Sunday, April 21, 2019

The Story of Our Research


SCIENCE OF THE BLUE WALLEYE OF CANADA by Dr. Wayne Schaefer


As a fish biologist and university professor, I had worked with walleye in Ontario for nearly 10 years but had never seen a blue one.  While a student, I was taught the importance of blue pike (really a walleye) in the history of Lake Erie where commercial catch of that species was a major part of the market in the early 1900s.  However, the species had been driven to extinction by over-fishing and pollution by the mid-1950s and no one had seen one since that time.  Therefore, I was greatly surprised to see a blue colored walleye on the end of my line while fishing the Papaonga River System in Northwestern Ontario.  The riiver is near my summer cabin on Pakwash Lake near Ear Falls (Figure 1).  



It was a warm August day in 1993 when we slipped a canoe into McKim Lake.  The lake seemed perfect for a canoe.  It was small (only 2 miles across) with a medium sized river going through its eastern basin and with easy access from a gravel logging road just 35 miles east of Ear Falls.  We worked the canoe to the east side of the lake where the river entered and started to catch walleye that had a beautiful turquoise blue color on their dorsal (upper) and caudal (tail) fins (Figure2).  

Closer examination of the fish showed the blue color to be present only on the dorsal part of the fish, above the lateral line (Figure 3).  



The belly of the fish was white with no yellow pigmentation.   Could this be a remnant population of blue pike?


The next spring I started asking Canadian outfitters at sport shows in Wisconsin (my home state), Illinois and Minnesota if they had any blue walleye in their lakes.  Most outfitters responded with “There is no such thing as a blue walleye”.  However, the guys at Wilderness North Outfitters said they had blue walleye in a small lake out of Armstrong, Ontario, just north of Lake Nipigon.   I made arrangements to fly into Dawn Lake the next May.  The single cabin on the lake was well cared for and the boats and motors were in good shape.  It wasn’t difficult to locate walleye but none were blue in color.  Instead they were unusually gray in color with very little, if any, yellow coloration.  They tasted just as good as any walleye I had ever eaten.  Out of desperation, I put a couple whole fish into the cooler in zip lock bags, with a little lake water, and brought them back to my lab at the University of Wisconsin Milwaukee – Washington County.  To my surprise, upon opening the cooler a couple days later, the water in the bags was colored bright blue.  The color could only have come from the mucus of the fish.  After searching the scientific literature, I found no other report of color in the mucus of any fish.  The most logical explanation was a symbiotic micro-organism in the skin mucus of the fish.  Surely it would be easy to isolate the organism and produce blue color on a nutrient substrate.  I enlisted the help of many students, and a few colleagues, in an effort to isolate the suspected bacterium or algae that must be producing the blue color, but we were unable to identify any such micro-organism that produced blue color on any type of nutrient agar, and we tried them all.  About that time, I took on a Ph.D. student, Mark Schmitz, who was enrolled at the University of Wisconsin – Milwaukee.  I was his co-advisor and his dissertation would be “the biology of blue walleye in Canada”.


After talking to several well-known microbiologists at big universities we learned of a professor at the University of Iowa who was working on blue color in bacteria.  His name was Dr. David Gibson and he was a world renowned scientist.  With a little bit of timidity, I emailed the expert and, to my surprise, he replied, “I have two passions in life – microbiology and fishing”.  That was the start of one of the most fulfilling collaborations of my life.  Dr. Gibson put a bright young female postdoc student on the project.  Her name was Chi-Li Yu.  Since Chi-Li was from China, she had never seen a walleye before in her life but she did know protein chemistry.  Within two months Chi-Li had isolated the blue pigment and determined it to be a medium-sized protein, secreted by the fish, with molecular weight 87,850 Daltons.  The protein was a tetramer, consisting of 4 identical sub units and belonged to a family of proteins called lipocalins which were known to act as carriers of specific smaller molecules referred to as ligands.  A couple months later Chi-Li and David had identified the ligand as biliverdin, a common excretory product in vertebrate animals.  We named this previously unknown protein, “sandercyanin”.  Sander is the genus name for walleye and cyanin means blue in Greek.  Sounded pretty scientific.  We published that early work in 2008 in the Journal of Fish Biology (82(1): 51-58).  After David’s retirement in 2004, Dr. Ramaswamy Subramanian (Rams), continued working with Chi-Li in Iowa until he transferred to India to work as a supervisor in the world renowned Institute for Stem Cell Science and Regenerative Medicine (inStem) in Bangalore.  He, and one of his doctoral students, Swagatha Ghosh, continued to work on the chemistry of sandercyanin and accomplished remarkable things, which we will discuss later in this article.


