N I N E

In Cold Blood:
The Tale of the Icefish
In all things of nature there is something

of the marvelous.
-Aristotle

It was a long way just to go fishing.
The us-foot converted wooden sealing boat Norveg/a put to sea out of
Sandcford Harbor, Norway on September 14,1927. Its primary destination was perhaps the most remote piece of land on the planet. Tiny Bouvet Island, a speck in the vast Southern Ocean, lay more than six thousand miles from
Norway, sixteen hundred

miles from the tip of Africa, and more than three

thousand miles from South America.

In the mid-1920S, commercial invention whaling was booming. The Norwegian

of factory ships allowed greater numbers of animals to be taken

without relying on shore facilities. Finding new stocks of whales was a priorHCllRE 9.1 The Non'egia a/ Bouvet Island.
Photo (rom F;1I1gstOg Forskning r Sydish,lVct by Bjame Aagaard, Volume
"N)le Tider." Published bv Cyldelldai Norsk Foriag, Oslo. 1930.

ity for the entrepreneurs
2,

who went to sea, and establishing

tory and w.llers was a priorit) for the countries government claims to terri-

involved. The Norwegian

wanted to stake a claim to this icc-covered volcanic rock with

167

168

PART IV

EVOLUTION

IN ACT

CHAPTER

ON

FIGURE
9.2
DitlefRlIstad 011 the Norvegia foredeck.
Photo from Fangst Og Forskning I Sydishavet h)' 13,Clme i\C1gclClrd,volume
"Nye Tider." Published by Cyldendal Norsk Forlag, Oslo, 1930.

9

IN COLD BLOOD

rx, tz u

OF THE ICEFISH

169

FIGURE
9.3 Claiming Bouvet /slcllld (or NOMa),.
'Iakcn on December I, 1927. Dillcf Rustad is at the far IcR. Photo from Fangst Og
Forskning I Sydishavet b» Biame AagacJrd, Volume 2, "Nye Tider." Published by
Cyldenda/ Norsk Forlag, Oslo, 1930.

2,

thc aim of establishing some kind of outpost there that could assist the sacks prevented drainage, so l11an~ had to be thrown overboard. As the

whal ing fleet.
The Norregta thus had a three-pronged mission - commercial, political,

Norvegta made its \yay south, the sea temperature dropped quickly and very

and scientific. It was outfitted and financed by a leading whaling businessman,

soon another hazard - icebergs - appeared. On November 30 the crew got

but equipped

a first glimpse of Bouvet Island, but a snowstorm prevented the crew from

largely for research. Thc young zoologist aboard, Ditlef

Rustad, was to carry out investigations of marine life, paying close attention to

rowing ashore. Finally, on December

whale populations (Figure

erect a pole bearing the Norwegian flag (Figure 9.3)· The first mission had

9.2).

1,

it was possible to land a party and to

where it was prepared for the more treacherous leg of the voyage to Bouvet.

been accomplished.
Surveying of the waters around the island was constantly interrupted by

The ship was equipped with a coal-fired steam cngine, but could do just seven

storms, rough seas, and hazards to navigation. On some occasions it was very

knots at bcst on a calm sea. The "Roaring Forties" and the "Furious Fifties,"

difficult for the shore boats just 10 return to the mother ship. On December 3,

the names sailors had given to the wind- and storm-battered latitudes that lay

during a storm, the Norvegta struck a rock off thc coast of the island, which

further south, were anything but calm. The ship needed to take on all the coal

tore off some of her icc protection, damaged the keel, and opened a leak.

After two months at sea, the Norveg/a reached Cape Town, South Africa

Despite the hazards, the ship spent about a month around Bouvet. Rustad

it could in order to face the uncertain weather and to maximize its range as it

spent as much time as he could trawling for plankton and fish with a net. On

explored BoU\ et and its environs,

the day after Christmas. at a depth of about one hundred feet, he caught some

Sixty tons of coal were placed in sacks on deck, which soon created probthe rolling in

unusual looking fish he ca11 "crocodilc-fish'' on account of their large. procd

large waves, When water poured onto the ship's deck in rough seas, the

truding jm\'s full of teeth. About twenty inches long, they had large pectoral

lems. The sacks made the ship top-heavy and exacerbated

_ .............

