DNA Barcodes Methods and Protocols 2012th Edition W. John Kress
DNA Barcodes Methods and Protocols 2012th Edition W. John Kress
DNA Barcodes Methods and Protocols 2012th Edition W. John Kress
DNA Barcodes Methods and Protocols 2012th Edition W. John Kress
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9. DNA Barcodes
Methods and Protocols
Edited by
W. John Kress and David L. Erickson
DepartmentofBotany,NationalMuseumofNaturalHistory,
SmithsonianInstitution,Washington,DC,USA
11. v
Foreword
The diversity of life in a hectare of reef, a county of grassland, or a shipload of imports
challenges biologists called to identify the species comprising biodiversity, functioning as
ecosystems, or invading ports. The sequences of black-and-white barcodes that empower a
newly hired clerk to wave a wand over a cart full of goods swiftly, print an itemized receipt
infallibly, and order replacements invisibly call forth a vision of an analog for identifying spe-
cies. The resemblance of barcodes on commercial products to sequences of DNA shown as
black-and-white bars on electrophoretic gels reinforced the vision back in 2003 in the found-
ing meetings of the barcode of life movement. This book edited by early adopters of DNA
barcodes, John Kress and David Erickson, proves the barcode of life has arrived in environ-
mental science. In less than a decade, they and the other authors in this volume have realized
the vision of a short DNA sequence on a uniform locality of the genome to identify species
rapidly and accurately.
Because the currency in biology is species, their identification is no academic diversion.
Biologists count the rise and fall of biodiversity in species. Regulators designate endangered
species by their identified populations and reserve land where they identify the endangered.
Governments appraise the success of preservation in the currency of species. Inspectors
define quarantines in identified species. Biologists carry the weight of these consequences
as they select an exact name from almost two million known species names or conjecture
that a specimen may belong to one of millions more unknown species.
Written as a sequence of four discrete nucleotides—CATG—along a uniform locality
on a genome, a barcode of life provides a “digital” identifying feature, supplementing the
more analog gradations of shapes, colors, and behaviors. A library of digital barcodes will
provide an unambiguous reference that will facilitate identifying species invading and
retreating across the globe and through centuries.
Making a difficult task harder, many species metamorphose into different forms as they
cycle through stages in their lives. Eggs may become caterpillars and caterpillars become
butterflies but, of course, all remain the same species carrying the same genes. Different
species may resemble one another or be too small to distinguish easily but each carries dif-
ferent alleles and thus barcodes, which can unmask their identity. Furthermore, an inspec-
tor of unloaded cargo on a dock or an analyst of the remains of diets in a stomach may be
called to identify species from only a snippet, a hair, or a fin. The fragment may be unrec-
ognizable, but it will faithfully carry the identifying barcode of the source.
Since Carl Linnaeus (1707–1778) developed systematic naming, ranking, and classify-
ing of organisms, biologists have produced master keys to all knowledge about a species in
the form of binomial names. Biologists use distinguishing features, such as shape, color, or
number of legs in taxonomic keys first to assign binomials, like Homo sapiens, and then to
associate the names of the organisms with biological knowledge about the species and its
relatives. Of course, the bank of names suffers some problems, such as when several names
are applied to one species [1]. And, biologists continuously debate criteria for species. The
diversity of life from bacteria to whales renders any single rule inadequate for defining all
species. Nevertheless a few basic criteria, such as that distinct species do not interbreed and
meld their genetic sequences, serve for many groups.
12. vi Foreword
Since Charles Darwin (1809–1882) proposed a branching pattern of evolution in On
the Origin of Species, biologists have sought to arrange a phylogenetic system of species on
an evolutionary tree of life. A tree of life illustrates every introductory biology text.
Barcoding will reveal whether a newly collected specimen belongs to a species already on
the tree. Or if a specimen is a truly new species, barcoding will help place it as a new leaf
among known species on the proper branch of the tree of life.
Whatever the criteria for defining and recognizing species, their inheritance and their
genes must differ to maintain species distinctions generation after generation. Since the
molecular discoveries of the mid-twentieth century, genes intimate a code comprising
sequences of the four nucleotides that constitute DNA. Even before the barcoding move-
ment now embodied in the 200 member organizations from 50 countries of the Consortium
for the Barcode of Life (CBOL), scientific revisions of species boundaries included DNA
analysis, and the ability to distinguish new species included DNA divergences.
The product barcode analogy leant impetus to the continuing matching of species and
genetic differences. Commercial barcodes must be uniform across shelves and warehouses.
For animals, concentration on the single segment of the mitochondrial COI gene across the
far wider shelves of life imparted the necessary uniformity to avoid a Tower of Babel.
Conceiving the series of nucleotides CATG as bars and their presence and order as digital
bars opened the door to rapid and unambiguous connection of specimens. Instead of con-
necting biological specimens to shelves and suppliers, the DNA barcode of life connects
them to curated collections in museums and herbaria, lifting their utility. It would also con-
nect specimens to the biological literature of binomial names. DNA barcodes offer a glob-
ally consistent way to propose provisional or candidate species that experts have not yet
honored with a full description and binomial name.
Worries at first evoked by DNA barcoding have not been realized. It has heightened the
nuance of the species concept, not diminished it. It has widened humanity’s view of diver-
sity, not reduced diversity to ciphers. It has excited wonder at the knowledge hard-won
through earlier techniques and accessible through the master key of binomial names. It has
enhanced the need for systematists to match the flood of barcodes with a sound array of
binomial names. Barcoding is not a mere slogan and an inadequate analogy. It is a now
proven tool for understanding biodiversity.
Recurring to the need for uniformity to avoid a Tower of Babel, the choice of a seg-
ment of the mitochondrial COI gene has excelled for almost all animal taxa. This barcode
region meets four basic specifications: the locality must be present in all barcoded species;
it must be shaved as short as possible; the locality must have sequences stable within a spe-
cies through many generations; and it must nevertheless have sequences variable enough to
distinguish species. As this book reports, botanists have now also found barcode regions
that are proving successful from carrots and chamomile to oats and pines. Fungal barcodes
are not far behind.
Some observers do ask a single, searching question about the barcode of life arriving in
environmental science: When will it be small, cheap, and convenient enough for nonex-
perts, even children? In particular, when will the needed equipment shrink to the size of a
laptop or a handheld barcoder? In fact, even today the key machines have shrunk until they
fit comfortably on a desk or tabletop.
