Pond-based aquaculture, which is  by far the most important way in  which people raise fish in the  world.  Raceway aquaculture, most of the  trout in the United States, in  fact 75% of all the rainbow  trout you eat are raised in one  small valley in south-central  Idaho.  And that's what that's a  photograph of.  The Snake River actually comes  pouring out the side of a  mountain, it's exactly the right  water temperature, excellent  quality, and they just raise  lots of fish there.  On the bottom left you see  netpen culture, this is where a  lot of marine aquaculture takes  place.  And on the right is a system  called recirculation aquaculture  where the fish are actually  contained in tanks and the water  is reused, passes through  biofilters, cleaned up and  passes right back through the  fish.  Each of these has advantages and  disadvantages, there's no one  right way to do it, it depends  on the fish that you're rearing,  the conditions you're under and  so on.  So just to put everything in  perspective, before I get into  Wisconsin aquaculture, by far  most of the fish raised in the  world are produced in China. 

 As you can see in this upper  right-hand graph, almost 67% of  all the fish raised in the world  today are produced in China.  23% roughly of the fish are  produced in Asia and the Pacific  region.  Leaving about 10% in the rest of  the world and in North America  we only produce about 1.2%  of all the fish that are  reared in aquaculture at this  point.  These are the countries where  aquaculture really takes place,  you can see China produces about  30 million tons of fish a year,  followed by India, Vietnam,  Thailand, and so on.  I've actually worked in both  Japan and the Philippines, two  countries which have a very  large aquaculture industry.  America's actually not far  behind the Philippines, we're  roughly about a half million  tons right now.  And it's expected that  aquaculture in the US could grow  to be as high as 1.5 million  pounds by 2025 if some  things happen that allow some  off-shore netpen culture to take  place.  The fastest growing, the one  shown on the right, areas of  aquaculture growth are actually  primarily in Africa and South  America.  Okay, in Wisconsin, the  aquaculture industry here is  characterized by its diversity.  About a third of the fish are  raised to enhance sports  fisheries, so we raise  Northerns, walleyes, that sort  of thing, by the DNR. 

 About a third are food fish, the  biggest species raised in  Wisconsin is rainbow trout.  And then there's a lot of  farmers that are trying to raise  yellow perch and walleyes and so  on.  My fish farm was a yellow perch  fingerling production farm.  And the finally, bait fish is a  very big industry.  The single most well-to-do fish  farmer in the state is actually  somebody that raises bait fish.  There's about 2,300 registered  fish farms in the state, but  that's kind of a misnomer  because somebody that has a  little pond in their backyard  that has a couple croppies would  qualify.  From an economic impact,  aquaculture in the state is  roughly between the sheep and  goat industry and the horse  industry at about $14 million in  total sales of fish.  When you do the economic  multiplier stuff like feed and  consultants and stuff, it's  about a $24 million industry in  the state.  Wisconsin is actually a regional  leader, and if you look at this  graph you can see that we are  the largest producer of  aquaculture products in our  region.  Okay, so that's sort of the  quick introduction to  aquaculture I wanted to give  you. 

 Now I'm gonna spend some time  talking about four projects that  we've accomplished in the last  four to five years.  That's not right, I'm wrong.  I'm gonna give you an  introduction to my lab here,  real quick.  What I call the Lab of  Endocrinology and Aquaculture.  So we have a lab mission  statement and this is it.  I am an endocrinologist, a fish  physiologist, and so our goal is  to conduct basic and applied  research in fish endocrinology,  with the goal of increasing fish  production, of understanding the  impact of endocrine disruption  on fish, and also to gain  insights into basic biological  questions using fish as research  models.  I can talk more about that last  point, fish have some very  unique attributes that allow you  to do things with fish that you  can't do in any other vertebrate  species.  One quick story, I worked on  sharks for several years in  Maine.  The testes of a shark is  organized in such a way that you  can literally cut the testes in  half and you'll see a bunch of  concentric rings, and these  represent the spermatogenic  wave, so you can actually go in  and dissect out pure  spermatocytes, pure secondary  spermatocytes, pure sperm and so  on.  Can't do that with any mammalian  species where all these cell  types are all intermingled. 

 I also worked on the pituitary  of the fish where the cells  really aggregate so you get pure  populations, I worked on a  hormone called prolactic.  You can actually literally  micro-dissect, under a  microscope, a section of the  pituitary, it's pure, 100%  prolactic cells.  You can't do that with any  mammalian species either.  One of our other methods is to  develop, improve fish rearing  methods, and I formed a company  called Aqua Most, you'll see  about that in a minute.  Also to train students including  next generation of  physiologists, and to share the  information we learn with the  citizens of Wisconsin and the  world.  As you can see, most of the  aquaculture, most of the need is  actually worldwide, a global  problem.  All right, so in our program,  actually there's two main  facilities.  We have a facility located in  Lake Mills at the Lake Mills  State Fish Hatchery.  These big ponds that you see  here are actually mostly run by  the DNR, and these ponds are set  aside for the production of game  fish.  Mostly walleyes and Northerns. 

