Abstract
Intertidal organisms can spend a great deal of time out of water and must be
physiologically adapted to tolerate terrestrial conditions. I hypothesized that one of the
physiological adaptations made by intertidal organisms is reduction of metabolic rate
during the low tide period. To test this hypothesis, I investigated the ability of the
intertidal porcelain crab Petrolisthes cinctipe, to regulate its heart rates during the
transition from water to air. Heart beats were monitored by impedance electrodes at
10°C, 15°C, and 20°C during one hour of immersion followed by one hour of emersion.
Heart rates were recorded using a PowerLab data acquisition system and were averaged
for each 10 minute period of the experiment. Linear regression analysis was used to
determine if heart rate varied with body size using crabs with carapace width ranging
from 9mm to 16 mm. A significant decrease in heart rate with increasing body size was
found. Heart rates were corrected for body size in comparisons of the effects of
emersion. No significant effect of emersion was found (p-value of 0.079.) The
temperature dependence (Qio) of heart rate also did not differ between immersion or
emersion. In future studies, variables such as size should be more strenuously controlled
for, and this may yield clearer results.
Introduction
An important theme in evolution is the phenomenon of terrestrialization, whereby
aquatic species developed abilities to live on land and respire in the air. The intertidal
zone is an ideal location to explore mechanisms of terrestrialization because organisms
inhabiting this habitat must be adapted to both land and water.
Potential challenges that these animals face during transition from water to land
include heat stress, desiccation, and difficulties in breathing because properties of air and
water are vastly different (Burggren and McMahon, 1981). One possible adaptation to
such stressful conditions made by marine organisms when adapting to land would be
reduced respiration as a means of reducing loss of water of the respiratory surfaces,
especially the gills. Lowering heart rate could be one mechanism for reducing the threat
of desiccation.
Arthropods have been used as model organisms for many studies of
terrestrialization (Burggren, 1992). In this experiment, intertidal porcelain crabs will be
explored. The effect of marine organisms adapting to land could be observed in the
alternating tidal cycles, where twice daily low tides simulates the process of
terrestrialization.
The intertidal porcelain crab Petrolisthes cinctipes can be emersed during every
low tide, and spend over half its life out of water. They are found on the Northeastern
Pacific coastal shores living underneath rocks and in mussel beds at the mid intertidal
zone. P. cinctipes are also regularly subjected to large changes in temperature during
tidal cycles where, during emersion, temperatures may read 31’C. Their Arrhenius break
temperature of heart rate in response to temperature is 31.5°C (Stillman and Somero,
1996).
One adaptation to emersion in response to the difficulties of respiration in P.
cinctipes is that these crabs possess a membraneous structure on the ventral merus on
each walking leg, which functions as a respiratory structure (Stillman and Somero, 1996).
What I want to investigate is how P. cinctipes are affected by alternating between
air and water in these microscale simulations of terrestrialization. Are their metabolisms
lowered? Cardiac output is a good indication of the rate of metabolism, and it has been
seen to be generally lower in terrestrial crabs relative to marine species (Burggren, 1992).
Thus, I asked if an effect of emersion in P. cinctipes could be a lowered metabolism
indicated by a decrease in heart rate.
Materials and Methods
Collection and Care
Specimens were collected during low tide from two intertidal sites, Fisherman’s
Wharf in Monterey, CA and Hopkins Marine Station in Pacific Grove, CA. Exposed
rocks that had crevices underneath were selected and turned over to collect crabs. The
crabs were placed into a small cooler filled with seawater and transported to Hopkins
Marine Station, where they were then placed into aquaria with flowing ambient seawater
at 13+2’C. They were kept in a rectangular Rubbermaid tupperware container
(21x13cm) that had its bottom and sidewalls cut out so that only its frame was being
used. The walls were replaced with plastic wired mesh, which allowed flow of seawater
through the system and provided crabs with support. The crabs were allowed at least 24
hours to acclimate before use in experimentation. Crabs were fed Cyclop-Eeze (Argent
Laboratory, Redmond, WA) cultured micro-crustaceans every Monday, Wednesday, and
Friday.