The answer to the most important question still had eluded us, “Could these fish represent a remnant population of the extinct blue pike of Lake Erie”?  Maybe we could restock Lake Erie and bring back this important species.  The only true test would be genetic analysis.  Fortunately, an expert in walleye genetics was only a couple hundred miles away from my home campus and taught at the University of Toledo.  Dr. Carol Stepien was excited to see the fish.  Not only had she worked on present-day walleye but had also done genetic analysis on museum specimens of extinct blue pike housed at the University of Michigan.  If anyone could get the answer, it would be Dr. Stepien.  After careful analysis of the DNA from our Canadian walleye, Dr. Stepien determined that the fish were not the same subspecies (glaucus) as the extinct blue pike of Lake Erie.  In fact they were simply a color variant of regular, run of the mill, walleye (Sander vitreus).  That shot down our second hypothesis, the first one being the expected presence of a blue symbiotic micro-organism in the mucus of the fish.  We had, however, determined two possible reasons why the fish were blue in color.  First, they were albino for yellow color in their skin and, second, they produced a blue pigment in their skin mucus that was particularly evident on the dorsal side of the fish.  Also, the production of blue pigment (sandercyanin) seemed to be seasonal, peaking in late summer. 


We decided to document intensity of blue color in the mucus of the fish over all four seasons, which was particularly challenging in winter.  We used my cabin on Pakwash Lake as a base camp and traveled 50 miles, one way, to McKim Lake each day, sometimes through snow and ice or mud, and in the summer, always with mosquitos and black flies, and sometimes bears, on gravel logging roads to get specimens.  However, I always did say that the best part of this project was our method of sampling fish – hook and line!  Not only that but, unlike most researchers in biology, we got to eat our specimens!  Blue walleye taste just as good as regular walleye!  Another interesting observation was that the blue color does not come off the fish onto your hands when you handle them, however, it does come off onto a knife blade when you scrape the dorsal (upper) side of the fish from head to tail (Figure 4).  

The blue color is readily apparent against a white background of snow (Figure 5) and is also seen in snow around the fish when you are ice fishing.  

The author is shown in Figure 6 holding a blue walleye on the ice of McKim Lake.  

Eventually we did obtain samples of walleye mucus from over 300 fish across all four seasons during a period of six years and, with the help of other researchers from the University of Wisconsin Milwaukee, tested those samples for seasonal intensity of blue color.  The data turned out perfect (Figure 7).  

The intensity of blue color in the mucus peaked dramatically each year in late summer.  The pieces of the puzzle, as to why Canadian walleye produce sandercyanin, were beginning to fall into place.


In an effort to determine exactly where on the fish sandercyanin was being produced we examined fresh specimens of blue walleye under the microscope.  In Figure 8 is shown a close up of the spines in the anterior dorsal fin.  

Notice the blue line behind each spine which runs parallel to a blood vessel in each spine.  Clearly sandercyanin was being produced just behind each dorsal spine in the walleye fin.  As noted previously, blue color was only produced above the lateral line.  Closer examination of the blue color showed that sandercyanin was packaged in cellular vesicles in the mucus of the fish (Figure 9).  

Blue walleye are the only fish in the world, so far as we know, that produce color in their skin mucus.  Further, no other fish species, that we could find, packages pigment in cellular vesicles.  These results caused us to make histological cross sections of the skin and examine them under a microscope at higher magnification (Figure 10).  

Dr.Vicki Blazer at the National Center for Fish Disease in Kearneysville, West Virginia was gracious enough to help us with that project.   In Figure 10, the area above the surface of the skin appears white in the upper left.  The blue ovals represent the cells producing sandercyanin.  The white spaces, just below the surface of the skin, are the locations of empty mucus-producing cells.  The cells producing sandercyanin are just below the mucus cells and are blue in color.  They are a type of “sacciform” cell.  Sacciform cells occur rarely in fish species and generally store important chemicals needed by the surrounding tissue.  We have found no other report in the literature of a sacciform cell that stores pigment.


From the start of this research, I had wondered if the blue color of sandercyanin might fluoresce in ultraviolet (UV) light.  All attempts we made to “light up” whole specimens of blue walleye in UV light failed.  However, in June of 2008, while working in the microscope lab at the University of Iowa, we radiated samples of blue mucus, from fresh Canadian walleye, with UV light under the microscope.   To our amazement, the cellular vesicles containing sandercyanin lit up red like a Christmas tree.  Sandercyanin was a fluorescent protein (Figure 11)!  