The analogy of the newly hired clerk faultlessly pricing the cart of goods suggests the
ability to make taxonomic expertise go further, and very far if a handheld barcoder were
present. Clues that such a goal will be achieved lie in reports of students detecting endan-
gered marine species on sale in supermarkets, identifying insect traces in their homes, and
13. vii
Foreword
analyzing tea leaves with inexpensive equipment. As well as enabling specialized scientists
to do more and lift the value of specimen collections, barcoding promises to enable laymen
to appreciate the diversity of life.
The array of opportunities offered by DNA barcodes must rest on a sound foundation
of binomial names with associated, vouchered, and identified specimens—housed in readily
accessible museum collections. A sound foundation of binomials based on new and existing
natural history collections stands as the first priority for the success of DNA barcodes.
Fortunately, the Global Names Architecture project associated with the Encyclopedia of
Life has already amassed 19 million common and scientific names and is reconciling them
for the two million or so species estimated to be known already. Within 5 years, we could
celebrate the achievement of the international Barcode of Life (iBOL) project: access to the
barcodes of an array of five million specimens sequenced from 500,000 species. Voucher
specimens, which are prepared, curated, databased, often digitally imaged, and stored in
natural history collections, will support this effort.
Already, in just a handful of years, the DNA barcode of life database (www.boldsystems.org)
has soared above 1.2 million specimens from about 150,000 species. Already, as the chap-
ters in this book show, the library of barcodes linked to names and curated specimens is
multiplying the knowledge of a marine ecologist about a reef, the quality of surveillance for
invasive species, and the accuracy of labeling of food products. Such successes will motivate
and sustain the further building of the reference library of barcodes and the removal of
obstacles for its quick, frugal realization and use.
Our vision, first inspired by a barcode wand in the hand of a supermarket clerk, is com-
parable magic for an ichthyologist on a research vessel with featureless fish larvae, a child on
a woodland trail, or an inspector at a port infallibly identifying a species. Reading this book,
we learn that science can make magic.
New York, NY, USA Jesse H. Ausubel
Alfred P. Sloan Foundation
Reference
1. Patterson DJ, Cooper J, Kirk PM, Pyle RL, Remsen DP (2010) Names are key to the big new biology.
Trends Ecol Evol 25:686–691.
16. “Perhaps some one had better remain at the camp,” said
Merriwell, with a wink. “Otherwise the wolves will come and eat up
our provisions.”
Hans came out from under the blanket as if he had been suddenly
stung by wasps.
“Vollufs!” he gasped. “Meppe dey voult ead der brovisions instit uf
me, t’inkin’ I vos dhem! Shimminy Gristmas! Vollufs! Vy didn’t you
tolt me dere vos vollufs on dis islant?”
Merriwell did not answer. Having sent back that call to Diamond,
he hurried into his clothing. Then he ran from the tents in the
direction of the calls, with John Caribou running at his side, and the
other members of the party trailing behind.
“Vait!” Hans was bawling. “Vot made me in such a hurry do run
avay from you?”
Then he heard the crashing of the bushes, and, thinking the
wolves were coming, he picked up a gun and a heavy case of
ammunition and hastened out of the tent.
“Vait!” he screeched. “Vait! Vait!”
He was in his white nightshirt, and his head and feet were bare.
With the gun in his right hand and the heavy ammunition case
tucked under his left arm, he was as comical a figure as moonlight
ever revealed, as he wallowed and panted after his comrades.
“This way!” shouted Diamond, hearing their movements.
The big cats began to grow uneasy, for they, too, heard that rush
of footsteps across the island, though the sound was still some
distance away. One of them got up and walked to the foot of the
ledge, as if it had half a notion to climb up and try conclusions with
Diamond at close quarters. But it merely stretched up to its full
height against the rock and drew its claws rasping down the face of
the rock as if to sharpen them.
“Not a pleasant sound,” was Diamond’s grim thought.
17. The loup-cervier retreated, after having gone through this
suggestive performance, and again sat bolt upright beside its mate
and stared at the prisoner with shiny bright eyes.
But they became more and more uneasy, as the sounds of
hurrying feet came nearer and nearer, and at last rose from their
sitting posture.
Once more Diamond funneled his hands.
“Don’t come too fast,” he cautioned. “There are some wildcats
here that I want you to shoot. You’ll scare them away.”
“All that scare for that!” laughed Merriwell, dropping into a walk. “I
thought he was in some deadly peril.”
“I’m just wanting a wildcat,” said Hodge, pushing forward his gun
to hold it in readiness. “No close season on wildcats, is there,
Merry?”
“Think not,” Merriwell answered. “You go on that side with
Browning and Caribou, and I will go on this side. Look out how you
shoot. Don’t bring down one of us, instead of a wildcat.”
“Vait! Vait!” came faintly to their ears from Hans, who was
struggling through the bushes, having fallen far behind in spite of his
frantic haste. “Vai-t-t!”
As a seeming answer came the report of Merriwell’s gun.
One of the cats, scared by the noise of the approaching force,
sprang away from the foot of the rock and scampered toward the
cover of the trees. Merriwell saw it as it ran and fired.
Instantly there was an ear-splitting howl.
The other cat leaped in the other direction and was shot at by
Bart Hodge.
The young Virginian descended from the ledge in anything but a
pleasant mood.
18. “They’re loup-cerviers, and they had me treed nicely,” he said;
“but you got one of them, for I heard it kicking in the bushes after it
let out that squall. I tumbled into their nest a while ago and that
seemed to make them more than ordinarily pugnacious. I came——”
He stopped and stared. At Merriwell’s side he saw John Caribou,
and he had been about to announce that he had followed Caribou
and seen him row out into the lake. Clearly he had been mistaken.
“What?” asked Merriwell.
“Better see if I’m right about that cat,” suggested Diamond, his
brain given a sudden and unpleasant whirl.
He was not in error about the cat, whatever he had been about
the guide. The biggest of the loup-cerviers was found dead in the
leaves, where it had fallen at the crack of Merriwell’s rifle.
While they dragged it out and talked about it, the young Virginian
gave himself up to some serious thinking. If that was not John
Caribou he had followed—and he saw now that it could not have
been—who was it?
The question was easier asked than answered.
However, he decided to speak only to Merriwell about it for the
present, and began to frame some sort of a story that should
satisfactorily explain to the others why he had left the camp.
