INTRODUCTION
The cirripedes of the intertidal region are frequently
subjected to widely fluctuating environmental conditions which
can bring about dessication during tidal exposure. Barnes and
Barnes (1957) reported that littoral barnacles appear to have
a "controlled behavior" to cope with dessication, whereas sub-
littoral forms 'struggle upon exposure and soon become dessica-
ted.'
In Monterey Bay, California, three species of acorn barn-
acles are found which vary in size and vertical zonation. The
smallest of these, Balanus glandula, weighs from one to two
grams and is found intertidally from about two to ten feet
above mean lower low water (Doty, 1946). The other two species,
Balanus nubilis (weighing up to two hundred grams) and Balanus
intinnabulum (weighing to about twenty grams), are found in
lower intertidal regions, from about plus two feet to the sub-
littoral. The local ocourence of three species of the same ge-
nus which differ in size and inhabit distinctly different in-
tertidal zones provided an opportunity to make a comparative
study of the effects of dessication in relationship to tidal
height.
The barnacles Balanus glandula, B. tintinnabulum, and
B. nubilis were investigated with regard to three aspeots of
dessication; (1) the behavior of the animal upon exposure,
(2) the rate and percent of water loss from the whole animal,
-1-
the body, and from the shell, and (3) the respiration rate
of barnacles during periods of exposure.
METHODS AND MATERIALS
Specimans of B. glandula were removed intact from their
granite substrate with a scalpel. Excess water was blotted
off and each animal was weighed to the nearest milligram on a
Mettler balance. Specimans of B. nubilis from wharf pilings
and B. tintinnabulum removed from a float in the bay were
collected and each weighed to the nearest O.1 gram. All speci-
mans were numbered and put in a saltwater aquarium at 13°0.
until needed.
To observe the effeot of dessication under field cond-
itions, a screened-over, open-sided box was placed in the
intertidal, just above the highwater mark. Weighed and number-
ed animals of all three species were placed in this "natural
dessicator" and were subsequently sampled over a period of
seven days. Since wide variations in temperature and humidity
occured during this experiment, a similar artificial dessica-
tion experiment was conducted in the laboratory under more
controlled conditions. Samples were placed in a commeroial
dessicator (Drierite was used as a dessicant) for forty-eight
hours at room temperature. The system was flushed by a cont-
inuous stream of air run into the dessicator through a tube of
calcium chloride. The behavior which the barnacles demonstrated
was noted periodically through these experiments.
-2-
the body, and from the shell, and (3) the respiration rate
of barnacles during periods of exposure.
METHODS AND MATERIALS
Specimans of B. glandula were removed intact from their
granite substrate with a scalpel. Excess water was blotted
off and each animal was weighed to the nearest milligram on a
Mettler balance. Specimans of B. nubilis from wharf pilings
and B. tintinnabulum removed from a float in the bay were
collected and each weighed to the nearest O.1 gram. All speci-
mans were numbered and put in a saltwater aquarium at 13°0.
until needed.
To observe the offeot of dessication under field cond-
itions, a screened-over, open-sided box was placed in the
intertidal, just above the highwater mark. Weighed and number-
ed animals of all three species were placed in this "natural
dessicator" and were subsequently sampled over a period of
seven days. Since wide variations in temperature and humidity
occured during this experiment, a similar artificial dessica-
tion experiment was conducted in the laboratory under more
controlled conditions. Samples were placed in a commeroial
dessicator (Drierite was used as a dessicant) for forty-eight
hours at room temperature. The system was flushed by a cont-
inuous stream of air run into the dessicator through a tube of
calcium chloride. The behavior which the barnacles demonstrated
was noted periodically through these experiments.
-2-
For the field experiment water loss measurements were ob-
tained by weighing the samples at the beginning of the experi-
ment and then twice daily over a period of a week. In contrast
the more severly dessicated laboratory animals were weighed be-
fore being put into the dossicator and then every two hours
over a period of forty-eight hours. Two to five animals of each
species were removed once daily (in the evening) from the field
experiment and every four hours in the case of the laboratory
experiment. After the animals had been weighed, the bodies of
each of the specimens were carefully separated from their shells
and individually weighed. The shells were then cleaned of lamal-
lae and opercular plates and they too were weighed. The total
water loss was taken as the ratio of the total weight after des-
sication to the initial total wet weight and expressed as a
mean percentage loss. The average wet body weight to wet total
weight was calculated for a series of wet control samples (fig.1).
Following dessication the body water loss was determined by
comparing the body weight after dessication to the average wet
body weight derived from the previously calculated ratios.
Shell water loss was calculated in a like manner.
In this study,the presence of lactic acid, a major end-