Several of these remarkable proteins have been found in simple animals around the world, including the Nobel Prize winning, and very useful, green fluorescent protein from jelly fish.  Who would have guessed that walleye in Canada might produce a fluorescent protein?  Rams and Swagatha cloned the gene for sandercyanin and we patented it through the University of Wisconsin.  The gene has been transferred to bacteria which now produce the blue colored Sandercyanin.  However, to our great disappointment, the recombinant sandercyanin does not fluoresce well in UV light like the native protein does in vesicles.  Rams and his colleagues in India are attempting to engineer the recombinant protein to fluoresce brighter but, so far, have been unsuccessful.


Another big question loomed over our research.  I had established a blog website (bluewalleye.com) where sport fishermen could report the geographic location of blue walleye.  Hundreds of sightings came into the site from across Ontario and Quebec but very few from south of the Canadian border.  Why were blue walleye mostly found in the northern part of the range of walleye, above the Canadian border, and only rarely in the United States, and did that observation go along with the observations that sandercyanin was produced more in the summer and only on the dorsal part of the fish?  The answer to that question came from a high school student, one of over three hundred, who helped us with chemical analysis of the blue mucus.  He simply stated “Dr. Schaefer, maybe it is related to the ozone hole over the North Pole”.  As soon as he said it, I knew it was true.  It was the most reasonable hypothesis to tie together all three of our major observations – sandercyanin was only produced on the dorsal side of the fish where the sun hits it.  It was produced seasonally during the period of longest day-length and it was produced only in the northern part of the hemisphere where an ozone hole existed in the earth’s atmosphere.  Ozone there is destroyed by chlorofluorocarbon (CFC), an air pollutant.  Ozone, which naturally exists in the upper atmosphere of the earth, absorbs harmful UV radiation from the sun, thereby protecting life on earth.  With an ozone hole over the North Pole, walleye in that area would be exposed to higher levels of harmful UV radiation than their cousins to the south.


How did UV radiation cause increased production of sandercyanin?  The answer to that important question is in the fact that UV radiation causes the breakdown of heme, the red protein of blood, into biliverdin, the ligand carried by sandercyanin.  Excess biliverdin is excreted from the skin of the fish and combines with the protein Sandercyanin causing it to turn blue in color.  Further, analysis of the photo-absorption spectrum of Sandercyanin revealed that its highest absorption of light was in the UV range (300-400 nanometers), thereby protecting deep skin tissue from the harmful effects of UV light.  It was all starting to come together.  Walleye in the upper part of North America were producing sandercyanin to protect themselves from exposure to high levels of UV light.  Ironically, the fish were using the very product (biliverdin), resulting from damage to their skin by exposure to UV radiation, to protect them from the cause of that damage (UV radiation).  This is a beautiful story of nature protecting itself from human interference. 


Perhaps, this is how science is done.  You ask questions and follow the answers until the truth reveals itself.  Most science seems simple, once you know the answers.  The answers to these questions came only by diligent research from many tough students and many tough colleagues, to which I am most grateful.  It has been my joy to work on blue walleye for over 16 years, if you can really call “fishing” work!  We were privileged to work in some of the most beautiful wilderness country in the world (Figure 12) and were treated many times to northern lights at night (Figure 13).  




It has been said that if you enjoy your work, you will never work a day of your life.  Such has been my happy lot.  It is my hope that this article will instill within walleye fisherpersons even a greater appreciation for this beautiful fish.


To those who would like more detailed information on the science of blue walleye, please check out the links below to our two most recent publications:



Schaefer, Wayne F., Schmitz, Mark H., Blazer, Vicki S., Ehlinger, Timothy J., Berges, John A.  2015.     Localization and seasonal variation of blue pigment (sandercyanin) in walleye (Sander vitreus).  Canadian Journal of Fisheries and Aquatic Sciences 72(2): 281-289.


http://www.pnas.org/content/early/2016/09/28/1525622113.full

Ghosh, S.; Yu, C.; Ferraro, D.J.; Sudha, S.; Samir, K.P.; Schaefer, W.F.; Gibson, D.T.; Ramaswamy, S.  2016.  A Blue Protein with Red Fluorescence.  Proceedings of the National Academy of Sciences of the United States of America, 113(41):11513-11518.


To those who would like to catch a blue walleye, just contact the outfitters below and they will set you up in a remote camp in the wilderness of Canada where blue walleye are present.


Excellent Adventures in Ear Falls, Ontario.  Exc-adventures.com.  807-662-5292.

Northern Wilderness Outfitters in Thunder Bay, Ontario.  Wildernessnorth.com.  888-465-3474.

Ghost River Outfitters in Sioux Lookout, Ontario.  Ghostriverlodges.com.  888-446-7874.


Next time you catch a walleye, scape the skin from head to tail on the dorsal side of the fish with a knife and see if blue color is present.  If so, please report the finding on my website at bluewalleye.com.  Good fishing!

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