 You can't really see it but  there's a little raceway up  there.  They don't do any more of it,  but for many years they raised  Coho salmon that they used to  stock the Great Lakes.  And all the salmon that I use in  my research that I'll talk about  later actually came from this  facility.  We also have a very nice  facility with lots of tanks and  a small set of small research  ponds that are being used in our  research there.  A lot of the practical work we  do is done at this facility.  There's also a very nice fish  rearing facility located in the  basement of the Water Science  and Engineering building.  You can see that's the red roof  building there, right on the  lake, right next to the Helen C  White library and the Union.  A nice place to work, 'cause I  just go over and have my lunch,  right?  There's another picture, the  black and white one there shows  the old building, it was just  called the "hydraulics lab" and  it was built in 1929, which is  the same year my mother was  born, which is how I know it's  80 years old.  My mom is here today, actually.  (laughs)  Happy birthday, Mom. 

 Public TV.  Okay, so I'm gonna spend a  little time.  Even when fish scientists,  people who really know what  they're doing, come to my lab,  they're actually quite astounded  by the quality of our facility,  and like I say, one of the  best-kept secrets.  So you start at the top right.  The water that we use, and water  is extremely important for  raising fish.  High-quality water is the key to  raising healthy fish.  So we use city of Madison water,  which is well water, and it's  chlorinated.  So it passes through these blue  cylinders up on the left which  contain carbon which removes the  chlorine from the water.  We also heat the water, in the  bottom picture there are  heaters.  We've got two industrial steam  heaters that run off university  steam, where we can heat the  water. 

 The water then is just pumped  upstairs to the second floor  where, I wish I had a pointer,  but see these little columns?  It says L1 above those little  columns there?  Those are actually filled with  these little bio-balls, they're  called.  So the water comes in and it  trickles down over these  bio-balls, and it gases off  nitrogen and carbon dioxide and  takes on oxygen.  So it becomes saturated in  oxygen.  Now, the water input into that  system, it's usually constant  cold on, and then we've got hot  water which is pulsed in to  maintain any temperature I set  it at.  On the wall here on the top  middle one, we've got a bunch of  solenoids, which just are  electronic valves.  And there's a control box that  just senses the water  temperature and the hot water is  pulsed on continuously to  maintain the temperature.  So then that water feeds down  into the basement where we have  a whole bunch of tanks.  Right now we're set up with  dozens of 30-gallon tanks,  dozens of 60-gallon tanks, a few  of these large 200-gallon tanks.  I've got a visiting scientist,  Dr Shi in the lab, he's raising  some frogs and some zebra fish.  We've got a bunch of smaller  aquariums all set up. 

 And the beauty of our system is  that it's completely  flow-through water.  The water quality is always  absolutely excellent.  Control the temperature, it's  fully oxygenated, it allows us  to do all kinds of work.  I've literally had experiments,  I told you about, we had an  Amazonian catfish at 32 degrees  and right next door we had a 5  degree lake trout.  Side by side tanks.  You can see there's three lines  along the wall up there?  Those head tanks, I can maintain  the temperature in three  different tanks.  And conduct experiments in that  way.  You guys are welcome to come  visit, too, basement of the  Water Science building.  All right, now I'll talk a  little bit about some work we've  done recently.  Now, I've been here for 20  years, so this is work we've  done over the last four years. 

 So the first thing I'll talk  about is a novel  growth-promoting feed additive.  So growth of the aquaculture  industry is constrained, one of  the biggest constraints, besides  land and so on is the high cost  of feed.  In fact, the catfish industry in  the south is suffering now  because of the rise in the cost  of feedstuff.  So anything we can do in order  to reduce the cost of feed or to  increase the growth rate is  gonna benefit aquaculture no  matter what species of fish, no  matter what kind of method we  use to raise those fish.  So there's a lot of different  ways that we can improve fish  growth rates.  The most obvious way, and the  way it's been done a lot is  through natural selection.  Just like raising better cows  and so on.  We can genetically select for  superior growth in fish.  And in fact, the species that  are raised primarily in China,  the carp, highly domesticated  animals, very low disease rates,  very rapid growth rates.  Rainbow trout is a species in  the US which has a very rapid  growth rate, it's been selected  for about 100 years. 

 But a lot of fish we're raising  now, an example would be yellow  perch or walleye, haven't been  selected for at all.  These are essentially wild  animals that we're growing in  the tanks and they don't grow  very quickly so a lot of extra  work needs to be done into  these.  You can use biotechnologies to  improve the growth of the fish.  One example I'll talk about  later is to produce mono-sex  populations.  In some species of fish the  males grow bigger than the  female, so if you grow all-male  stocks you would enhance your  overall production and we'll  talk about that.   Improved feeds.  The problem with fish in one way  is that there's so many species  every species has to be  independently researched to find  out the optimal diets for those  animals.  But that research goes on, there  are a lot of fish nutritionists,  believe it or not.  And finally, there's things like  feed additives, flavor  enhancers, that make the fish  want to eat the feed that's  artificial more.  An example of that would be to  take rinses of krill and add  that to the feed so there's a  flavor they enjoy. 