product of anaerobisis, was detected by a modified enzymatic
technique (cf. Bergmeyer, 1965). For each test a body sample
of known weight was homogenized in cold distilled water and
the homogenate was centrifuged for thirty minutes at 6500 R.P.M.,
at 0°C. The supernatant was then deproteinized with O.5ml. ZnSO
-3-
20
and O.5ml. Ba(OH),, at a pH of 7.0, followed by centrifugation
for thirty minutes. O.5ml. of the resulting supernatant was
added to 1.4ml. of a glycine-hydrazine buffer (pH9.4) and O.lml.
of a 0.025M solution of DPN. The absorbance of the solution was
read on a spectrophotometer at 340mu. 5Yof lactic dehydrogenase
was added at room temperature and the change in optical density
(AOD) was read after thirty minutes. This test measures the
shift of DPN to DPNH' as a result of the following reaction:
DPN + lactate
pyruvate + DPNH' + H'. The shift is related
to lactic acid by the formula, moles lactic acid - 2
X 10
031
The result was calculated as molarity of lactic acid per gram
of body weight.
Oxygen consumption was measured by the WINKLER method
(of. Welsh, 1953). Speoimens of the three species were scraped
and brushed to remove algae and then put in sealed, volume-
controlled plexiglass respiratory chambers. Eleven B. glandula,
six B. tintinnabulum, and two B. nubilis were used, one species
per chamber. The chambers were filled with millipore-filtered
seawater with an oxygen content of 5.1ml. 0/liter. A lmg./1.
solution of streptomycin was added to inhibit bacterial resp-
iration. The oxygen decrease in each chamber was measured after
the apparatus had been placed in the dark at 13'C. for two
hours. From these results a standard rate of respiration for
the three species was calculated. The chambers were then filled
with water that had been bubbled with nitrogen for twenty-four
hours in order to remove the oxygen and the apparatus was again
-4-
20.
placed in the dark. After the barnacles had been exposed for
eighteen hours to these essentially anaerobic conditions, the
deoxygenated water was drained off and carefully replaced
with filtered seawater of a known oxygen concentration. After
two hours the rospiratory rate in the latter solution was cal-
culated and compared to the initially established rate to
see if there had been an increase which might imply that an
oxygen debt had been accumulated.
In addition the respiratory rates of B. glandula out of
water and in water were determined on a WARBURG respirometer.
(cf. Umbrett, Burris, and Stauffer, 1964). Four specimens were
put into 18ml. manometric flasks with jml. of filtered seawater
at 15°0. O.2ml. of a 20% KOH solution was placed in the center
vessel of the reaction flasks to absorb C0,. Four animals
that had been freshly removed from seawater were placed in a
similar flask without any water and the respiratory rates com-
pared. The experiment was repeated after the latter sample
had been exposed to air at 15'0. for six hours, and again after
it had been dessicated for thirty-six hours.
OBSERVATIONS AND RESULTS
Behavior
Under natural conditions the behavioral responses of the
three species of Balanus to exposure and dessication differ
markedly. The high intertidal B. glandula ceases all cirral
activity immediately upon exposure. The operculum closes with
-5-
20.
the exception of a small, micropylar opening which forms be-
tweon the turgum and the scutum. After exposure of from three
to four days, however, the micropylar opening becomes grad-
ually smaller until by the sixth or seventh day it has almost
disappeared. The animal generally dies after a week of exposure.
The lower intertidal species, on the other hand, show no
adaptive responses to exposure and continue their normal cir-
ral pulsing. Most of the B. nubilis pulse at from four to
seven beats per minute, while those animals not beating remain
open with their valves slightly parted. By the third or fourth
day of exposure, the cirral pulse rate diminishes and most of
the animals withdraw back into their shells, closing the valves.
For the next few days there is only occasional cirral activity
until the animal dies (usually by the seventh day).
B. tintinnabulum is even more active than B. nubilis, pul-
sing from seven to twenty times per minute for the first few
days. Thereaftor the pulse rate lessens but rarely does the
animal ever stop its cirral beating. The pulsing becomes slow
and spasmodic and B. tintinnabulum eventually dies by the
fourth day of exposure, with its dried oirri still partially
extruded.
Water loss
Fig, 2a. illustrates total water loss under natural cond-
itions (expressed in mean %-total weight loss per day) and
fig.2b. shows the results of tho same experiment run under
-6-
2
laboratory conditions (mean %-weight loss per hour). B. nub-
ilis loses water at a slow, steady rate until approximately 15%
of its total water has been lost, at which time it dies. Al-
initialy
though B. glandula loses water faster than does the larger
B. tintinnabulum, it can withstand a loss of 25-35% whereas
up to about 30-40% of the total water content is lost in B.
intinnabulum. In all three specios sholl water loss is great-
est during the first few hours of exposure, but thereafter the
rate is markedly reduced (fig.3a). In general body water loss
is slight immediately following exposure, but then it increases
until shortly before death. The rate then levels off until
B. glandula has lost about 55% of its body weight at death,