Hans Dunnerwust came flying into their midst, dropping his gun
and the case of ammunition.
“Vollufs!” he gurgled. “One py my site peen shoost now! I snapped
his teeth ad me. Didn’d you see him?”
Hans’ wolf was the loup-cervier, which had run close by him as it
scampered away.
“Only a wildcat,” Merriwell explained, as he turned to Diamond.
“A viltgat!” screamed Hans. “Dot vos vorser yit. Say, I peen doo
sick do sday on dis islant any lonker. Vollufs mid wiltgats! Dunder
und blitzens! Dis vos an awvul blace!”
19. CHAPTER III.
HANS GOES FISHING.
The next morning the ledge of rock was visited where Diamond
had his adventure with the big cats, and he and Merriwell searched
along the shore for some marks of the canoe in which the nocturnal
visitor had made off. No young loup-cerviers were found, though a
hole was discovered between some roots near the base of the rock,
which the cats had no doubt used.
“I don’t understand it,” the young Virginian admitted, referring to
the man he had seen sneak away from the camp. “The only thing I
can imagine is that it must have been some one who hoped to steal
something.”
“Yes,” said Merriwell, thinking of the suspicions Diamond had
harbored against the guide. “Do you suppose Caribou could give us
any ideas on the subject, if we should tell him about it?”
“Don’t tell him,” advised Diamond, who still clung to the opinion
that John Caribou was not “square.”
The coming of daylight drove away the terrors that had haunted
Hans Dunnerwust during the night. He became bold, boastful,
almost loquacious.
When the sun was an hour high and its rays had searched out and
sent every black shadow scurrying away, Hans took a pole and line
and some angle worms and went out to a rocky point on the lake,
declaring his intention of catching some trout for dinner. He might
have had better luck if he had pushed off the shore in one of the
canoes and gone fly fishing: but no one wanted to go with him just
20. then, and he was afraid to trust himself alone in a canoe, lest he
might upset. This was a very wholesome fear, and saved Merriwell
much anxiety concerning the safety of the Dutch lad.
“Yaw!” grunted Hans, after he had found a comfortable seat and
had thrown his baited hook into the water. “Now ve vill haf some
veeshes. I don’d peen vrightened py no veeshes. Uf I oben my
moud and swaller do dot pait mit der hook on id, den der veeshes
run mit der line and blay me!”
He had slipped a cork on the line. The cork gave a downward bob
and disappeared for a moment in the water.
But before Hans could jerk, it came to the surface; where it lay,
without further movement.
“Dose veeshes vos skeered py me, I subbose!” soliloquized Hans,
eying the cork and ready to jerk the moment it appeared ready to
dip under again.
Finally he pulled out the hook and, to his delight and surprise,
found that the bait was gone.
“Hunkry like vollufs!” he said; then glanced nervously around, as if
he feared the very thought of wolves might conjure up the dreaded
creatures. “Vell, I vill feed mineselluf again mit anodder vorm.”
Baiting the hook he tossed it again.
Hardly was the cork on the wave when it went under. Instantly
Hans gave so terrific a jerk that the hook went flying over his head
and lodged in the low boughs of a cedar.
“A troudt!” gurgled Hans, in a perfect spasm of delight. “Vot I tolt
you, eh? A trout gid me der very virst jerk! Who vos id say dose
veesh coultn’t gadch me?”
A little horned pout, or catfish, about three inches long, was
dangling at the end of the line. It had swallowed the hook almost
down to its tail.
21. Hans Dunnerwust’s fat hands fairly shook as he disengaged the
line and tried to get the hook out of the pout’s mouth.
“Wow! Dunder und blitzens!” he screeched, dropping the pout
with surprising suddenness and executing a war dance on the shore,
while he caressed one of his fingers from which oozed a tiny drop of
blood.
“Shimminy Gristmas! I ditn’d know dot dose troudt had a sdinger
like a rattlesnake. I vost kilt!”
He hopped up and down like a toad on hot coals.
“Hello! What’s the matter?”
Frank Merriwell came round the angle of the rocky shore at that
moment, seated in one of the canoes.
“Why are you dancing?” he asked.
Hans subdued the cry that welled to his lips, trying to straighten
his face and conceal every evidence of pain.
“I shust caught a troudt,” he declared, with pride, “und scracht
mineselluf der pushes on.”
He held up the little horned pout that was still on the hook.
Merriwell propelled the prow against the shore and leaped out,
drawing the canoe after him.
“Yes; that’s a fine fish,” he admitted, trying to repress a smile of
amusement.
Hans was so jubilant and triumphant that it seemed a pity to
undeceive him.
“Und dot hunkry!” cried Hans, forgetting the pain. “He vos more
hunkrier as a vollufs. See how dot pait ead him und den dry do
svaller der line. I don’d know, py shimminy, how I dot hook gid oudt
uf my stomach!”
“Cut it open,” Merriwell advised.
22. “My stomach? Und me alife und kickin’ like dot? Look oudt! Dot
troudt haf got a sdinger apout him some blace.”
Merriwell gave the pout its quietus by rapping it with a stick on
the head, and then watched Hans’ antics during the cutting out of
the hook.
“Uf dey are hunkry like dot,” said Hans, tossing the line again into
the water, “ve vill half more vor dinner as der troudt can ead.”
“Spit on the bait,” suggested Merriwell. “It makes the angle worm
wiggle and that attracts the fish. If you had some tobacco to chew I
expect you could catch twice as many.”
Hans made a wry face.
“Oxcoose me! I vos—— Look oudt!” he squawked, giving the line
another terrific jerk. “Shimminy Gristmas! Did you seen dot? Dot
cork vent oudt uf sight shust like a skyrocket.”
The bait was still intact and he tossed in the line again.
“Dere must be a poarting house down dere someveres dot don’t
half much on der taple, py der vay dose troudt been so hunkry,” the
Dutch boy humorously observed. “Look oudt! he vos piting again!”
He gave another jerk, and this time landed a pout double the size
of the first.
His “luck” continued, to his unbounded delight; and in a little while
he had a respectable string of fish.
“Who told me I couldn’t veesherman?” he exultantly demanded,
struggling to his feet and waddling as fast as he could go to where
the last pout was flopping on the grass. “He haf swallered dot hook
again clean to his toes. Efery dime I haf do durn mineselluf inside
oudt to gid der hook!”