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PART IV

EVOLUTION

CHAPTER

IN ACTION

9

IN COLD BLOOD

THE fALE Of THE

CEflSH

111

and tail fins, but were very pale, almosttransparCIll. When he sliced one open,
Rustad immediately noted their most peculiar feature of all - their blood was colorless. He took some photographs but did not keep the fish.
The leak in the Norveg/a grew to the point where hand pumps had to be used to keep her afloat. As the pack ice increased, and the coal supply decreased, the decision was made to head to South Georgia Island for repairs.
Rustad eventually made his way back home to Norway. The strange fish were well out of his mind, until two years later, when another zoology student brought them up.

A Bloodless Fish?
Johan Ruucl also traveled to the Antarctic, in 1929, aboard the factory ship

li'lCUR~;

Adult

Vikillgen. One day on deck, one of the old hands said to him, "Do you know

9.4 An ice(ish.

mackerel icefish, Cham/J$ocephalus

gllmwri.

there are fishes here thai have no blood?" Ruud knew that was impossible because he had learned from textbooks that one trait all vertebrates share is red

A few more samples trickled back to Norway, but these were always stored

blood, the color of which is due to the presence of oxygen-binding hemoglobin protein in red blood cells.

in preservatives that limited the kinds of information Ruud could extract. He

Thinking that the veteran was just having fun with him, Ruud replied, "Oh

himself. In 1953, nearly twenly-live years after his first journey and the first

needed fresh fish, and the only way to get those was to go back to the Antarctic rumors of the icefish, Ruud went to the Southern Ocean as the leader of an

ycs? Please bring some back with you." conversation as just shipboard lore. But when he returned to Norway in 1930

international whale-marking expedition.
A few days before Christmas, Ruud landed at South Georgia Island. He was

and mentioned the tall tale to Rustad, he was shocked when Rustad told him,

anxious to get some icefish and find out once and for all what was clifferent

"J have seen such a fish" and produced his earlier photographs to prove it. The

about them. In the first two days on South Georgia Island, before he could

crocodile fish or "icefish," as the whalers also called them, were very real.

even set up his makeshift laboratory, three specimens were brought to him.

When the shipmate rctumed empty-handed, Ruud dismissed the whole

Ruud, who subsequently became a professor of marine biology at the

l lc drew their blood and noticed right away that it was almost transparent

University of Oslo and leader of the Norwegian Institute for Whale Research,

(Figurc 9.5). Ile popped the samples into a refrigerator for later study, figuring

heard nothing more about the bloodless fish for nearly twenty years. Then in

that he would have all the fresh icefish whenever he wanted. When he cen-

1948, one of his students returned from an Antarctic expedition with some ice-

trifuged the blood, he obtained a pellet of cells comprising less than

fish he had caught in the Straits of Magellan. The student noticed that the gills

of the total blood volume. The plasma above was as "clear as water." More-

were also \\ hite, unlike the red gills of other fish. I Iis curiosity reignited, Ruud

over, he could not detect a singlc red blood ccllunder

asked other colleagues voyaging to the Antarctic to be on the lookout for, or better yet, to bring back more icefish (Figure 9.4).

icefish, unlike all other vertebrates, completely lacked the pigmented oX'ygencarrying cells that had, until then, been found in

I

I

~

every

I

percent

the microscope. The living vertebrate.

172

PART

IV

EVOLUTION

CHAPTER

IN ACTION

red-blooded

9

IN COLD BLOOD

THE TALE OF THE ICHISH

relatives. The icefish blood contained

l1J

no more oxygen than

would be carried by liquid plasma alone.
Ruud had his answers and reported his findings in Nature in 1954· Even more than fifty years later, his discovery is a shock for any biologist reading it for the first time. The icefish, a group of sixteen species, are the only "bloodless" vertebrates to have ever been discovered.
As Ruud noted in his paper, "The fact that a good-sized vertebrate can exist without any oxygen-binding blood pigment raises some interesting questions."
These questions included where, when, why, and how did these fish evolve?
What happened to their hemoglobin? And, how could these fish survive without hemoglobin or red blood cells?