 There's a start-up technology  company formed by a professor in  my department named Mark Crook.  And he's actually a chicken  immunologist.  And he has a patented  technology, which is very  interesting, and I'll tell you  about the work that we did.  The technology is that you  inject a chicken with an  antigen, the chicken will make  an antibody to that, whatever  you've injected it with, right?  And then what happens, when  chickens do this, is they'll  accumulate those antibodies in  their eggs.  So all you do is you harvest the  eggs from the laying chicken,  you make egg powder, and what  you've got is this really rich  source of antibody.  So the antibody that we use is  an antibody to an enzyme called  phospholypase A2.  And what this is, is an enzyme  that mobilizes lipids from the  membrane to create something  called arachidonic acid which is  then further metabolized by a  bunch of enzymes to produce a  hormone called prostaglandin.  And prostaglandins are hormones  that mediate the gut  inflammatory response.  So by making an antibody to this  enzyme and feeding this to the  fish, the fish no longer has a  gut immune response.  This is kind of interesting, I  didn't know this until I did  this work. 

 But one of the largest immune  systems in your body is your  gut.  It's adapted to respond to  ingestion of pathogens in your  food, and that makes a lot of  sense, right?  If you're a wild animal, you're  eating this half-decayed zebra,  if you're a lion or whatever.  So you want to protect your gut  from this and the gut is ready  to have this major immune  response.  But obviously when you're  feeding these very careful, very  clean diets, that's unnecessary  and in fact it's a waste of  quite a bit of energy.  So that's the approach.  What we did is we used these  tanks in my lab and we used  rainbow trout as a model species  and we fed three levels of this  antibody stuff to the fish.  And what we found was extremely  surprising.  Like I said, rainbow trout are a  very selected fish.  They've been raised for 100  years.  They grow very quickly in  captivity. 

 And yet we were able to increase  the growth of this fish by about  30% by giving it 0.3%  of this antibody-coated feed.  So this is the first time that  Aovatech, that's the name of the  company, had tested their  product in fish.  Up to that time they had been  really trying to enter the  market for poultry, for swine  and so on.  Very difficult markets to enter.  And now they've signed  contracts, they've got major  contracts to sell this product  in Thailand, Japan, India, and  so on.  So I'm fortunate to have done  the first study in that.  Okay, next study.  Sex reversal in aquaculture drug  development.  So just like in any other, like  humans or terrestrial vertebrate  animals, to get a new drug into  the market requires a lot of  research that has to be done in  a lot of different areas before  that drug can be approved by the  FDA.  One of the most important drugs  that is currently being tested  for use in aquaculture is a  steroid hormone called 17  alpha-methyltestosterone, or MT. 

 And the reason it's important is  because it allows us to make  mono-sex populations of fish.  Now in the case of tilapia, the  males grow much faster than the  females.  So you really want to raise  all-male populations of fish.  The males grow faster.  The other reason is that if  you've got females in with the  males, so you've got a pond of  these fish, the females will  come into reproductive  maturation and the males will  spend all their time courting  the female and she'll be putting  on eggs instead of meat, which  is a detriment, so you're  wasting feed that way.  So what you do, fish are very  plastic.  So even if you have a genetic  female, an XX female fish, if  you treat that XX female fish  with very low levels of  testosterone at a critical  developmental stage, it's  usually about one to two weeks  after hatching, you can convert  that XX female into a phenotypic  male, grows up to be a male  fish.  So there's some controversy  about that because you're  literally treating the fish  you're gonna eat with a hormone  when it's young.  However, all the work that's  been done, and a lot has been  done, there's no residual  leftover hormone at that time.  Yeah. 

 >> (inaudible)  >> Barry: They are, yes.  The female actually develops a  testes.  And that's one of the reasons  they grow faster because the  hormone antigen produced by that  testes actually is a growth  promoting anabolic steroid.  That's interesting, this same  hormone can actually be used to  make all-female populations, as  well.  In the case of yellow perch,  which is a species that's really  well, it's a delicacy in the  Midwest.  Most of you guys remember the  Friday night fish frys.  A lot of research has been done  on this campus to try to bring  this fish on as an aquaculture  animal.  But you can produce all female,  but it's a different approach.  What you do there, you take the  XX female fish, you treat her  with testosterone and turn her  into a male, right?  So she's XX, but she produces  sperm.  So now you take that fish and  you cross it with a normal  female, right, it's a male, so  it's producing sperm, and you've  got a normal female. 

 XX, XX, the only sperm you've  got are XX.  Every baby is a female.  So that's how we do that.  So what we do, the work that I  did in my lab to try to get this  drug approved is a bunch of  stuff, there's many types of  studies you do, but what I did  was the environmental safety.  There's concern about putting  steroids into the environment  that could potentially change  the sex of the endogenous fish,  jeopardize human health and so  on.  These are called endocrine  disrupters.  This is clearly an endocrine  disrupter because you're  actually adding this stuff to  make this fish change sex.  So we did experiments, I worked  in collaboration with some  scientists in Henry Lardy's lab  who are experts on HPLC -- and  we developed an assay to measure  methyltestosterone and a lot of  its degradation products.  So we incubated carbon 14  labeled methyltestosterone under  aerobic and anaerobic conditions  and just tested at different  times to look at the effect, the  longevity of the testosterone.  It actually degrades very  rapidly, within a couple of days  under both conditions, all of  the testosterone is gone, which  makes sense. 