The horns of the pout got in their work this time, and Hans
stumbled about in a lively dance, holding his injured hand.
“Dose troudt sding like a rattlesnake,” he avowed. “Der peen leetle
knifes py der side fins on, und ven he flipflop I sdick does knifes indo
23. him. Mine gootness! Id veel vorser as a horned!”
“Shall I send for John Caribou?” asked Merriwell. “He has some
tobacco.”
Hans glanced at him in a hurt way, then extracted the hook, put
on another worm, and resumed his fishing.
A pout bit instantly, and Hans derricked it out as before; but the
line flew so low this time that it caught Hans about the neck, and
the pout dropped down in front, just under his chin, where it flopped
and struggled in liveliest fashion.
“Dake id off!” Hans yelled. “Dake id off!”
Merriwell tried to go to his assistance, but only succeeded in
drawing the line tighter about Hans’ neck.
“If you’ll stand still a minute, I can untangle the line, but I can’t
do anything while you’re threshing about and screeching that way,”
he declared.
The pout flopped up and struck Hans in the face, and thrust the
point of one of its fins into his breast as it dropped back.
This was too much for the Dutch boy’s endurance, and the next
moment he was rolling on the ground, meshing himself more and
more in the snarl of the line, and getting a fresh jab from one of the
pout’s stingers at each revolution.
“Hellup! Fire! Murter!” he yelled.
Finally the pout was broken loose, and Merriwell succeeded in
making Hans understand that the dreaded stingers could no longer
trouble him.
Hans sat up, a woe-begone figure. He was bound hand and foot
by the line as completely as Gulliver was bound by the Lilliputians.
“Are you much hurt?” Merriwell asked.
“Much hurted?” Hans indignantly snorted. “I vos kilt alretty! Dose
knifes peen sduck in me in more as sefendeen hundret blaces.
24. Bevore dose troudt come a-veeshin’ vor me again I vill break my
neck virst.”
It was impossible to untie the line, so Merriwell took out his knife
and cut it.
“This was an accident,” he said. “I shan’t say anything about it to
the others. Take the fish to camp, and we’ll have them for dinner.
They’re good to eat.”
As indeed they were.
Thereupon Hans’ courage came back. He washed his hands and
face in the lake, carefully strung the pouts on a piece of the severed
line, then waddled to camp with them, with all the proud bearing of
a major-general.
Frank Merriwell sat for a time on the point of rock, looking out
across Lily Bay. Then he started, as the sound of the deep baying of
hounds came to him from the mainland.
“They’re after some poor deer, probably,” was his thought. “The
only way to make a deerhound pay attention to the close season is
to tie him to his kennel.”
Though the sounds drew nearer, the dogs were concealed from
view in the woods of the mainland by a bend of the island.
At last there arose such a clamor that Merriwell entered the canoe
and paddled quickly round the point in the direction of the sound.
He came on a sight that thrilled him. A large buck, with a finely-
antlered head, had taken to the water to escape the hounds, and
was swimming across an arm of the bay, with the dogs in close
pursuit. Only the heads of the dogs were visible above the water, but
he saw that they were large and powerful animals.
At almost the same moment Merriwell beheld John Caribou rush
down the opposite shore and leap into a canoe—the other canoe
belonging to the camping party.
25. “What can Caribou have been doing over on the mainland?”
thought Frank. “Oh, yes; probably looking for another camping
place, for we were talking about changing last night.”
Caribou cried out to the hounds, trying to turn them from their
prey; and, failing in this, he pushed out in the canoe and paddled
with all speed toward the buck.
The hounds had overtaken it, and it had turned at bay, having
found a shallow place where it could get a footing.
The largest hound swam round and round it, avoiding its lowered
head; then tried to fasten on its flanks.
The buck shook it off, and waded to where the water was still
shallower, in toward the shore.
The dogs followed, circling round and round.
Caribou shouted another command and paddled faster than ever.
The shout of the guide and the buck’s deadly peril now caused
Frank Merriwell to push out also, and soon he was paddling as fast
as he could toward the deer and the dogs. But the separating
distance was considerable.
The shallower water aided the biggest hound, for it got a footing
with its long legs and sprang at the buck’s throat. The buck shook
the hound off and struck with its antlers.
“That’s it!” Merriwell whispered, excitedly. “Give it to them!”
The attacks of the three dogs kept the buck turning, but it met its
assailants with great gallantry and spirit. When the big hound flew at
its throat again, it got its antlers under him and flung him howling
through the air, to strike the water with a splashing blow and sink
from sight.
“Good enough!” cried Merriwell. “Do it again!”
The other hounds seemed not in the least bit frightened by this
mishap to their comrade, but crowded nearer, trying to get hold of
the buck’s throat.
26. The big hound came to the surface almost immediately, none the
worse for its involuntary flight and submergence, and swam back to
the assault.
Merriwell looked at Caribou, who was now standing up in the
canoe and sending it along with tremendous strokes.
“Hurrah!” Merry cried, not taking time to stop, however. “I’m
coming, Caribou, to help you.”
The largest hound again flew at the buck’s throat, while one of the
others, getting a foothold, climbed to the buck’s back.
But the advantage of the hounds was only temporary. The big
hound was again caught on those terrible antlers, impaled this time,
and when it was hurled through the air to sink again on the lake it
did not rise.
The hound that still remained in the water in front of the buck,
now caught the latter by the nose, and the buck fell with a threshing
sound. It rose, though, shaking off both hounds.
“Hurrah!” screamed Merry, sending his canoe skimming over the
water. “Hurrah! Hurrah!”
So admirable and plucky was the fight the buck was making that
he was fairly wild with admiration and delight.
John Caribou was close to the buck, and still standing up in his
canoe.
The hound that caught the buck by the nose now received a
thrust that tore open its side and put him out of the fight; but the
other one again leaped to the buck’s hip and hung there, refusing to
be dislodged.
At this hound John Caribou struck with his heavy paddle.
The blow was a true one. It tumbled the hound into the water,
where the guide came near following.
While Caribou sought to recover his balance, the buck, mistaking
him for a new enemy, turned on him and made a savage dash that
27. hurled him from the canoe.
Frank Merriwell was now so near that he could see the buck’s fiery
eyes, note the ridging of hair along its spine, and could hear its
labored and angry breathing. Its tongue protruded and was foam-
flecked.
Caribou tried to seize the sides of the canoe as he went down, but
the effort only served to hurl it from him, and send it spinning out
into the lake.