B. tintinnabulum 35% and B. nubilis 15% (fig.4).
Lactic acid production
Fig.5 illustrates that the concentration of lactic acid
in all three species is low, however, there is a difference in
concentration of three orders of magnitude between the species
(fig.5a). B. nubilis and B. tintinnabulum showed a slight in-
crease after thirty-six hours of dessication in the field, with
a maximum lactic acid concentration of 3.Ox10M/g and 3.5110 M/g.
respectively, by the fourth day. After two days B. glandula in-
creased its lactic acid concentration to a maximum of 3.Ox10 2M/g.
By the fifth day a slight deorease in concentration was shown
in all three species.
In an additional experiment barnacles were subjected to
-7-
20
submergence in deoxygenated seawater for forty-eight hours.
Noincrease of lactic acid could be found in the lower inter-
tidal species, B. nubilis and B. tintinnabulum, whereas
B. glandula showed a small increase to 2.3x10"2 M/g.
Respiration
The normal respiratory rate for B. glandula was calculated
to be 180 ul 0/g/hr., while rates of 96 pl 0,/g/hr. and 260 pl 0,/g/hr.
were found for B. tintinnabulum and B. nubilis respectively.
After having been subjeoted to the deoxygenated water for eighteen
hours, the rate increased to 925 ul 0,/g/hr. for B. glandula
while B. tintinnabulum only increased its rate to 400 ul 0/g/hr.
and B. nubilis to 310 ul 0/g/hr. In comparing the rate of resp-
iration in water to that out of water, it was found that B. glan-
dula decreases its rate with increasing exposure. The rate was
the same for animals that had been freshly removed from the sea-
water but after six hours of exposure declined to 92 ul 0/g/hr.
and to 10 pl 0/g/hr. after thirty-six hours of dessication.
DISCUSSION
The micropylar opening seen to form when B. glandula was
exposed to dessication is similar to that which Barnes and Barnes (1957)
described in Balanus balanoides, but the pulsing of the micropy-
lar opening which was observed in the latter species was absent
in B. glandula. The two lower intertidal species studied, B. nub-
ilig and B. tintinnabulum, however, exhibited cirral pulsing even
under the conditions of severe dessication. As the ratio of ex-
-8
a
posed surface area to body volume in larger animals is less
than that in smaller ones, B. nubilis and B. tintinnabulum
would be expected to have proportionately less area devoted
to respiratory surfaces than would the smaller B. glandula.
Both of the larger intertidal spocies have gill-like fila-
ments attached to the wall of the mantle cavity, which effect-
ively increase their respiratory surfaces, whereas B. glan-
dula lacks these structures (G. Hilgard, 1967). Daniel (1955)
found that the constant beating of the opercular muscles in
B. tintinnabulum was a respiratory adaptation serving to main-
tain a uniform flow of water past the respiratory tissues
of the body and the gills. It thus appears that the cont-
inued cirral activity of exposed B. nubilis and B. tintin-
nabulum is a continuation of their normal respiratory act-
ivity which, undor these circumstances, is non-adaptive.
On the other hand, when B. glandula is exposed, it minimizes
its oxygen needs by remaining motionless (von Brand, 1946)
and so reduces its respiratory rate, as seen both in this
study and by Kreps and Borsuk (1929) in their study on
Balanus crenatus, a barnacle very similar to B. glandula
On exposure then, B. glandula seemingly modifies its
respiratory rate, whereas B. tintinnabulum and B. nubilis
do not.
A high ratio of surface to air may increase the resp-
iratory capabilities of an animal, but it also promotesmore
rapid evaporation. The continued cirral activity of the
-9.
20.
lower intertidal species during exposure, though it may be an
important respiratory funotion, exposes the moist body parts and
thus hastens dessication. B. glandula is smaller than the other
two species and rapidly loses a large percentage of its body
water (fig.3b), however it appears to be able to tolerate this
loss, maintaining itself during prolonged exposure. The re-
sults of the rapid cirral activity of B. tintinnabulum is re-
flected in fig.2. This organism shows the greatest total water
loss, yet it can tolerate less body water loss than can the
smaller B. glandula (fig.4). On the other hand B. nubilis loses
the least amount of water, but this is probåbly a function of
its size and not of any adaptive characteristics. This was borne
out in a study that indicated that immature B. nubilis, of the
same size as B. glandula, could tolerate only two days of ex-
posure, whereas B. glandula could tolerate seven.
Shell water loss apparently occurs mainly by the evap-
oration of water from the shell surface and appears to be
of minimal importance. Body water loss, however, appears to be
of great significance in determining the survival of intertidal
barnacles (cf. fig.4). It is interesting to compare the result
of Edney's (1967) investigation of the desert cockroach, Arnev-
igo sp.,under xeric conditions to the results obtained in this
study. The cockroach can tolerate a loss of from 25-30% of its
body water,before it dies, whereas the intertidal barnacles
studied can tolerate a 15-50% loss, depending on the species,
before death ensues. The observations of this study therefore
agree with those of Suzuki and Mori (1963), who stated that
-10-
20.
tolerance of body water loss was most significant in deter-
ming the adaptation of an intertidal barnacle to exposure.