Ruud surmised that the answers to these questions would be linked to the icefishes' unique ecological setting: the very cold and stable temperature of the food- and oxygen-rich waters of the Antarctic. He believed that only in such a unique combination of ciTcurnstances "is the survival of these peculiar animals possible." Answers to the mysteries posed by the icefish would not unfold for decades, until a new generation of scientists traveled to the Antarctic and brought new
F I GU RE

9· 5 Ice{1sh blood.

approaches to studying icefish history - by analyzing not just their blood, but

Compared with the red blood of a rock cod (left), icefish blood is white (right) and contains only a small volume of white cells and no red cells. Photograph from
B. D. Sidell and K. M. O'Brien, Journal of Experimental Biology 209:1791-1802
(2006), used by permission.

their DNA. For the DNA of icefish and their relatives harbors a detailed record of how these remarkable animals have evolved - of the genetic changes that have shaped their unique history. These genetic changes offer some remarkable glimpses into "evolution in action."

Ruud was eager to measure the oxygen-carrying capacity of fresh icefish blood and to compare it with red-blooded fish in the same waters. But, as luck

A Matter of Degrees

would have it, no more icefish beyond the first thrce appeared in his traps or were hooked.

Day after day passed as Ruud's scheduled

departure

Before confronting

date

the mystery of the anemic icefish, biologists first con-

Desperate, he sent out an appeal to all of the whaling stations on

fronted the mystery of why (and how) there are any fish at all in Antarctic

the island. To his delight and relief, a doctor at one of the two stations showed

waters. It came as a big surprise to some of the first explorers of the region that,

up in the nick oftime with a live fish in a barrel of sea water.

contrary to the inhospitable

approached.

habitat they imagined, the coldest waters in the

and found that icefish blood had an oxygen capacity of

world are alive with fish.
How cold are the Antarctic waters? The waters of McMurdo Sound have

0·77 percent by volume, compared with capacities of around S.o percent for its

been found to average about -1.9°C (28.6°F), and to vary by only about o.r'C

Ruuc! went right to work and drew blood from the heart. He took eight separate measurements

...........

174

PART IV

EVO.UTlON

IN A(' TION

CHAPTLR 9

IN COLD BLOOD

THE lAt (OF

THE I([FISH

175

with depth or through the seasons. Most tropical and temperate fishes freeze at around -o.8°C, so Antarctic fish must have evolved some means to cope with the icy Southern Ocean.
One group of fish, the suborder Notothenioidei, of which icefish constitute one family, dominates Antarctic

waters. Notothenioids

comprise about

one-third of all Antarctic fish species and about 90 percent of Antarctic fish biomass. Notothenioids are unknown from the Antarctic fossil record as recently as forty million years ago, when the Antarctic coastal waters abounded with

Antarctica

sharks, rays, catfish, and other groups of fish that arc now vanished. This dramatic turnover in fish fauna is associated with dramatic changes on land and in the ocean. About thirty-three to thirty-four million years ago, Antarctica became fully separated [rom South America and completely surrounded by deep ocean. Ensuing changes in ocean currents formed what is called the
"Antarctic Convergence," which isolated the waters around the Antarctic and limited the inflow of warm waters and the migration of fish from northern waters (Figure 9.6). It is thought that over the span of twenty million years or so, the water temperatures around Antarctica plummeted from roughly

woe

(5 °F) and reached belov, freezing by about fifteen million years ago. The
0

notothenioids have thrived in this icy habitat, which leads to the question,
"How do these fish endure the extreme cold and avoid freezing?"
The biologist who has made the most contributions to our understanding of

.. ~ ~

the adaptation of notothenioids to the cold Southem Ocean isArthur DeVries.