 The other thing we did is a  bunch of work on just product  manufacture.  This is quite a bit easier work.  The producer of the food, in  this case it's a company called  Rangen in Idaho.  They have to prove that when  they mix up their feed with the  testosterone that, they've got a  conveyor belt of the food and  they fill up one bag and then an  hour later the last bag, they've  got to show that the first bag  has the same concentrations as  the last bag.  They've got to prove that the  hormone doesn't migrate down the  bag in storage.  They want to show how long it  lasts in storage.  So a bunch of studies like that  we did using our assay.  And this is one of the results.  This is just feed storage for  three months at room  temperature, refrigerator,  freezer, and we saw that it  degrades rather rapidly at room  temperature but when it's held  in the fridge it maintains.  So all that means is when they  label the feed they're gonna say  it must be stored in the  refrigerator. 

 Okay, novel mechanism of  endocrine disruption.  There's a lot of chemicals in  our water.  These come from industry,  agriculture, they come in from  the air, PCBs, -- these are  things you maybe have heard  about, they're in the news a  lot.  A lot of these things are  hormones in a sense that they  can bind to receptors for normal  steroid hormones and mimic the  actions of these steroids.  And by definition hormones are  chemicals that have a very  profound effect at very low  concentrations.  The typical way in which a  hormone acts is that it binds to  a receptor, can either be on the  surface of the cell or it can be  in the nucleus of the cell, and  that turns on a machine, a lot  of different mechanisms.  Endocrinologists study the  mechanisms by which these  hormones act and they have some  effect on the body.  If you look at estrogen here,  you can see that it actually  looks pretty similar to some of  these potential endocrine  disrupters.  There's kind of a mystery about  this.  There's a lot of reports that  certain kinds of these chemicals  have had strong effects that  were estrogenic. 

 I'll talk about what that means  in a minute with fish.  But yet, endocrinologists can  actually do studies where they  show how strongly these  chemicals bind to the receptors  on the cell.  And a lot of these things that  were shown to be pretty  estrogenic did not bind to the  receptor well at all.  Not well enough to explain the  potency, the estrogenic potency.  So we wanted to investigate  alternative mechanisms by which  these things could act.  So I'm gonna quickly show you  this.  This is the life cycle of an egg  in the ovary.  It just shows that you've got an  immature fish, and as their eggs  grow they get bigger and bigger.  Now what's interesting is that  at some point the egg starts to  really grow as it accumulates  yolk.  Yolk protein is called  vitellogenic. 

 The thing about vitellogenic  which is interesting is that  it's actually produced in the  liver.  So the egg itself at some  developmental stage receives a  signal from the pituitary gland  and that egg starts to produce  estrogen.  So estrogen travels in the body,  here's the egg and the liver,  travels through the body through  circulation, binds to estrogen  receptors in the liver, and the  liver begins to produce  vitellogen, this yolk protein.  And that circulates in the blood  and gets accumulated by the egg.  So the egg talks to the liver,  the liver makes a protein that  gets incorporated into the egg.  So one of the genes that's  really very useful in studying  endocrine disruption in the  environment is the gene that  regulates the transcription of  the yolk protein vitellogen.  And there's an estrogen receptor  there and you put a little bit  of estrogen or any of these  estrogen mimics and you'll get a  very big increase in vitellogen,  it's very powerful, very useful  assay.  So when you do that with some of  these things, we're not seeing  much, we're seeing good  vitellogen induction but we're  not seeing good estrogen  receptor binding.  So my student, her name is Gail  --, she hypothesized that maybe  what these things are doing is  acting through a different  mechanism.  To make a long story short, she  studied the metabolism of  estrogen. 

 Estrogen circulates and at some  point you've got to get rid of  it.  So the body has mechanisms to  metabolize the estrogen to make  it into a form where it can be  gotten rid of from the body.  Basically there's three ways to  do that.  You stick a bunch of hydroxyl  groups on the estrogen, you  stick something like a sulfur  group on it, or you stick this  little sugar group called a  glucuronic acid.  Just three ways.  You put these groups on it, it  becomes inactive.  So long story short, Gail showed  the metabolic pathways of  estrogen metabolism in the lake  trout, first to do that, and  then she showed that a variety  of these endocrine disrupters  are potent inhibitors of  estrogen metabolism.  In other words, if this is  present in the fish, this  estrogen which normally would go  away sticks around for a lot  longer.  And I think that explains the  result.  So it's actually estrogen  hanging around longer which  causes these effects. 