The buck put down its head for a rush; while the hound that the
guide had struck with the paddle blade did not try to renew the
fight, but began to swim toward the shore, which was not distant.
“Look out!” cried Merriwell, warningly.
Caribou heard the cry, saw the antlers go down and tried to dive.
But he was not quick enough. Before he was under water the buck
struck him a vicious blow.
Though half stunned, he clutched it by the antlers, to which he
clung desperately, while the buck struck him again, this time with
one of its sharp hoofs.
Caribou, realizing that his life was in peril, tried to get out his
knife, but the enraged and crazed buck bore him backward with so
irresistible a rush that Caribou was kept from doing this. Then he
went under the water again.
This time the buck seemed determined to hold him down till he
was drowned. Merry saw the guide’s hands and feet beating the
water, and knew from their motions that he was rapidly weakening.
“I’m coming!” he shouted, though he must have known that the
guide could hardly hear or comprehend.
With one deep pull on the paddle he put the canoe fairly against
the buck; then rising to his feet, he brought the blade down with
crushing force across the animal’s spine.
The buck half fell into the water and the antlered head was lifted.
28. When John Caribou came to the surface Merriwell clutched him by
the hair and pulled him against the side of the canoe, regardless of
the buck’s threatening attitude. Then, seeing that Caribou was
drowning, he lifted him still higher, so that the water no longer
touched Caribou’s face and head.
The buck put down its horns as if it meditated another rush.
Merriwell remained quiet, holding the guide’s dripping head. He had
a rifle in the bottom of the canoe, but he did not wish to use it
unless driven to kill the buck in self-defense. More than all else he
did not want to let go of the guide.
The buck stood for a moment in this pugilistic attitude; then,
understanding it was not to be attacked, it turned slowly and waded
toward the land.
The hound that had preceded it had disappeared, and the other
two were dead.
“How are you feeling, Caribou?” Merry anxiously asked, drawing
the guide’s head still higher.
There was no answer, and Merriwell lifted the guide bodily into the
canoe. Great caution was required to do this, together with the
expenditure of every ounce of strength that Merriwell possessed.
A ringing and encouraging cheer came from the shore of the
island, where the other members of the party had gathered, drawn
by the baying of the hounds and the noise of the subsequent fight.
Merriwell had no power of lung to send back a reply. Instead he
sank down by Caribou’s side and began an effort to restore him to
consciousness.
This was successful in a little while. The guide opened his black
eyes and stared about, then tried to get up. He comprehended at
once what had occurred, and a look of gratitude came to his dark
face.
“You’re worth a dozen drowned men,” announced Merry, in his
cheeriest voice. “If you can lie in that water a little while without too
29. much discomfort, I’ll try to catch your canoe with this one. The
waves are carrying it down the bay.”
John Caribou did not seem to hear this. His eyes were fixed on
Merry’s face.
“Caribou, him not forget soon! Not forget soon!”
Only a few words, but they were said so earnestly that Merriwell
could not fail to understand the deep thankfulness that lay behind
them.
30. CHAPTER IV.
HANS DUNNERWUST SHOOTS A DEER.
Two days later Merriwell’s party moved from the island to a high,
dry point on the mainland, where the tents were repitched and
where they hoped to spend the remainder of their stay on Lily Bay.
It was an ideal camping place, and freer from mosquitoes than the
island had been.
Hans told Merriwell quite privately that the stings of those island
mosquitoes were almost as bad as the stings of the “trout” he had
caught.
Except that the sun was torridly hot during the midday hours, the
weather was almost perfect. The skies were clear and blue, the bay
placid. Trout, genuine trout, took the hook readily. The canoeing was
all that could be desired. Merriwell, too, had secured some splendid
views of wild life with his ever-ready camera. One of the finest of
these was a trout leaping. When developed, the photograph showed
the trout in the air above the surface of the lake, with the water
falling from it in silvery drops, and its scales glinting in the sunlight.
Another fine view was a moonlight scene of a portion of Lily Bay,
from the headland where Hans had done his fishing.
“I shall always regret that I didn’t snap the camera on that buck
while he was making such a gallant fight against those dogs,”
Merriwell often declared. “That would have been great. But really, I
was so excited over the buck’s peril that I entirely forgot that I had a
camera.”
31. But he had caught other scenes and views, that were highly
satisfactory, if they did not quite compensate for the fine scene of
the combat between the hounds and the buck. Whose the hounds
were they had no means of knowing, but Caribou suggested that
they probably belonged to a gentleman who had a cottage not far
from Capen’s.
Highly as Merriwell regarded John Caribou, there could be no
doubt that there was something mysterious about his movements.
Merriwell had once seen him steal out of camp in the dead of night,
an act for which the guide had no adequate explanation when
questioned. In fact, Merriwell’s questioning threw Caribou into
singular confusion.
The day the camp was moved, Jack Diamond saw the guide meet
a stranger in the woods, to whom he talked for a long time in the
concealment of some bushes, in a manner that was undeniably
surreptitious. Still, Merry clung to his belief in the guide’s honesty.
Hans Dunnerwust had become valiant and boastful since his great
success at catching “trout.” He wanted to further distinguish himself.
“Uf I could shood somedings!” Merriwell once overheard him say
in longing tones.
This remark, which Hans had only whispered to himself, as it
were, came back to Merriwell with humorous force a couple of days
after the setting up of the camp on the mainland.
“If only Hans could have come across this!” he exclaimed.
It was a dead doe lying in the woods not far from the camp. It
had been shot, and after a long run had died where Merriwell had
found it, nor had it been dead a great while.
“The work of poachers,” said Merriwell, with a feeling of ineffable
contempt for men who could find it in their hearts to slaughter deer
in this disgraceful and unlawful manner. “I wish the strong hand of
the law could fall on some of those fellows.”
32. This was not the first evidence he had seen that poachers were
carrying on their dastardly work around that portion of Moosehead
Lake known as Lily Bay. A wounded deer had been noticed and
distant shots had more than once been heard. He was beginning to
believe that the dogs which had followed and attacked the buck
belonged to these poachers.
After pushing the deer curiously about with his foot, Merriwell was
about to turn away, when he chanced to see Hans Dunnerwust
waddling down the dim path, gun in hand. It was plain that if Hans
continued in his present course he could hardly fail to see the dead
deer.