Antarctica

Now a Professor at lhe University of Illinois. Devries has ventured to the
Antarctic more than forty times since 1961, beginning when he was a graduate student. DeVries grew up on a Montana farm \\ hich, while it did not prepare

)!!o,~r:'I':'I':' "' ' IB=-

\

L/

him for long sea voyages, gave him plenty of experience of working in the cold and snow.
From Ross Island, a small volcanic island forty miles from mainland
Antarctica, Devries and his colleagues launched a series of studies into the temperature tolerance of Antarctic fish. They caught the fish in traps and

with conventional hooks and lines in holes cut through fifteen feet of sea ice.
They had to make their holes over deep water to avoid the Weddell seals that

FICLH
96 The changing geology of Antarctica.
When Antarctica became fully separated from South America, this opened Drake's

passage and changed ocean currents such that Antarctic waters became \ cry cold and isolated. illustration b)' lack Cook, \-"oods Hole Cceanograplnc lnstitution.

Redrawn bv Leanne Old.~.

176

PART IV

EVOLUTION

CHAPTER

IN ACTION

DeVries

9

IN COLD

and his colleagues

BLOOD

THE

ICEFISH

TALE OF- THE

then analyzed

notothenioid

177

sera in order to

identify the components responsible for the freezing resistance. They found that a glyeoprotcin fraction of the serum was responsible for about 50 percent of the freezing point depression. wilh dissolved salts (NaCl, urea, and other compounds) responsible for the remainder. The notolhenioids arc chock full of these antifreeze glycoproteins (AFCPs), containing twenty lo thirty-five milligrams of antifreeze protein per millililer of serum (about half of all serum protein).
Subsequent structural studies revealed that AFCPs have a very unusual and simple structure. They are composed of four to fifty-five repeats of just three amino acids, threonine-alanine-alanine

(sometimes threonine-proline-alanine),

with a disaccharide (sugar) of N-acetylglucosamine

and galactose attached to

the threonine residue.
The main enemy of fish in polar waters is not so much lhc cold, but ice. In the -1.9°C water column there are small crystals of ice that, if they enter the fish via the gills or by ingestion, can nucleate the formation of larger ice crystals and freeze the fish. The AFCPs work by adsorbing to small ice crystals and lowering the temperature at which crystals can grow.
Warm-water fish do not have AFCPs, so the antifreeze genes must have been somehow invented by notothenioids.

One of the major quests in evolu-

tionary biology has been to understand how new gene functions arise, and the
AFGPs presented a perfect opportunity to explore this question. It took more
9.7 Fishing in the Antarctic.
7op, Art Devries standing on the ice sheet over McMurdo Sound, Antarctic. The
FIGURE

fishing hut is behind him, and Mt, Erebus is in the background.
(checkered

Bottom, Art DeVries

jacket), fishing through a large hole for antifreeze-bearing

fish.

than twenty-five years after the discovery of AFCPs to find out, but it was well worth the wait.
When a team at the University of lllinois, including Chi-Hing

Arthur DeVries, and Liangbiao Chen, isolated the AFCP genes from a giant

Photos circa 1961, courtesy of Art Devries, University of Illinois.

Antarctic toothfish (D;ssostichus ruined holes made in shallower

notothenioids,

water (Figure

9.7). Several species of

froze al=-z.o to

2.1oC,

just a fraction of a degree below the freezing temperature of the sea water, and well below the freezing point (-o.8°C) of temperate fish. This extra clegree or so of freezing-point depression means that notothenioids do not freeze at water temperatures that would freeze other fish.

171(/IIISon;), they noticed that parts of one

AFCP gene had striking similarities to parts of another gene that encodes trypsinogen, a digestive enzyme. The first exon of the AFCP, including the

some reaching more than fifty pounds, were lancled.