 >> (inaudible)  >> Barry: Yeah, --  hormones are in the same  super-family, they bind to  receptors that are very similar  to steroid hormones.  Okay, endocrine mechanism  meeting the death of salmon.  Like I said, this is my life's  greatest work right here and I'm  going to share it with you  really quickly.  This is a couple of years of  work.  Salmon, as you know, migrate  upstream and then they die.  And it's been known really since  the '60s, '50s, why they die.  The reason they die is that a  stress hormone called cortisol  is highly elevated in these  fish.  We've known that.  A lot of studies they've done,  if you inject a fish with  cortisol it'll die.  Some guy literally was able to  remove the adrenal gland of a  fish, it's not easy, those fish  didn't die. 

 A bunch of studies like that  have been done.  So people know that cortisol  killed them but what no one  really could understand is,  cortisol is high in fish at  other times.  If I stress any fish, he's gonna  get cortisol levels that are as  high as the fish when they die,  but they don't die.  Or rainbow trout, which are very  closely related to salmon, same  genus, oncorhynchus, they don't  die after they spawn.  And yet they have highly  elevated levels of cortisol.  So these sort of mysteries  surrounding the actual  mechanism.  So we went to work to try to  figure out what that is.  Just a little bit on basic  stress physiology.  The thing about stress which is  interesting is it's a  neuro-endocrine mechanism where  multiple types of sensory inputs  result in about the same  response.  So a lion attacking you,  or you're getting bled,  or you watch a horror movie,  your wife yells at you. 

 A lot of different, really  unrelated sensory inputs.  My wife is here, that's why I  have that there.  They cause a pre-determined  response which is an elevation  in the levels of cortisol.  And I'll show you that here.  This is called the HPI axis  which stands for the  hypothalamic pituitary  interrenal axis.  In us, it's the same thing in us  except we have an adrenal gland  instead of a fish has a  homologue called the interrenal  tissue.  It's the same exact thing, cells  that produce the steroid hormone  cortisol.  So H stands for hypothalamus,  it's a part of the brain, P  stands for pituitary,  interrenal.  So the sensory inputs come into  the brain, brain releases a  little hormone that stimulates  the pituitary, just basic  endocrinology here.  The pituitary releases another  little hormone that stimulates  the interrenal to produce  cortisol. 

 So the cortisol then binds to  some target tissue, the little  dot here is the receptor for  cortisol.  Cortisol also rises in the blood  and it reaches a certain level,  it turns off this signal and  turns off that signal.  That's called negative feedback,  very important concept.  So cortisol stimulates the  target tissue, negative  feedback, so cortisol goes up,  it feeds back, it'll go down.  So this is actual data from our  lab that shows the standard  cortisol stress response.  And what we did there, we just  took a salmon and we held it up  out of the water for 30 seconds  in the net and stuck it back in  the water and then we just bled  fish at different times, that's  all that is.  You can see that cortisol goes  from essentially zero in a  resting fish to extremely high  levels of about 200 nanograms  per mil, in some fish they went  up as high as 400 to 500  nanograms per mil.  And then as the negative  feedback kicks in, the levels  fall back to baseline in about  three to six hours.  Now what's interesting is that  if I were to maintain the level  of cortisol really high like  this in a fish that wasn't  reproductively mature, it  wouldn't die.  But yet it's known that levels  of cortisol this high are  sufficient to kill spawning  salmon. 

 So there's a mystery there.  The other thing that's  interesting is that in spawning  salmon cortisol levels are this  high and they don't come down,  they stay up.  Which suggests that they've lost  the ability to have negative  feedback, right?  So cortisol is continually being  produced and it stays high.  So we came up with a hypothesis  and it's this.  That the cortisol levels, the  high levels of cortisol, and the  death of salmon is mediated by a  steroid produced by the gonads,  abbreviated 17 20-P.  All that is is progesterone with  a couple hydroxyls on it, the 17  position and the 20 position.  That's a real common steroid  hormone, progesterone, and fish  make this special hormone called  17 20-P, it's actually a really  famous and important hormone in  fish.  It's the hormone that controls  the final maturation of the  gametes, both the sperm and the  eggs.  So for example, this is an egg  that was taken out of a walleye,  that's the nucleus right there.  If I were to culture those eggs  in vitro and then add that  steroid, the nuclear membrane  would break apart, would open  up, and then the chromatin would  spread out throughout the egg. 

 And that egg cannot be  fertilized, that can.  So it's the last step before the  eggs are spawned and fertilized  and it's absolutely essential in  the life of the fish that this  event occurs, otherwise you  wouldn't have any fertilization.  Now what's interesting is that  the only time in the life of any  fish, most fish have this  hormone, not all, is this very  giant rise in the levels of this  steroid.  This is actually rainbow trout  but it's pretty similar in  salmon.  So we hypothesized that the fish  obviously has to spawn before it  dies, it can't, evolution could  never select for a mechanism  that occurred where they could  possibly die before they  spawned, so we postulated that  this event occurs, 'cause  spawning happens very soon after  the rise in this hormone.  That that's what triggers the  death of the fish.  So to make a long story short,  our data supports this model,  I'll show you a little of the  data that supports it.  But basically what we found was  a couple of things.  It's possible that negative  feedback is intact.  We don't really have data but  I'm suspecting that the  pituitary signal is turned off. 

