E. Kincannon Temm rature Response in Tonicella Page 0
Abstract
Two vertically separated populations of Tonicella
neata (Wood) were tested for acute and acclimated res¬
piratory rates at a series of five temperatures. Oxygen
uptake was considered as a function of both body weight
and temperature. Q in these chitons was not affected
by body weight. Intertidal Tonicella were shown to have
a greater ability to compensate for temperature changes
than the subtidal population; they showed better acclimation
patterns after three weeks in the laboratory and showed
decreasing 0's at higher temperatures while 0 of the
subtidal population remained constant. These results are
related to stability of temperature regimens in each habitat.
Temperature Response in Tonicella
E. Kincannon
page 2
INTRODUCTION
Intertidal molluscs are known to show compensatory
changes in metabolic rate in response to different temperature
regimes. For example, some species from high latitudes have
the same rates of oxygen consumption as do related tropical
species at warmer temperatures (see reviews by Bullock,
1955, and Segal, 1961). Furthermore, this compensation, or
acclimation, has been shown between two spatially separated
populations of the same species. Low level populations of the
limpet Collisella limatula (Carpenter, 1864) show cold
acclimation of heart rate while high intertidal C. limatula
from a few feet away show warm acclimation (Segal, 1956).
Kenny (1958) suggests that this should apply to the Poly-
placophora as well; the chiton Clavarizona hirtosa (Blain¬
ville) from low intertidal zones shows a lower upper lethal
temperature than those from comparatively higher, warmer
zones. However, no definitive work on acclimation as a
function of microgeographic separation has been done on the
Polyplacophora.
This study was designed to answer the following questions:
(1) Can chitons acclimate to altered temperatures
within a few weeks?
(2) If so, are subtidal animals acclimated to a
lower temperature than intertidal ones of the same species;
(3) Are intertidal animals, which are exposed to
E. Kincannon
Temperature Response in Tonicella
wide fluctuations in habitat temperature, better able to
acclimate to experimental temperatures than subtidal ani¬
mals, which experience a more limited temperature range?
The species Tonicella lineata (Wood, 1815) was selected
for study because of its wide vertical distribution. In
addition to being abundant subtidally (Barnes, 1972, p. 67).
the species has been found by the author to occupy sides in
the intertidal up to three feet above mean lower low water.
Exchange of individuals between deepwater and shallow pop¬
ulations is probably small or nonexistanat (Demopulos, 1974).
Acute respiratory rates (oxygen consumption rates of
animals collected and held at constant temperature for
one day before measurement) were compared with rates of
acclimated aniamls (held three weeks at constant temperature
prior to measurement), at a series of five temperatures.
Rate of oxygen consumption was used as an indicator of
metabolic rate. Since many factors other than temperature
affect respiratory rate, notably body weight and activity
level, an attempt was made to study and correct for these
variables. There is evidence (Bullock and Rao, 1954) that
temperature affects the relationship between body weight and
respiratory rate. Consequently, weight-rate regressions were
done at each temperature for each population, using the
formula M = k-W. Where M = metabolic rate, K = a constant,
W - weight of the organism, and b - the regression coefficient.
page 3
E. Kincannon
Temperature Respe
in Tonicella
page 4
MATERIALS AND METHODS
All intertidal Tonicella were collected from Carmel
Point, Carmel, California, between O and 2 feet above mean
lower low water. Subtidal Tonicella were collected at a
depth of 20 to 26 feet off Mussel Point, Pacific Grove,
California. In the laboratory, all were held under constant
light in aquaria equipped with air stones. Constant temperature
was maintained by means of running sea water and immersion
heaters. Three holding tanks were maintained in this manner
at 8°C, 13°C, and 18° C; temperature was checked daily and
fluctuated + 2°C in the two warmer tanks, less in the 8°C
tank. Large glass jars covered with plastic screens served
both to keep the chitons submerged and to keep the popula¬
tions separate.
Rates of oxygen consumption were expressed on the basis
of wet weight which was measured by blotting the Tonicella
with paper towels before weighing. Fourteen chitons were
weighed after 29 hours in an 80°C oven. They were then
combusted at 600°C for 48 hours. Those initially weighing
over 2 g were combusted 6 days and reweighed. Percentages
of dry to wet weight, and ash-free to dry weight, were
subsequently calculated and plotted as a function of wet
weight.