Experimental Materials and Design
Effects of emersion were observed at 10'C, 15°C, and 20'C. The crabs were
placed into an experimental chamber constructed with a rectangular Rubbermaid ice
chest (36x25cm). A refrigerated water bath (RC6 CS Lauda, Brinkmann, Germany) was
used to keep the water temperature constant inside the chamber. Styrofoam lids were
devised to loosely cover the water chamber to control the air temperature and keep the
specimens inside moist when suspended in air by metal clamps. The inside of the
chamber was lined with pipes and tubes in which anti-freeze circulated to control the
temperature of the water bath. An aquarium air stone at the bottom provided oxygen.
The chamber also contained two thermocouples calibrated to each other, one to measure
the temperature inside the chamber, and one that was inserted into the crab. Before each
run, the crabs were left undisturbed in the chamber in water for at least 15 minutes to
exclude the influence of preparation activities and stimulation. Each run consisted of six
crabs immersed in filtered seawater. Heart rate of immersed crabs was recorded for one
hour, at which time the water was siphoned out to a set level to simulate low tide. The
crabs were suspended in air for another hour. 12 crabs were used for each temperature,
making a total of 36 runs. An experimental run with a fake "crab" crafted with a sponge
whose soft side was cut into the average size of a crab was done to test for evaporative
water loss. The imitation "crab" was weighed, left suspended in air for six hours, and
then weighed again.
Preparation of crabs
Only male crabs were used. Females were not used because differences in the
brooding cycles might have complicated the results. Small numeric paper tags were
glued to the left claws of the crabs for identification. The tags did not hinder the crab's
natural mobility or ability to pinch. The widths of carapaces were measured with a
Vernier Caliper Type 6914 (Scienceware Bel-Art Products, Pequannock, NJ). Some
crabs sustained sustained injury to their exoskeleton before the entire sequences of runs
were performed, so replacement crabs were used. SuperGlue gel was used to attach a
thin band of cork onto the back of each crab to allow metal clasps to grasp and suspend
the specimen. Shaped like a long trapezoid, the narrow end of the cork was attached to
the crab, whereas the broad and flat end was clasped. Pin-sized holes were made through
the carapace on either side of the heart and impedance electrodes made of 0.025mm
diameter ceramic-coated copper wire, whose tips had about Imm of the ceramic
insulation removed, were inserted through each opening and glued in place (Stillman and
Somero, 1996). Of each of the two sets of six crabs, number three received an additional
pin-sized hole near the previous right one, into which a thermocouple was inserted to
measure temperature inside the crab. The impedance electrode wires were connected to
an impedance converter model 2991 (UFI, Morro Bay, CA), and the heart rate was
monitored with PowerLab/16sp (AD Instruments, Pty, Ltd, Castle Hills, Australia).
Data Analysis
Heart rate was monitored continuously for the duration of each experimental run
and expressed as average beats min“. Data were then averaged over ten minute intervals
and graphs against time were generated. To better compare heart rates between
immersion and emersion, the averages for each hour in air and water were calculated. To
test for a correlation between crab size and heart rate, linear regression analysis was
employed to generate regression lines for each temperature. The average of the three
regression lines was then used to correct the heart rates for effects of size. An Analysis
of Covariance was performed with the corrected data. The factors temperature and
treatment (air or water) were orthogonal to each other, and n = 12 replicates. The heart
rates were transformed into square roots before this test was performed to gain
homoskedasticity. Qjos were also calculated and compared between 10°C to 15°C and
15°C to 20°C in air versus water.
One other possible statistical test that had been considered was a Chi Square.
However, this could not be applied to a two by three model, in this case two treatments
(air or water) x three temperatures. If performed, the difference in heart rate between air
and water would have been compared within each temperature separately. This would
have necessitated three separate Chi Square tests, one for each temperature. I understood
that this would have a lower statistical power than an Analysis of Covariance. To get an
idea of what the results might have been, single factor Anovas were performed with each
temperature orthogonal to treatment, and no statistical significance was seen in any of the
three temperatures. Therefore, an Analysis of Covariance was chosen.
Results
Temperature
The thermocouple inside the chamber showed no change in temperature
throughout each experiment, whether done in water or in air. Similarly, the thermocouple
inserted into the crab revealed that temperature remained constant. When evaporative
cooling was explored, the initial weight was 4.2340.2g, and after six hours suspension in
air, 4.7740.2g. This demonstrates no net water loss by the crabs during the experiments.