 But what we found was that the  gonad, which is producing, go  back here.  It's producing a lot of this  steroid here.  So the blood levels of this  steroid are very high, turns out  that that is converted by the  adrenal gland of the fish, the  interrenal tissue, into  cortisol.  So even if we're not getting a  stimulation of the adrenal by  the pituitary, we're feeding in  all this steroid precursor which  the interrenal converts into  cortisol.  It's substrate for cortisol  synthesis, so that's one thing.  So that doesn't explain, though,  why the salmon die and the  rainbow trout don't die.  So what we found was that the  target tissues are protected by  an enzyme.  So even if I had all the  cortisol in the world, this  target tissue would never see  the cortisol because there's an  enzyme between it and the  receptor that turns the cortisol  into something else which is  inactive.  Does that make sense?  It's a little bit complicated,  it's not that complicated,  though.  Cortisol, which is right here,  would normally get in and kill  the thing. 

 But instead I got this enzyme  right there that protects the  cell, it turns the cortisol into  something else.  But when 17 20-P is high, that  turns off that enzyme, which  means the cortisol's really high  now and it's not protected any  more, it gets in and kills the  fish.  What we found was that the  enzyme in rainbow trout and coho  salmon were different.  And the enzyme in rainbow trout  is not turned off by 17  20-beta-P, and the one in coho  salmon is.  So here's a little bit of data.  Cortisol and 17 20-beta-P, so  you can see that just a couple  of steps change this into this.  So we did studies where we used  radioactive-labeled this stuff  and we found radioactive-labeled  that stuff.  We know for sure that this  steroid gets converted into that  steroid.  And this is, yeah.  >> (inaudible)  >> Barry: Exactly. 

 Tried three times to get funding  from NSF to do just that.  But it's, yeah, exactly.  And that was actually the  motivation for this work.  There were actually stocks of  fish where there were just  dozens of individuals going to  some tributary.  They all died.  If we could intervene with some  shot or something to keep them  alive, that was one motivation.  Another idea is that in  aquaculture you actually do  genetic selection for these  fish, right?  And you've got this great strain  of fish that's disease  resistant, fast growing, it  dies.  You don't have eggs for the next  generation so we want to  intervene there to keep them  alive for that purpose.  So there is a practical side to  all of this besides the basic  understanding of it.  This is another piece of data, I  won't go into it too much, but  you can see that if you add 17  20-beta-P the production of this  cortisol sulfate, which is what  the coho salmon produce, is  inhibited. 

 The other thing I found which is  kind of interesting is that this  is rainbow trout and that's coho  salmon, we produce some of these  other, when I inhibit the  production of cortisol sulfate,  we get a shift.  So cortisol normally would go  from here to cortisol sulfate, I  turn this off, so now the  cortisol turns into some other  stuff.  And we're not sure what those  other things are, it's these  compounds, we really don't know  what they are, but maybe they're  the killer steroids, we don't  know, that's a possibility.  Enough of that.  One other thing, this is kind of  interesting.  I don't have a lot of data on  this, but I was really, this is  one of those things.  I was sitting having lunch on  the Union terrace, one of the  advantages of working where I  work, and right next door is the  limnology center and there was a  scientist from the University of  Washington, who's really an  expert on salmon, he was there  visiting the limnology guys and  we just had lunch together.  Turns out he was down in New  Zealand doing a study of really  kind of an interesting problem.  It took one strain of Chinook  salmon from the state of  Washington about 100 years ago,  one strain, and they put it in  New Zealand.  And in 100 years, this one  strain has radiated into what we  would call in the US maybe two  dozen strains. 

 Some migrate in the fall, some  migrate in the winter.  Some don't migrate at all.  Interestingly, one strain that  evolved was a strain of salmon  that don't die after they spawn.  And that's what this guy was  studying.  So we were able to get some  blood samples from those fish  and what we did is we tested the  hypothesis that a difference in  the level of the binding  protein, see, steroids don't  circulate in the blood as the  free steroid, they're actually  attached to proteins called  binding proteins.  And there's specific binding  proteins for each class of  steroids.  So there's an estrogen binding  protein, an antigen binding  protein.  There is something called the  CBG, cortisol binding protein.  And it's thought that only the  steroid that's not stuck to the  protein works.  So what we think happens is,  remember the big rise in 17  20-beta-P, we think it knocks  the cortisol off the binding  proteins and it binds instead. 

 And what we would have is more  free, more biologically active  steroid.  And we have evidence showing  here, this -- are the ones that  die, exact same fish, you can't  tell the difference, except  these guys don't die.  And look at the difference in  both total and free cortisol  fitting our hypothesis.  But we haven't yet tested the  hypothesis that 17 20 would take  cortisol off the binding  protein.  All right.  So now, I'm about wrapping up  here.  So now a little bit about our  ongoing research.  We've got two major research  projects going on right now.  One, we're working on a device  that we hope is gonna help  recirculate some aquaculture to  keep the water cleaner, and  another project funded by the  EPA looking at steroid hormones  associated with concentrated  animal feeding operations and  their effects on fish.  So device that converts aqueous  ammonia to nitrogen gas. 