The respiratory rates observed in this study are comparable
to those reported for other species of barnacles. Barnes and
Barnes (1959) found a rate of from 140 pl 0/g/hr. to 160 ul 0/g/hr.
in B. glandula and a rate of from 94 ul 0/g/hr. was found for
Balanus amphitrite communis by Ganapati and Rao (1960). The
large increase in the respiratory rate of B. glandula after
exposure to deoxygenated seawater was somewhat surprising, how-
ever Lund (1921) reported an increased rate in Planaria agilis
of about 85% under similar conditions. He also found that the
greatest increase occured after starvation. As filtered sea-
water was utilized in this study starvation might also have been
a factor in the large respiratory increase of B. glandula. In
any oase it would appear that B. glandula had accumulated an
oxygen dobt after having been subjected to essentially an-
aerobic conditions. There were also indications of similar
but much reduced oxygen debts in B. nubilis and B. tintin-
nabulum. B. glandula, which exhibited the greatest increase
in respiration following anaerobic conditions, also showed the
greatest lactic acid concentration, while both B. tintinnabulum
and B. nubilis had smaller lactic acid concentrations and
lesser oxygen debts.
The pattern of oxygen consumption and lactic acid con-
centration refered to above was emphasized by leaving the barn-
acles in deoxygenated seawater for forty-eight hours.
-11
305
B. nubilis and B. tintinnabulum both died with no production
of lactio acid, On the other hand B. glandula lived and a slight
increase in lactic acid was demonstrated. This would imply
that B. glandula can perhaps metabolise food reserves in the
absence of oxygen, while tho two lower intertidal species
cancnot. This is further indicated in a study made by Augen-
feld (1967), who found that the level of stored glycogen in
the high intertidal B. glandula was two to three times greater
than similar stores in the lower intertidal species, such as B,
nubilis. Augenfold reported that the glycogen level was halved
in B. glandula and exhausted in B. nubilis after several days
of exposure. He also found a trace of lactic acid in B. glan-
dula while none was demonstrated in B. nubilis. However, Aug-
enfeld agreed with Barnes and Barnes (1957) in that he didn't
think that anaerobic respiration, if even present, was ever
significant as an adaptive mechanism to dessication.
Other marine organisms when placed under stress often
show the capability of being able to mako at least a partial
reversion to anaerobic respiration. For example, Mysa relicta
is normally found in water with an oxygen content of about
4ml. 0/1., but Thieneman (1928) found it partially utilising
anaerobisis in the depths of the Black Sea, where the oxygen
content is 1.6ml. 0/1. Likewise Kreps (1929) found the occurence
of an oxygen debt in the barnacle Balanus crenatus along with
partially oxidized anaerobic byproducts, while Dugal found an-
aerobic glycolysis in the clam Venus mercenaria.
-12-
2/0
or it is affected by lack of food and it reverts at least
in part to anaerobio respiration. By the fourth to sixth day
of exposure most of the stored glycogen has been utilized
and after about a week, the animal dies from the combined
effects of dessication and possible starvation.
The story differs somewhat for the lower intertidal
species, B. nubilis and B. tintinnabulum. Upon exposure
they continue their cirral activity (this seems to be a
respiratory mechanism and also a possible feeding reflex).
Little water is lost during the first few hours, after which
time they are normally resubmerged. If the barnacles should
continue to be exposed, however, rapid dessication occurs.
These organisms apparently cannot get enough oxygen through
their dessicated respiratory surfaces and since their glycogen
supply is small, it is quickly exhausted and they die after
from four to seven days. The two lower intertidal forms exhibit
no adaptive characteristics to cope with dessication. The
high intertidal B. glandula has greater tolerance limits to
water loss and a respiratory mechanism which allows partial
reversion to anaerobisis.
-14.
2
SUMMARY
1. A comparative study of dessication in three species of
Balanus from different intertidal zones was undertaken.
2. It was found that the high intertidal B. glandula is
adapted to dessication by being able to tolerate large
body water losses. Though B. glandula normally respires
aerobically it can revert to partial glycolysis under
conditions of extreme stress.
3. The lower intertidal species B. nubilis and B. tintin-
nabulum were found not to possess any special adaptive
mechanism that would enable them to cope with extreme
dessication.
C
FIGURE LEGEND
fig.1. Ratio of body weight and shell weight to the
total weight of the barnaole.
fig.2b. mean-% of total weight loss under natural
conditions.
fig.2b. mean-% of total weight loss under laboratory
conditions.
fig.3a. mean-% of shell water loss under laboratory
oonditions.
fig.3b. mean-% of body water loss under laboratory
conditions
fig.4. average-% of weight loss at death (of 50%
of the animals).
fig.5a. relative magnitudes of laotic acid concentration
in the three speies.
fig.5b. increase in lactic acid under natural conditions.
fig.6. increase in respiration after eighteen hours in
deoxygenated seawater. "After" = respiration after
anaerobic conditions, "Before" - normal rate of
respiration.
2
C
—