“Just the thing!” Merriwell whispered, while a broad smile came to
his face. “If I don’t have some fun with Hans I’m a Dutchman
myself!”
He put down his camera and rifle, and, lifting the body of the doe,
stood it up against a small tree. By means of ingenious propping, he
contrived to make it stand on its stiff legs and to give it somewhat of
a natural appearance.
“It’s natural enough to fool Dunnerwust, anyway!” he muttered,
picking up the camera and gun and sliding into the nearby bushes.
Hans came down the path, carrying his rifle like a veteran
sportsman. He was looking for game, and he found it. His eyes
widened like saucers when he saw the deer standing in the bushes
by the tree.
“Shimminy Gristmas!” he gurgled. “Id don’d seen me, eidher! Uf
dot deer don’d shood me, I like to know vot vos der madder mit me,
anyhow! You pet me, I pud a palls righd t’rough ids head und ids
liver. A veller can shood a teers dot don’d ged any horns, I subbose,
mitoudt giddin’ arresded py dose game vardens! I vill shood him,
anyhow, uf I can. Yaw! You pet me!”
He dropped to his knees, then began a stealthy approach, for the
purpose of putting himself within what he considered good shooting
distance. He was less than eighty yards from the game when he first
33. saw it, but he knew so little about rifles that he doubted if his gun
would carry so far. It is not easy for a fat boy to crawl stealthily sixty
yards on his hands and knees, dragging a gun along the ground, but
that was the task that Hans Dunnerwust now set for himself.
Merriwell, hidden in the bushes, shook with laughter, as Hans
began this cautious advance. When half the distance was passed,
Hans rose to a half upright posture and stared hard at the deer. This
was an opportunity for which Merriwell had been waiting. He drew
down on Hans the camera, but scarcely able to sight it accurately for
laughing. The picture caught, showed Hans all a-tremble with
eagerness, his mouth wide open, his eyes distended and staring.
Assured that his game was still in position by the tree, Hans got
down on his hands and knees again and made another slow
advance.
When no more than twenty yards separated him from the deer, he
lifted himself very cautiously and drew up the gun to take aim. He
was shaking so badly he could hardly hold the weapon. Merriwell
focused the camera on him at this instant and caught another view
of this great hunter of the Moosehead country.
As he took the camera down, he saw Hans trying to shoot the gun
without having cocked it. Again and again Hans pulled the trigger,
without result.
“If only some of the other fellows were here!” Merriwell groaned,
fairly holding his sides. “He’s shaking so I’m afraid he won’t hit the
deer, after all.”
He had arranged the deer so that the slightest touch would cause
it to fall.
Hans put down the gun and anxiously turned it over. Then
Merriwell saw his puzzled face lighten. He had found out why the
weapon would not go off.
This time when he lifted the rifle it was cocked. Then he pressed
the trigger.
34. When the whiplike report sounded, the deer gave a staggering
lurch and fell headlong.
Hans Dunnerwust could not repress a cheer. He sprang to his feet,
swinging his cap, and ran toward the fallen doe as fast as his short,
fat legs would carry him.
“Id’s kilt me! Id’s kilt me!” he was shouting.
Fearing it might not be quite dead, he stopped and drew his
hunting knife. It did not rise, however, it did not even kick, and,
made bold by these circumstances, Hans waddled up to it and began
to slash it with the fury of a lunatic.
“Whoop!” he screeched. “I god id! I shooded id! I vos a teer gilt!
Who said dot Hans Dunnerwust coult nod shood somedings, eh?”
Merriwell trained the camera on him once more, as he stood in
this ferocious attitude, with the knife extended, from which no blood
dripped, and looked triumphantly down at the deer at his feet. Then
Merry rose and advanced.
Hans turned when he heard the snapping of the bushes, and was
about to bolt from the place, but, seeing that it was Merriwell, he
changed his mind and began to dance and caper like a crazy boy.
“You see dot?” he screeched, proudly pointing to the dead doe.
“Dot vos a teer vot kilt me shust now. Tidn’t you heered id shood
me?”
Merriwell’s face assumed a look of consternation.
“I’m very sorry you did that,” he declared.
“Vy? Vot you mean py dot?” Hans gasped.
“The game wardens are likely to hear of it.”
The face of the Dutch boy took on such a sickly look of fright that
Merriwell relented.
“But you didn’t think, I suppose?”
35. “Yaw! Dot vos id.” Hans asserted. “Id shooded me pefore I know
mineselluf.”
“Perhaps it will be all right for you to take the head in to show the
boys what you have done,” Merriwell suggested.
This was pleasing to Hans, and so in line with his heart’s desire,
that he immediately decapitated the doe, and proudly bore the head
into the camp, as proof of his skill as a deer-stalker.
36. CHAPTER V.
MOOSEHEAD EXPRESS.
“A moose!”
“Cricky! Isn’t he a fine one?”
“Him plenty big!”
The first exclamation was from Merriwell, the second from Bruce
Browning, the third from John Caribou, the guide.
The three were in a canoe, which had been creeping along the
wooded shore of a narrow arm of Lily Bay.
“Reach me the camera,” whispered Merriwell.
The camera was at Browning’s feet and was quickly handed up.
John Caribou was sitting in one end of the canoe as silently as an
image of bronze.
The big moose that had not yet seen them, stepped from the
trees into full view, outlining itself on a jutting headland, as it looked
across the sheet of water.
Even the impassive guide was moved to admiration. A finer sight
was never beheld. The moose was a very giant of its kind. With its
huge bulk towering on the rocky point, its immense palmated antlers
uplifted, its attitude that of expectant attention, it presented a
picture that could never be forgotten.
Frank Merriwell lifted the camera, carefully focused it on the big
beast and pressed the button.
37. He was about to repeat the performance when something stirred
in the trees a hundred yards or more to the left, and Hans
Dunnerwust came into view.
He did not see the canoe and its occupants, but he saw the
moose, and he stopped stock still, as if in doubt whether to retreat
or proceed on his way.
The moose had turned and was looking straight at him, with
staring, fear-filled eyes. Then it wheeled with surprising quickness
for so large a beast and shambled off the headland toward the
water’s edge.
This increased Hans’ courage. He was always very brave when
anything showed fear of him. He had been on the point of turning in
flight, but now he sprang clear of the trees, and ran toward the
moose with a shout.
“A teer! A teer! Another teer!” he screeched, waving has hand and
his gun.