 This is one of those interesting  stories.  I work in the water sciences  building, I'm the only guy from  the College of Agriculture over  there, most of the people in  that building are engineers and  chemists and so on.  And I've been there for many  years, and over the years you  gradually get to know one  another.  And there's a professor, his  name is Mark Anderson.  I think he's got the third most  patents of anybody at UW,  something like that, top five at  least.  He's a material scientist, civil  engineer, kind of a nutty  professor kind of guy who keeps  inventing stuff, I think he  talked here before.  Anyway, he's been working for  years on these photo-activated  catalysts.  You shine a light on them and  they become very active  catalysts.  So one day over beers on the  terrace, I said, what do you  really do?  And he told me and I said, I  wonder if that would work to get  rid of ammonia in water.  And we tried it and the results  were amazing, we formed a little  company. 

 Ammonia is one of the biggest  problems of raising fish in  closed systems like aquariums or  recirculation aquaculture  systems.  Fish excrete ammonia the same  way we excrete uria, or pee.  So the nitrogenous waste  products of a fish are actually  released primarily through the  gills into the environment.  In nature it's just dissipated  and goes away very quickly, but  in these closed systems, ammonia  can build up rather rapidly and  it's very toxic to fish,  outright kills them.  So a big problem.  The traditional solution to  ammonia toxicity in these closed  systems is biofiltration.  And most of you have probably  kept an aquarium, but the  filter, the under-gravel filter  or the little thing with the  glass -- in the back of your  tank, what that is is a  substrate on which bacteria  grow, two kinds of bacteria,  that convert ammonia, one kind  converts it to nitrite, which is  also pretty toxic to fish, not  quite as toxic as ammonia, but  close.  And then another set of bacteria  convert nitrite into nitrate.  These are very slow growing  bacteria so if you go to an  aquarium store and you buy a  brand new aquarium, you really  can't stock that with new fish  for roughly a month without  risking ammonia toxicity.  You don't have enough bacteria  to make that conversion and the  ammonia will build up and kill  it. 

 It's a horror story.  Many kids will take their dads  to the store and say, I want to  put a fish in now.  And they die, and then they lose  interest in the hobby.  You have to wait that month.  So there's that lag period.  The other problem is that  there's no solution to nitrate  accumulation.  Eventually the nitrates reach a  point where you have to change  the water in your tank to dilute  the nitrate.  The problem of algae growing in  your aquarium is because of the  excess nitrate.  Even large aquariums, like the  Shedd Aquarium, it's the largest  right out here, have that  problem.  And at some point they've got to  dump all that seawater somewhere  to get rid of the nitrate. 

 I know the guys at the Shedd and  they move the water around, go  from the fish tank to the whale  tank where it's not an issue,  and they do their best, but at  some point they've got to dump  all that saltwater, it's really  expensive.  It's vulnerable to change.  These are living organisms that  if you, like Sarah for example,  if you overfeed the fish and all  of a sudden there's a big spike  in ammonia, you don't have  enough bacteria to keep up with  it, you can kill your fish that  way.  You can't treat sick fish in  your aquarium with antibiotics  because it'll kill the bacteria  that do this necessary job.  The biofilters tend to be fairly  large so you've got space issues  and it's hard to clean these  filters without knocking off the  bacteria and reducing their  numbers and putting fish at  risk.  So the solution is our gizmo,  which is a device that works on  a principle called  photoelectrocatalytic oxidation.  We call it the PECO unit.  PECO, photo-electro-catalytic  oxidizing.  It's a molecule that when light  shines on its surface, it's a  semi-conductor, electrons go  from a resting to an active  state.  And on the molecule itself,  there's two sites, one that's an  electron acceptor, and an  electron donor. 

 So we get oxidation and a  reduction reaction occurring on  the surface of this  light-activated catalyst.  It turns out that ammonia coming  in contact with that surface is  converted not into nitrate which  is oxidized all the way up to  the +5 state, here's ammonia  here at the -3 state, but it  gets oxidized partially to  nitrogen gas.  So using our gizmo, you just  plug it into the wall, you don't  have to wait for bacteria to  grow, you don't have to worry  about nitrate accumulating  because what we've got is  nitrogen gas just bubbling out  of the top of the tank.  So it kind of looks like this,  it looks like a normal UV light  and then lining the inside of it  is a foil that's got the coating  of our special catalyst on it.  This is our prototypes that  we've built and this just shows  the way in which ammonia can be  destroyed.  If we spike, an aquarium with a  pretty high level of ammonia,  it's removed.  But in an aquarium the fish are  just gradually producing this  ammonia and we just built a  machine to kind of keep up with  the fish's ammonia production.  This is one of our first fish  tests.  You can't really seem them but  there's a bunch of little  tilapia in there.  And we had over here controls  that were just normal  biofilters, and you can see,  they were biofilters that had a  lot of bacteria on them. 