Rates of oxygen consumption were measured at experimental
temperatures of 3°c, 8°c, 13°C, 18°C and 23°C using the direct
E. Kincannon
Temperature
sponse in Tonicella
page 5
Warburg method of constant volume manometry as described by
Umbreit et al. (1972). After collection, Tonicella were
kept in the holding tank closest in temperature to that at
which they would be tested. Before the experiment they were
weighed, placed in the Warburg reaction vessel, and covered
with a measured volume of sea water filtered through a 0.22
u Millipore filter. Oxygen consumption by particles less than
0.22 u was assumed to be negligible. Two-tenths of a ml of
10% KOH was added to a filter paper wick located in the side¬
arm. After one hour of equilibration in the water bath,
manometer readings were taken at one-hour intervals for six
to twelve hours, depending upon the time required for
respiratory rates to stabilize. Hourly rates of oxygen uptake
were calculated and averaged over the time interval showing
the most consistent rates.
Notes on the animals' activity and degree of submersion
were kept throughout each experiment. Only the rates of
those chitons which appeared unhurt, remained submerged,
and did not move more than a few cm were used in further
calculations. Each chiton was used in only one experiment,
after which it was liberated.
Acute respiratory rates were measured 22 to 26 hours
after collection for six Tonicella at each temperature.
Rates of acclimated animals were measured after three
weeks using two specimens for each combination of acclimated
and experimental temperatures. A regression analysis was
performed on each set of acute rates measured, using a double
E. Kincannon
Temperature Response in Tonicella
page 6
logarithmic transformation (Sokal and Rohlf, 1969). The
method of least squares (Ibid., Box 14.1) determined the
regression coefficients, and Fisher's F-test (Ibid., Box 8.1)
tested the significance of the lines. In the comparison
of regression lines with each other, the F-test was used to
compare slopes (Ibid., Box 14.8), while Student's T-test
(Ibid., Box 9.6) compared the means after a determination
that the variances were similar (Fisher's F-test as above).
The mean weight of all chitons collected was 0.70 g.
Rates of oxygen consumption for this weight were obtained
from the regression line at each temperature and plotted
on a rate vs. temperature graph. Assuming that b, the
slopgof the regression line, remains constant with
acclimation, rates of acclimated animals were standardized
to this same weight and plotted with the same procedure as
the acute rates. Since all rate-temperature curves were
plotted semilogarithmically, their slopes are directly pro¬
portional to Qjo as calculated by the formula:
Ri
10/t-t)
210 - R9
(Prosser, 1973, p. 363).
RESULTS AND DISCUSSION
Wet weight as a measure of metabolizing tissue
Figure I shows dry weight as a percentage of wet weight
and ash-free dry weight as a percentage of dry weight for a
nearly fifty-fold range of weights of Tonicella. Apparently
Temperature Response in Tonicella
page 7
E. Kincannon
the proportion of water (upper graph) and ash-weight (lower
graph) in Tonicella does not depend upon total wet weight.
Ash-free dry weight excludes the bulk of a chiton's
normetabolizing tissue—the shell plates. However, other
non-metabolizing elements such as fats and connective tissue
will combust and thus form part of the ash-free dry weight.
These could conceivably be present in larger amounts in larger
animals, leaving less metabolizing tissue in proportion, and
thus contribute to an overall decrease in weight-specific
metabolism in larger creatures. However, ash-free dry weight
has commonly been used as a measure of the amount of metabolizing
tissue in an organism. Since ash-free dry weight represents
a fairly constant proportion of weight weight. I chose to
compute weight-specific respiration in terms of wet
weight.
Stability of respiratory rates
To determine whether respiratory rates measured over
a 6 to 12-hour period are representative of an entire 24-hour
period, five chitons were tested for 24 hours. Change in
manometric pressure is plotted against time in Figure 2.
Although many species show a diurnal or tidal respiratory
rhythm, this is clearly not the case for Tonicella. (The
irregularity near 0800 hours was reflected by the thermo¬
barometer and is not due to changes in respiration.)
E. Kincannon
Temperature Response in Tonicella
page 8
Effect of body weight upon respiratory rate
Rates of oxygen consumption as a function of body weight
at five different temperatures in subtidal and intertidal
populations are plotted in Figures 3 and 4 respectively.
Slopes and 95% confidence limits for each regression line
appear in Table 1. Correction for weight by means of regression
analysis greatly reduced the variance in even these small
samples of rates; this suggests that at any given temperature,
weight is a more important determinant of respiratory rates
in Tonicella than are other factors such as sexual and nutritional
states.
Statistical comparison of the regression lines revealed
no consistent pattern of change in the slopes (b-values) as
a function of termperature. The slopes of subtidal animals
(Table 1) suggest a pattern of decreasing b-values with
decreasing temperature, but this is not a statistically significant
trend and does not appear in the intertidal population. It
may be concluded from this that b for this species does not
change with temperature, large and small Tonicella are equally
affected by temperature, and Q0 does not vary with size.
These conclusions represent an exception to the general rule.