DeVries found that the sera of several notothenioids

Cheng,

5' untranslatecl sequence

and signal peptide, was 94 percent

identical

in

sequence to those of the trypsinogen gcne, the 3' end of thc AFCP gene was
96 percent identical to trypsinogen, and two introns were 93 percent identical.
These are extraordinary degrecs of identity among two functionally distinct genes and suggest that the AFCP gene evolved from an ancestral trypsinogen

PART IV

178

EVOLUTION

CHAPTER

IN ACTION

stop

Trypsinogen

.

.

IN COLD

BLOOD:

THE TALE OF THE ICEFISH

179

lrypsinogen gene and an evolving AFGP gene. This discovery

provided a very rare view of how a preexisting gene gave birth to a new gene and a new protein with a new function - one that ultimately allowed an entire group

',
~ A (it~:~~j:j"'~~l d
!17~yt~;'iT'41

of fish to invade and dominate perhaps the most challenging habitat on Earth.

\~",""'''...

AFGP

ancestral

9

The origin of the antifreeze proteins stands oul as a prime example of how evolution works more often by "tinkering" with materials that are available - in this case the sequence of an already existing gene - rather than by inventing new functions completely from scratch. It is also a stellar illustration of how

start

stop

Chimaeric gene ......

-

history. Examination of

the icefish DNA record would also deliver some big surprises .

.. _
................................

~n

species' DI'\A contains a record of their evolutionary

_ .... _-- ..

(87 ThrAlaAla)

Fossil Genes

9.8 The origin of the antifreeze genes.
The AIo'CP genes evolved from an ancestral trypsinogen-like gene.
FICURE

Top, the structure of a notothenioid trypsinogen-like gene. The first exon is shaded and stippled, the second through fiflh exons (E2-ES) are indicated, and Ihe start and stop codons are labeled. fl.liddle, the structure of an AFCP gene. The first and last exons bear very strong similarities Lothe first and sixth exons of the trypsinogen-like gene. The APCP repeats in exons l-.p are encoded by a nine base repeat that is homologous to a sequence spanning junction between the first intron and second exon of the trypsinogen gene. These sequence similarities indicate this AFGP gene was derived from a trypsinogen gene. Bottom, striking evidence of the origin of APGP genes from the trypsinogen-like ancestral gene comes in the form of a chimeric gene lhal contains all of the trypsinogen exons together with AFGP repeals inserted at the junction of intron I and exon 2. Figure is modified (rom C.-H. Cheng and L. Chen
(1999) Nature -f01: 4-+3-444. Drawn b)' Leanne Olds.

The riddle of what happened

to iccfish hemoglobin

also had to wait a long

time before it was solved. More than forty years after Ruud first analyzed and described icefish blood, teams led by two Antarctic veterans, Bill Detrich of
Northeastern University and Guido di Prisco of the Consiglio Nazionale delle
Ricerche (Naples, Italy), probed icefish DNA for their adult a-globin and
[3-globin genes. In red-blooded fish, these two genes are located next to one another in a head-to-head orientation. But in all icefish species examined, only a remnant of the a-globin gene was detected and no [3-globin sequences were detectable (Figure 9.9). The p-globin gene and all but the third exon and a bit of the second intron of the a-globin gene were gone - extinct. The remaining chunk of the a-globin gene is a molecular fossil, or pseudogene, a vestige of

gcne. Furthermore,

the learn noted a nine base pair segment straddling a

spl ice junction in the hypsinogen gene that encoded a Thr-Ala-Ala tripeptide,

the red-blooded way of life in icefish ancestors that was abandoned.
Red blood cells and globin genes are found in all other fish and vertebrates,

the repeat building block of AFGPs. The team surmised that the man)' tripep-

which tells us thal hemoglobin has nurtured vertebrate respiration for over five

tide repeats of the AFGP gene evolved by repeated

hundred million years. How and why can icefish do without it?

duplication

of DNA

As Ruud surmised, the answers to these questions pivot on the cold, oxygen-

containing this short segment.
This model for the origin of AFGP received the best confirmation imaginable when Cheng, Devries, and Chen later isolated another AFGP gene that was clearly a chimeric

gene that encoded

bol17 AFGPs and trypsinogen

(Figure 9.8). They had captured an evolutionary intermediate

..