 As you can see in time, no  nitrate accumulation in these  tanks, and then the ones that  have the biofilters in nitrate  are just --.  This is showing what's called --  stuff, I don't know if you guys  ever kept a seawater aquarium,  but broken down protein and  stuff, the water starts to turn  yellow and it oxidizes that as  well.  In fact, we were surprised.  This device of ours doesn't just  oxidize ammonia, turns out it  oxidizes basically anything  we've ever tested on it.  So this little aquarium product  of ours really took on a life of  its own and we formed a company,  it's called Aqua Most and we are  now in the environmental  remediation business.  It turns out that this device is  able to oxidize a lot of  pollutants in the environment  including a lot of the things we  show here.  Gasoline constituents like -- if  you've ever heard of that.  It oxidizes MTBE which is a fuel  additive.  These are things that are very  difficult to get rid of.  We recently had a strategic  partner who owns a big  environmental remediation  company in Pittsburgh, was in  town and these are our little  gizmos here, and we tested the  ability of this machine to get  rid of --. 

 Benzene, that's the real problem  here.  So after running this thing, an  hour, we really reduced benzene  to almost negligible levels.  All right, so the final thing,  and this is the end of the  story.  We're funded by the EPA, and  this is a very large process.  There's actually seven  universities in the country  doing similar work  simultaneously.  The EPA really wants to get a  handle on what is the impact of  hormones associated with CAFOs,  CAFO stands for Concentrated  Animal Feeding Operations, these  giant pig farms, or giant  chicken operations.  We're spending a lot of time on  a big beef operation in the  state.  They administer hormones to  these animals to promote growth,  they have their own endogenous  hormones.  I showed you how those steroids  get turned into sulfates and so  on.  Those actually pass out through  the urine into the environment. 

 So it's like a point source of  pollution of steroids.  And the question is, do these  things have an impact on the  environment or not?  So my role in this, I'm working  with a bunch of scientists at  the state lab of hygiene who are  chemists using very  sophisticated -- analysis  looking at the fate and  transport of these steroids.  Where do they end up, where do  they go, what are their  concentrations.  And then what I'm doing is  taking the steroids that we  find, the concentrations we  find, and exposing them to fish.  In this case we're using a  native fish called the fat-head  minnow.  It's an important toxicological  model fish organism.  And seeing what impact if any it  has on reproduction.  This is an example of one of the  CAFOs in Wisconsin.  We've actually collected  material from this basin.  They've spread this manure on  the fields, so we're looking at  tile drains on the field and  when the rain comes, the stream. 

 Like I said, this is happening  at seven other sites throughout  the country.  This is the fat-head minnow.  What's nice about this fish is  that the male will defend a  little nest.  This is just a piece of PVC  pipe.  And he'll coax the female in  there and she'll lay her eggs on  the bottom side of the tile.  So all we gotta do is pull out  the tile every day and we count  the number of eggs.  So we can estimate very simply  the fertility of the fish.  And then we can also set the  tile aside for a day or two and  we can look at the embryonic  development and see what the  percentage of those eggs that  were fertilized was.  So that's the exposure system we  have set up in the lab.  These are just small tanks that  have these pairs of fish in  them. 

 And here we've got concentrated  stock solutions of the steroid  we're testing and a big  peristaltic pump that then  delivers the steroid using this  dilution system that we built  and designed.  We actually copied the one from,  I don't know if you know this,  but Duluth has got one of the  top fish toxicology research  centers in the United States, in  Duluth, Minnesota.  So my grad student has been up  there a couple times and really  picked their brain.  We basically copied the system.  So we probably have the best  fish exposure system in the  state of Wisconsin.  So we're delivering different  doses of the hormone to  replicate tanks of the fish and  looking at a variety of  reproductive end points.  Some other things we're doing.  I've got a student, his name is  Pat -- and he's developing a  sperm motility assay.  And basically this is really  kind of nice.  Turns out that sperm have  receptors for steroid hormones  on their head and there's data  that shows that that receptor  mediates motility. 

 So we're hoping that we can  develop a very rapid, robust  assay to very rapidly screen for  endocrine disruption using this  assay.  All you've got to do is take the  -- from the fish and you just  put it on a microscope slide and  you film the sperm.  And then there's a sophisticated  computer program that you can  just download off the internet  called Casa and this thing  automatically spits out a whole  bunch of data.  Things like straight lines,  swimming velocity, -- velocity.  It tells you how much curlicue  is in the swim.  Every sperm that's in the field  of view.  And you get this big ream of  data.  It's very robust, works like a  charm.  We're not there yet, but we're  hoping we can just say, okay,  look, 20% of the sperm are going  in curlicue left, it must be  polluted with this steroid.  We're not there yet. 

 We had another group of  students, I had a visiting  student from Thailand in the  lab, and another girl from  China, and they worked on a  study where they took a variety  of different steroid hormones  that we suspected were  associated with CAFO.  Another nice thing about working  with fish is that embryonic  development occurs very rapidly  and you can actually culture  embryos right under a  microscope, hundreds of embryos,  no problem.  And you can actually look at the  development of these fish.  So we added the steroids at  different concentrations to see  what effect, if any, there is on  embryonic development.  Again, we are kind of working on  this data now, but yes, there  are effects of some of these  compounds on the development of  these fish.  I think that's it.  Thank you very much for your  attention.  (applause)