% WEIGHT
—
O
—
L
-

9
80
60
100
80
6O-

......... B. tintinnobulum
—0 — —0 — B. nubilis
-0—o-B. glandula


-0
-0
6
TIME- days

o0

-o
-0

48
40
32
24
16
TIME-houts
2/6
.....o.. B. tintinnabulum
-0 — —o- B. nubills
O
—0—0— B. glanduld
00
—
o
——9 ——
—
—9-
——3

80
§ 60
40
32
24
8
16
TIME-hours
100



80.

60


— O—
O
40
48
40
24 32
16
TIME-hours
48
2/6
S
— .
os
D
—
% WEIGHT LOSS

L
—0
—
o


O


—
8/7
— 4210-5
2x10-


4x1


2x10-7-
4x10-8
2x10-8

B. gländula
O —

B. tintinnabulum
—
B. nubilis
—
o B. glondulo-10-5M
9B. tintinnabulum-10-'N
o B.nubilis -10-8M


-0



5.
k -

—
—
TIME -days
2
0
—

—
Al 02
N
gram/hour
O
O
—
219
ACKNOWLEDGEMENTS
I am most grateful to Dr. Welton Lee for his
guidance and patience with this project. I
also wish to thank Dr. David Epel for his
help and the use of his laboratory, and
graduate students James Childress and Ray
Markel for their help and equipment.
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in barnacles occupying different levels of the
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BALDWIN, E., 1965. Dynamic Aspects of Biochemistry. Univ-
ersity Press, Cambridge.
BARNES, H., and M. BARNES, 1957. Resistance to dessication
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H., and M. BARNES, 1959. Studies on the metabolism
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BERCMEIER, H., 1965. Methods of Enzymatic Analysis. Academic
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C
UNBRETT, W., R. BURRIS and J. STAUFFER, 1964. Manometrig
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W
TERMAN, T., 1960. The Physiology of Crustacea, vol. 1.
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