between an

rich Antarctic waters. The frigid cold presents a variety of challenges to body physiology. At low temperatures, body fluids. Elimination

one difficulty is the increased viscosity of

of red blood cells certainly reduces the viscosity

of blood and makes il easier to pump. But there remains the problem of

180

A
(X-

EVOLUTION

INCOlDBLOOD.rHETALEOl'lHEI('HISH

CHAP'eR9

IN ACTION

hcmoglobinless

B

GLOBIN

nototheniold
Q,~

PART IV

hlood more often than their red-bloodcd

hearts diffcr in another obvious and peculiar way -

~ - GLOBIN

181

cousins. Icefish

they arc oftcn pale. The

reddish color of venebraLe hearts (and muscles) is due Lothe presence of myo-

icefish

-v.c ~c c~c~ C'f. -v.'a>

Q,~

-v.c ~c

globin, a protein which binds oxvgcn more lightly than hemoglobin

C~ C~ C'f. -v.~

and

sequesters it ill muscles so that il is available upon exertion. But unlike most other fish and vertebrates in general, myoglobin is absent from ieefish muscles

- 14.1
- 14.1

-6.4

and the hearts of several, but not all. iccfish species.
Another Antarctic veteran, Bruce Sidell, [rom the University of Maine, and

-4.8
-6.4

his colleagucs have [ound that the myoglobin genes of pale-hearted

-3.7

also bear crippling mutations. Curiously,

icefish

different mutations have been identi-

fied in different species: in two species there is a five base pair insertion thai
-4.8

-2.3

disrupts the reading frame of the gene; in another species there is a fifteen base

-3.7

-1.9

pair insertion upstream of the gene; and in a yel another species there is a

-1.4
-1.2

mutation

-2.3

-1.9
-1.4
-1.2

in the mRNA polyadenylation

signal sequence downstream of the

stop codon. In these species, the myoglobin gene is a molecular fossil.
Because the three myoglobin genc mutations arc different, it appears that myoglobin function or expression has been lost at least three separate times within the icefish family. The presence of myoglobin in the hearts of ten species reveals that the function of myoglobin in icefish is probably in the process of being lost. One explanation for the incomplete loss of myoglobin in

~·IGI RF 9.9 The loss of globin genes in icefish.
Southern blots of genomic DNA from three reel-blooded notothenioid species (Cg,
Nc, Pc) and three ice fish species (Ca. Cg, Cr) were probed with cD;>.J.i\s or a-globin f (A) or I3-globin (B). Size standards are on the right of each blot ('\1a). Note the absence of an}' signal with the j3-g10binprobe in the iccfish. Figure modified from
E. Cocca et al., Proceedings of the National Academy of Sciences LISA.92:d3'7-1821
(1995); copyrigh/1995 the National Academy o(Sciences, USA.

oxygen delivery -

how do the icefish get enough oxygen to their tissues?

Several features of the ieefish cardiovascular system seem to meet this chal-

icefish may be the relatively young age of the icefish family (estimates range from two to eight million years). Even if myoglobin is not essential to icefish physiology, there may not yet have been enough time for mutations to inactivate the genc in all icefish line,'ges.
Whatever the fate of the myoglobin gene in the remaining species of icefish, it is clear thai several species

or icefish

arc getting along [ust fine without

two ancient oxygen-carrying proteins that have nurtured vertcbrate life for over half a billion vears.

lenge. Icefish have relatively large gills and have evolved a scaleless skin with unusually large capillaries. These features increase the absorption of o:";ygen from the environment.

limes larger blood volumes than fish of comparable size. It appears that icefish compensate The DNA Record of Evolution

Icefish also have much larger hearts and about four

for the lack of active oxygen transport bv circulating their dilute

Paleontologist

George Gaylord Simpson

once staled that "species evolve

exactly as if the) were adapting as best tl1