Effects of emersion
Plots of heart rate versus time (figs 1, 2) generally showed a pattern in which the
initial heart rate under immersion (i.e. the very first data point) was higher than the final
heart rate in air at the end of the experiment. 86% (30 of 36 runs) indicated decreased
heart rates in the comparison, and of the six that did not, one came from 10°C, two from
15°C, and three from 20°C. There was also consistently a great of deal of variation in
heart rate, sometimes over a 3-fold difference both within each crab and amongst crabs
overall. These trends were seen at all temperatures.
To more easily compare differences, the heart rates for each hour in air and water
were averaged (figs 3, 4), and 86% (30 of 36 runs) decreased during emersion. Of the six
crabs’ whose heart rates increased, one was from 10°C, two from 15°C, and three from
20°C. These were not the same crabs that exhibited higher rates at the end of the
experiments when the initial and final rates were compared (see above).
The average heart rate in water was divided by the average heart rate in air to
obtain percentages of overall difference (fig. 5). The percentage differences amongst
crabs ranged from 30.69% increase in heart rate to a 38.48% decrease. When all 36
experimental runs were averaged, a net decrease of 9.22% was calculated.
Effects of size
The carapaces of the crabs ranged in width from 9mm tol6mm and averaged
13mm. Linear regression analysis comparing carapace size to heart rate showed a
significant relationship at all temperatures. For example, at 15°C, the P-value was 0.001,
and R°= 0.463 (fig. 6). To incorporate the effects of size into the analysis of the effects
of emersion on heart rate, the average of the regression lines for all three temperatures
was used to correct for size. Heart rate data were square root transformed for this
analysis. When size-corrected, the heart rate data did not show a difference between
conditions of immersion and emersion (P-0.079).
Effects of temperature
Looking for differences in temperature dependence of heart rate between
immersion and emersion, Qjos showed no differences for either 10°C to 15°C or 15°C to
20°C (fig. 7).
Discussion
It has been seen in previous studies that heart rate is directly related to
temperature in a substantial number of crustacean species (Stillman and Somero, 1996;
McGaw, 2002) This was one of the main reasons why the experiment was performed at
10°C, 15°C, and 20°C. Not only was I investigating whether or not heart rates decreased
as an effect of emersion, but also, if this were to occur, could the amount of heart rate
dependence upon temperature between air and water be different at different
temperatures? This relationship was analyzed with Qjos, and it indicated no such
difference.
Another aspect that arose due to the specimen’s heart rate dependence on
temperature was whether or not temperature was absolutely controlled for throughout
each entire experimental run. If not, this could have confounded the results. As
mentioned previously, a general decreasing, albeit non-significant, trend had been seen in
heart rate as the crabs were moved from water to air, and my hypothesis would suggest
that this was due to the effects of emersion. However, one alternative hypothesis is that if
the crabs had consistently experienced even a slightly lower temperature while suspended
in air, then this could very well have been responsible for the perceived decrease in heart
rate. Hence, controlling the temperature both in air and water was vital.
There were many things that could have affected the temperature that the crabs
experienced when suspended in air. A Styrofoam lid was crafted to cover the top of the
chamber, and in this way separate to some extent the air inside from out. The cover
should also have been effective in shielding most random breezes. However, it was
devised to fit loosely over the top because sufficient airflow was needed to facilitate
oxygen circulation to prevent hypoxia. To test the efficacy of the lid in controlling
temperature, a thermocouple was immersed in water, and then left suspended in air when
low tide was simulated during the experimental runs. Because no difference was seen
between immersion and emersion, effective control of temperature by the lid was
demonstrated.
One other concern regarding the temperature that the crabs experienced in the air
was whether or not evaporative cooling was occurring. If this were the case, then the
temperature that the crabs experienced would have differed from the set air temperature.
To test for evaporative cooling, the control experimental run was performed with the fake
crab". The lack of water loss verified that evaporative cooling was not a contributing
factor. One other affirmation that evaporative cooling was not occurring was that the
thermometer inserted inside the crab also showed no signs of change when emersion
occurred. Note that although evaporative cooling was controlled for in the experimental
runs, this does not preclude that crabs do undergo evaporative cooling in their natural
environment.