Merriwell snapped the camera on the moose as it scrambled down
the slope.
“Might have another negative of it standing, if Hans hadn’t put in
an appearance,” he declared, feeling at the moment as if he wished
he might give the Dutch boy a good shaking.
But he had reason in a little while to call down blessings on the
head of Hans for this unintentional intervention.
Frightened by Hans’ squawking and the noise he made in running,
the moose dashed up and down the shore for a few moments, then
took to the lake.
“There he goes,” whispered Browning, roused to a state of
excitement.
“Plenty skeer!” said Caribou. “Sometime moose him skeer ver’
easy.”
38. “He’s going to swim for the other shore,” declared Merriwell,
putting down the camera and then picking it up again.
For a few yards the frightened moose made a tremendous
splashing, but when it got down to business, it sank from sight, with
the exception of its black neck and head and broad antlers, and
forged through the water at a very respectable rate of speed.
Merriwell focused the camera on the swimming animal and was
sure he got a good picture, then put down the camera and picked up
his rifle. He wanted to get nearer the big beast, and he knew he
would feel safer with a weapon in his hands in the event of its
urgent need.
“Fun now, if want?” said the guide, suggestively, looking toward
the moose with shining eyes. “Much fun with big bull moose in water
some time.”
“A little fun won’t hurt us, if it doesn’t hurt the moose,” responded
Merriwell, who as yet hardly knew just what was in the guide’s mind.
“Eh, Browning?”
“Crowd along,” consented Browning. “I don’t mind getting close
enough to that fellow to get a good look at him. If it wasn’t out of
season I’d have that head of horns!”
“Aren’t they magnificent?” asked Merriwell, with enthusiasm.
The guide looked at Merriwell as if to receive his assent.
Hans Dunnerwust had rushed to the shore in a wild burst of
speed, and was now hopping wildly.
Suddenly he caught sight of Merriwell and the others in the canoe.
“A teer! A teer!” he shrieked. “Didn’t you seen him? He roon vrum
me like a bolicemans, t’inking dot he voult shood me. Put noddings
vouldn’t shood me oudt uf seasons!”
“I don’t know about that,” grunted Browning. “Fools, as game, are
never out of season, and the fool-killer is always gunning for them.”
39. “Yes; go on,” said Merriwell to the guide. “As I said, a little fun
won’t hurt us if it isn’t of a kind to hurt the moose. See how he is
swimming! That’s a sight to stir the most prosaic heart.”
John Caribou did not need urging. He dipped the paddle deeply
into the water, and the canoe shot away in pursuit of the swimming
animal.
The moose was already some distance from land, and forging
ahead with powerful strokes; but under the skillful paddling of the
guide the canoe quickly decreased the intervening distance.
It was worth something just to watch John Caribou handle the
broad-bladed paddle. He dipped it with so light a touch that scarcely
a ripple was produced; but when he pulled on it in a way that fairly
bent the stout blade, the canoe seemed literally to leap over the
waves. Every motion was that of unstudied grace.
Browning could not remain stolid and impassive under
circumstances that would almost pump the blood through the veins
of a corpse. He grew as enthusiastic as Merriwell.
“See the old fellow go!” he whispered, referring to the speed of
the moose. “He’s cutting through the water like a steamboat.”
The guide rose to his feet, still wielding the paddle.
“We’ll be right on top of him in a minute,” said Merriwell. “Look
out there, Caribou! He may turn on us. We don’t want to have a
fight with him, you know.”
Caribou did not answer. He only gave the canoe another strong
drive forward, then dropped the paddle and caught up an end of the
canoe’s tow line, in which he made a running noose.
He stood erect, awaiting a good opportunity to throw the line. The
canoe swept on under the propulsion that had been given it. Then
the noose left Caribou’s hand, hurled with remarkable precision, and
fell gracefully over the broad antlers. Instantly Caribou grasped the
paddle and whirled the canoe about so that the stern became the
bow.
40. “Hurrah!” cried Merriwell, half expecting that the moose would
now turn on them to give them battle. “That was a handsome throw.
I didn’t know you were equal to the tricks of a cowboy, Caribou.”
The guide did not answer. Very likely he did not know the meaning
of the word cowboy.
In another instant the line tightened, and they were yanked swiftly
along.
“Towed by a moose!” exclaimed Browning. “That’s a new
sensation, Merry!”
“Yes; this is great. This is what you might call moose-head
express,” laughed Frank.
“It’s enough to make a fellow feel romantic, anyway,” grunted
Browning. “Pulled by a moose on Moosehead Lake, with an Indian
guide to do the steering.”
The moose was now badly frightened, and showed signs of
wanting to turn around, whereupon the guide picked up the paddle
and gave it a tap on the side of the head.
This brought a floundering objection from the scared animal, but it
had, nevertheless, the desired effect, for the moose again started off
smartly for the opposite shore, drawing the canoe after it.
The big beast did not seem to be tired, but it puffed and panted
like a steam engine.
“That’s right, Caribou,” cried Merriwell, approvingly. “Just hang on
and let him go. I don’t mind a ride of this kind. It’s a sort of sport we
weren’t looking for, but it’s great, just the same.”
“Much fun with big bull moose in water some time,” Caribou
repeated. “Drive big moose like horse.”
Then the guide gave them an exhibition of moose driving. By
yanking this way and that on the line, he was able to alter the
moose’s course, and that showed that he could turn it almost at his
will.
41. Not once did the moose seek to turn and fight as Merriwell had
thought he would do if lassoed. It seemed only intent on getting
away from its tormentors, and appeared to think the way to do that
was to swim straight ahead toward the land as fast as it could.
Hans was still hopping up and down on the shore, and now and
then sending a screech of excitement and delight across the water.
After he had shown that the moose could be turned about if
desired, Caribou let the scared animal take its own course. The
distance across was considerable, and he knew the moose would be
tired by the swim.
He held the line, while Frank and Bruce sat in their places enjoying
the novel ride to the fullest extent.
Thus the canoe was towed across the arm of the bay, giving to
our friends an experience that few sportsmen or tourists are able to
enjoy.
As the moose neared the shore, Caribou severed the line close up
to its antlers and let it go. It was pretty well blown, as the heaviness
of its breathing showed.
Scrambling out of the water, it turned half at bay, as if feeling that,
with its broad hoofs planted on solid ground it could make a stand
for its life; but when the occupants of the canoe showed no intention
of advancing to attack it, it gave its ungainly head a toss and
shambled away, the severed end of the noose floating from its
antlers.
