Inside JEB
ii
says. Could this explain the intracellular
acidification seen in anoxic embryos?
To show that this explanation is plausible,
Covi and Hand first had to establish that
brine shrimp embryos have V-ATPase.
Scooping floating cysts out of Utah's Great
Salt Lake, they took the embryos back to
the lab. They compared brine shrimp
embryos' cDNA with known sequences of
V-ATPases from other animals. Sure
enough, they found that the embryos
possess V-ATPase. They also noticed that
it's expressed differentially as embryos
develop, suggesting that it plays a role
during development. To show that V-
ATPase is positioned to sequester protons
in the organelles, Covi and Hand used an
antibody to locate the proton pump in
isolated cell fractions. They discovered that
V-ATPase is distributed in various
membranes, including those of organelles.
But does V-ATPase set up a proton
gradient between an embryo's organelles
and its cytoplasm? To find out, Covi and
Hand tried to stop the proton pump by
incubating dechorionated embryos with
bafilomycin, a V-ATPase inhibitor. It was a
long shot; nobody had breached the cysts'
chitin layer before. To their amazement,
the embryos stopped developing. `I
dropped everything else I was working
on,' Covi recalls. He called in Dale
Treleaven, an expert in 31
P-NMR, a non-
invasive technique to measure intracellular
pH. Monitoring embryos' pH as they
recovered from anoxia, Covi saw that the
cytoplasm of bafilomycin-treated embryos
remained acidic. So the pumping of
protons from the cytoplasm into organelles
by V-ATPase is crucial to the reversal of
acidification, and is therefore a key factor
in an embryo's recovery from anoxia.
Finally, to confirm that the release of
protons stored in the organelles causes
intracellular acidification, Covi incubated
embryos in oxygenated seawater with
CCCP, a chemical that makes membranes
leaky to protons. Sure enough, the
cytoplasm acidified, just as it does in
anoxic embryos. `The ability to dissipate
internal proton gradients under anoxia
appears to set brine shrimp embryos apart
from other animals,' Covi concludes.
10.1242/jeb.01722
Covi, J. A. and Hand, S. C. (2005). V-ATPase
expression during development of Artemia
franciscana embryos: potential role for proton
gradients in anoxia signaling. J. Exp. Biol. 208,
2783-2798.
Covi, J. A., Treleaven, W. D. and Hand, S. C.
(2005). V-ATPase inhibition prevents recovery
from anoxia in Artemia franciscana embryos:
quiescence signaling through dissipation of
proton gradients. J. Exp. Biol. 208, 2799-2808.
forced to cope with chilly water in
summer?
To find out, Day and Butler collected
brown trout from a fish farm, but soon
discovered that they are not very
cooperative. `Adult brown trout are very
aggressive and initially were a real
challenge to keep in the lab,' Day says.
Once Day and Butler had worked out how
to stop the fish from attacking each other,
they were ready to see how trout cope with
a thermal challenge. They acclimated some
brown trout to normal seasonal
temperatures (keeping fish at 5°C in winter
and fish at 15°C in summer) and
acclimated others to reversed-seasonal
temperatures (keeping fish at 15°C in
winter and fish at 5°C in summer). To see
how this affects swimming ability, they
measured each fish's speed as it swam
against an increasing current. They were
surprised to find that seasonally-acclimated
fish swam faster than trout acclimated to
reversed-seasonal temperatures. Clearly,
exposing trout to reversed-seasonal
temperatures wreaks havoc on their
swimming prowess.
Eager to explain this, Day and Butler
examined trout tissue samples for
morphological and biochemical clues that
might reveal why brown trout don't adjust
to reversed-seasonal temperatures. From
previous studies on trout, they knew that
ammonia build-up in white muscle reduces
swimming ability. But when they measured
ammonia and another waste product
(lactate) in the trout's white muscle, they
found that fish swimming at reversed-
seasonal temperatures actually had lower
levels of both waste products than fish
swimming at seasonal temperatures.
Resting fish also had lower ammonia levels
in their white muscle at reversed-seasonal
temperatures than at seasonal temperatures,
in winter at least. So a waste build-up can't
be the reason for their lethargic swimming.
But the lower level of waste products does
suggest that trout may swim poorly at
reversed-seasonal temperatures because
they don't use their white muscle very
much.
Day and Butler were even more intrigued
to find that there were clear differences in
muscle morphology and biochemistry
between fish acclimated to 5°C in summer
and those acclimated to 5°C in winter.
Since both groups were acclimated to the
same temperature, these differences cannot
be due to temperature alone. There must be
other `seasonal' factors at work, and Day
and Butler list photoperiod, geomagnetism
and the internal biological clock as
possible suspects. It's clear that seasonality
PROTON PUMP IS KEY TO
SURVIVING ANOXIA
Picture by Cindy Henk
Safely ensconced in their protective cyst
shells, brine shrimp embryos are some of
the toughest creatures on the planet,
happily surviving for years where other
animals would suffocate. Researchers have
painstakingly worked out that a key factor
for the hardy little creatures' survival in
oxygenless water is acidification of their
cells. This triggers an almost complete
metabolic shutdown and stops an embryo's
development in its tracks, conserving
precious energy until it's safe to reverse the
acidification and kick-start development
again. But the tough shell that provides
such effective refuge makes it almost
impossible to study an embryo's insides, so
the mechanism that causes this acidification
has proved to be frustratingly elusive,
baffling researchers for 20 years. Joseph
Covi and Steven Hand set out to explain
the reversible acidification that's crucial for
anoxia tolerance (p. 2783 and p. 2799).
Covi explains that brine shrimp embryo
cells don't yet have fully formed cell
membranes, so an embryo is essentially
one big cell. Suspended inside it are
organelles like lysosomes and yolk
platelets. Covi and Hand suspected that
when embryos are afloat in comfortable
oxygen levels, a common proton pump
called V-ATPase pumps protons into these
organelles, turning them into proton storage
units. But when oxygen levels suddenly
plummet, `the embryo's ATP levels crash,
the ATP-dependent V-ATPase stops
working, and the organelles leak their
protons into the embryo's cytoplasm,' Covi
has complex physiological ramifications, so
Day and Butler have their work cut out for
them.
10.1242/jeb.01720
Day, N. and Butler, P. J. (2005). The effects of
acclimation to reversed seasonal temperatures on
the swimming performance of adult brown trout,
Salmo trutta. J. Exp. Biol. 208, 2683-2692.
THE JOURNAL OF EXPERIMENTAL BIOLOGY