There was a great deal of variation in heart rate: up to 3-fold differences were
recorded both within each crab and amongst different crabs. Variation amongst different
crabs tended to lower the power of statistical tests such as Analysis of Variance. One
way in which this was slightly corrected for was by performing instead an Analysis of
Covariance. This was only made possible by the fact that a correlation was found
between the sizes of the crabs and their heart rates. Linear regression analysis was
performed demonstrating this correlation’s statistical significance, and the average of the
regression lines was used to correct for the heart rates before analyzing the data and
square root transforming it. Although this new P-value of 0.079 was considerably lower
than the one obtained from a regular Anova, it is still not statistically significantly
different.
Conclusions
Although it has been seen that cardiac output in terrestrial crabs is generally lower
than in marine crabs, this decrease was not detected in P. cinctipes as an effect of
emersion. The natural variation in an organism’s heart rate was unavoidable, and this
made comparing heart rates between immersion and emersion a challenge.
Additionally, metabolic rates in general can be strongly influenced by other innate
properties such as body size, physiological state (e.g. molting stage), and activity level
(Burggren, 1991). This makes it difficult to conclude that because a statistically
significant decrease in heart rate was not found, the metabolic rate did not decrease
either.
Instead, it has been determined that more stringent size control for the crabs is
needed to weed out some of the natural variation in heart rate, and in future studies, a
larger number of replicates should also be employed to increase the statistical power of
the experiment.
Acknowledgements
I thank Prof. George Somero for his guidance and support. He asked just the right
thought provoking questions and gave a continuous string of intellectual insights. Special
thanks to Prof. Jonathon Stillman for his time and dedication, without which this never
would have been accomplished. Likewise, the patient assistance and encouragement
from Prof. James Watanabe was invaluable. This work was supported by Hopkins Marine
Station of Stanford Uiniversity, and so I would like to recognize all of its wonderful staff,
students, and professors.
Literature cited
Burggren, W. W. (1991). Does comparative respiratory physiology have a role in
evolutionary biology (and vice versa)? In A. J. Woakes, M. K. Grieshaber, and
C.R. Bridges (eds.), Comparative insights into strategies for gas exchanges and
metabolism, pp. 1-13. Cambridge University Press, Cambridge
Burggren, W. W. (1992). Respiration and circulation in land crabs: Novel variations on
the marine design. Am. Zool. 32, 417-427.
Burggren, W. W. and McMahon, B. R. (1981). Oxygen uptake during environmental
temperature change in hermit crabs: adaptation to subtidal, intertidal, and
supratidal habitats. Physiol. Zool. 54, 325-333.
McGaw, I. J. (2003). Behavioral thermoregulation in Hemigrapsus nudus, the
amphibious purple shore crab. Biol. Bull. 204: 38-49.
Stillman, J. H. and Somero, G. N. (1996). Adaptation to temperature stress and aerial
exposure in congeneric species of intertidal porcelain crabs (genus Petrolisthes):
correlation of physiology, biochemistry and morphology with vertical
distribution. J Exp. Biol. 199:1845-1855.
Figure legend
Fig. 1. Average heart rates versus time in a given experimental run for specimens 1 to 6
at 15°C.
Fig. 2. Average heart rates versus time in a given experimental run for specimens 7 to 12
at 15°C.
Fig. 3. Average heart rates during immersion compared to emersion for specimens 1 to 6
at 15°C. Error bars are standard errors of the mean.
Fig. 4. Average heart rates during immersion compared to emersion for specimens 7 to 12
at 15°C. Error bars are standard errors of the mean.
Fig. 5. Ratios of average heart rates during immersion divided by average heart rates
during emersion to display percent differences for specimens 1 to 12 at 15°C.
Fig. 6. Q1o values for air and water in 10°C to 15°C, and 15°C to 20°C. Error bars are
standard errors of the mean.
Fig. 7. Average heart rates in a given experimental run versus crab size for specimens 1
to 12 at 15°C and linear regression line.
5
3
8
8-
Heart rate (beats min *)
O
O

A-

S
14 8

0.
o
8
OGAON
3
8
100
)O
Heart rate (beats min *)
9 15
5
e
8aaaa-
Heart rate (beats min
o
5
wate
Heart rate (beats min *)
5
percent below 100 indicates decrease heart rate in air
8
Qjo values

8

8
20
0
Heart rate (beat min")
*. *
. *.. / .


4