Merriwell caught up his camera and snapped it on the moose
before it entered the woods, so getting a picture of a moose fresh
from a swim in the lake, with its shaggy sides wet and gleaming.
Then the moose broke into an awkward run, and was soon lost to
view.
A half hour later, while they were still paddling along the shore,
they heard a shot from the woods, in the direction taken by the
moose.
43. CHAPTER VI.
AN UNPLEASANT SITUATION.
That afternoon an eccentric figure came capering through the
woods, bearing a strange burden. Perhaps capering is not the exact
word to use, for the figure was that of a rotund and fat-legged boy,
and it is hard for such a person to caper. Ever and anon this figure
sent up a pleased exclamation or a cry of delight.
“Anodder teer’s head!” he shouted, when he came in sight of the
camp. “A moose’s teer head this dime, I pet you!”
It was Hans Dunnerwust, and the burden under which he waddled
was the head of a moose. He tried to hold it triumphantly aloft as he
shouted his announcement, and while making this attempt struck a
foot against a protruding root, and went down in a heap, the
antlered head falling on top of him.
“Mine gootness!” he gasped, sitting up and rubbing his stomach,
while he looked excitedly around. “I t’ought, py shimminy, dot
somepoty musd hid me, I go town so qvick!”
His eyes fell on the head, and the pleased look came again into
his face.
“I pet you, I vill pe bleased mit Merriwell, ven he seen dhis. Dot
odder teer got no hornses, und dis haf hornses like a dree sdick up.
Id must pe vort more as lefendeen tollar, anyhow!”
After climbing to his feet and assuring himself that he had not
sustained any serious injuries or broken bones, he picked up the
heavy head and again hurried on, giving utterance to many
exclamations of pleasure and delight.
44. Hans had found the head hanging in the branches of a tree, in a
way to keep it out of the reach of carnivorous animals. Had he not
been looking for a red squirrel, that had gone flickering through
these very branches, he never would have discovered the head, so
cleverly was it hidden.
“Dot is a petter head as dot odder vun I got,” he had whispered,
wondering dully how it chanced to be there, but not for a moment
thinking of poachers.
There were marks on the earth and grass showing where the
moose had been skinned and cut up.
“Dose vellers don’d vand der head,” was his final conclusion, “und
day chust hang id ub here. Vale, I vill dake id mineselluf, den!”
Then he had fastened his knife to a stick and, after many futile
attempts, had succeeded in cutting the string by which the head was
suspended from the bough.
“Whoop!” he screeched, when he drew near the tent. “Yaw. See
vot got me, eh? A moose’s teer head got me de horns py!”
It was a hot afternoon, and the sweat was fairly streaming from
his round, red face. He was panting, too, almost as loudly as the
moose had panted while it drew the canoe across the water.
Merriwell and Diamond came to the door of one of the tents, and
Browning, Bart Hodge and John Caribou looked from the other.
A more astounded party would have been hard to find.
“Where did you get that?” asked Merriwell, thinking at once of the
shot they had heard in the direction taken by the moose.
“Id is a moose’s teer head,” announced Hans, holding it up. “See
mine hornses?”
“I can see that it is a moose head; but where did you get it?”
The other members of the party were as surprised as Frank and
equally as anxious for an answer to his questions. The guide looked
as if he might have given an answer himself, but he only folded his
45. arms and stared at the head with shining eyes and impassive
features.
“Pushes vos hanging to him in a dree,” said Hans, and then, in his
own peculiar way, he proceeded to make them acquainted with the
manner in which he discovered it.
He put it down on the grass in front of the tent, where it was
closely scrutinized.
“Same moose we saw this morning,” declared Bruce Browning,
very emphatically. “Do you see that peculiar turn of the horn there? I
noticed that on the fellow that towed us. Some scoundrel has shot
him.”
“There can’t be any doubt of that, I guess,” admitted Merriwell, in
a grieved tone. “What a magnificent beast he was, too! It is a
shame. I hope the rascal will be caught and punished, but I don’t
suppose he ever will be. This is a pretty wild country out here.”
“I tell you what,” said Hodge. “Whoever killed that moose will
come back for the head. Those antlers are worth something, and he
won’t want to lose them. How would it do to hide out there and see
if we can’t capture him?”
“The only trouble about that,” objected Diamond, “is that we’d
have to take the scamp before some justice of the peace and waste
a lot of time in trying to get him convicted. Nothing is slower than
the law, you know.”
“See there!” exclaimed Merriwell, who had been closely examining
the head. “He was shot in the head, just back of this ear.”
John Caribou pressed forward and looked at the bullet hole. He
carried a rifle himself that threw a big ball like that.
Merriwell did not know whether to reprove Hans or not for
bringing the head to camp, and let the question pass, while they
talked of the dead moose and the poachers, and discussed the
advisability of trying to capture those slippery gentlemen.
46. John Caribou disappeared within a tent and came out shortly with
his long rifle.
“Where are you going?” Merriwell questioned. “Not after the
poachers now?”
Caribou shook his head and held up his empty pipe.
“Tobac’ all gone,” he said. “No tobac’, Caribou him no good. Friend
down here got tobac’. Come back soon.”
He waved the pipe toward the timber as if to point out the
direction of the home of this friend.
There was an unfathomable look on Caribou’s face which Frank
did not like. The guide had said nothing about being out of tobacco
before that time, and the conviction was forced that this was merely
an excuse to enable him to get out of the camp.
Jack Diamond, who had all along doubted John Caribou’s honesty,
gave Merry a triumphant and questioning glance.
“I don’t think you had better go just now,” objected Merriwell. “We
may need you here in the camp.”
“No tobac’,” said Caribou, doggedly. “Must have tobac’!”
He did not try to parley, but threw his gun on his shoulder and
struck out for the woods.
“That fellow is up to some dirt,” averred Jack Diamond. “You mark
my words now. He has plenty of tobacco. If I’m not mistaken, I saw
him have a whole pouchful this morning.”
Merriwell wanted to defend the reputation of the guide, but he felt
that he could not satisfactorily explain Caribou’s queer action.
“Let’s not judge him hastily. He has certainly been all that the
most exacting could ask of a guide, and I don’t see why we should
now conclude that he will act otherwise.”
That was as much as Merry could say.
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