For example, in intertidal invertebrates studied by Edwards
and Irving (1943), Bullock and Rao (1954) and Newell (1970).
b decreased with temperature and 9,
decreased with body
weight. The results presented here for Tonicella are
therefore quite significant, and need to be confirmed with
more data and clearer statistical conclusions.
Temperature Response in Tonicella
page 9
E. Kincannon
These data also suggest that vertical location does not
affect the relationship between body weight and respiratory
rate; there are no consistent differences in b between sub¬
tidal and intertidal Tonicella tested at the same temperature.
An average of all 10 slopes obtained yields a b-value of
0.73 for this species. This is similar to those of other
molluscs as reported by Prosser (1973, p. 193) and close to
Zeuthen's (1970) generalized metazoan value of 0.74.
Effect of temperature upon acute respiratory rate
Figure 5 shows acute rates of oxygen consumption for a
hypothetical 0.70 g chiton plotted against temperature. These
points are derived from the regression lines in Figures 3 and
4, and their significance is determined by the confidence
limits of those lines. It is apparent that the curve for
intertidal Tonicella does not lie below that for subtidal
Tonicella. Subtidal chitons therefore do not appear to be
cold-acclimated; these results suggest that they may even be
warm-acclimated since their rates are generally lower than
those of the intertidal population. Althought this seems
unlikely, the question cannot be resolved until it is known
to what aspect of the thermal regime these animals are
responding. According to temperature data from nearby
Moss Landing, California (Harrold, Christopher personal
communication, 1974), intertidal animals are frequently
exposed at low tide to air temperatures both higher and lower
E. Kincannon
Temperature Response in Tonicella
page 10
than that of the ocean. If they are acclimating to the lowest
temperature in their environment, they could indeed appear
cold-acclimated in comparison with subtidal chitons.
Vernberg and Vernberg (1970, p. 132) have suggested
that the temperate-zone crabs Uca pugnax (Rathbun, 1901)
are better able to adapt to temperature extremes than are their
tropical relatives Uca rapax (Rathbun, 1918) because they
are exposed to more temperature fluctuations. This
argument may apply to Tonicella in that subtidal chitons
are exposed to a more narrow temperature range and would be
expected to be less flexible in respiratory response than
intertidal ones. From Figure 5, subtidal Tonicella show a
a constant 0l0 near 2.5, while intertidal ones appear to
decrease their Olos in higher temperatures. Segal (1956)
has found similar patterns in his work with heart rate in
Acmaed, although Bullock and Rao (1954) stated that Oho
generally increases with temperature in molluscs such as
Mytilus salifornianus (Conrad, 1837). Since a lower 0
would be advantageous for an intertidal animal exposed
at midday low tide to the sun, this preliminary result should
be further tested by repetition of these experiments, including
teperatures higher than 239c.
kelation of acclimation to temperature
Rates for each population of acclimated Tonicella
corrected to a standard body weight using acutely measured
E. Kincannon Temperature Response in Tonicella
b values are plotted in Figure 6. Very little can be said
about the subtidal population (Figure 6-B) because of the
intertwining curves. However, it appears that acclimation
has occurred to some extent in the intertidal population
(Figure 6-A). The rate-temperature curve for 13°0
acclimated animals falls below that for 8°C acclimated
aniamls at all points, although the distinction between 13°C
and 18°C curves is less clear. This is probably due to
greater temperature fluctuations in the two warmer tanks than
in the 8°C holding tank. These results come after a
relatively short period of acclimation, and experiments
conducted for a longer time are necessary to determine
whether or not subtidal Tonicella in fact can acclimate.
Nonetheless, they support the argument of the preceding section
that intertidal Tonicella are capable of compensating for
changes in their environmental temperature and possibly
can do so better than the subtidal Tonicella.
The curves in Figure 6-A do not show the same pattern of
decreasing Q with increasing temperature as do the acutely
measured rate-temperature curves. However, the absolute
shape of these curves is not as accurate, since only one or
to experimental animals determined each point and b-values
were not measured for acclimated animals. The curves in
Figure 6 are useful only in comparison with each other.
There remains the possibility that acclimation observed
page 11
E. Kincannon Temperature Response in Tonicella
in the laboratory is due to factors other than temperature.
Starvation is an obvious possibility; Newell (1970, p. 391)
reports that this is common and results in a decrease in
respiratory rate. However, at least one group of intertidal
Tonicella (8°c-acclimated, tested at 23°C) had higher rates
than did thecorresponding acutely measured group, and the
Tonicella were observed to deficate fecal pellets through¬
out their three weeks in the holding tanks. Possibly more
variation would have been introduced by trying to feed the
Tonicella than by starving them.
Future studies
An interesting question remaining is whether or not b
changes with laboratory acclimation. For the purposes of
this project no change was assumed, but this need verification.
Another topic is that of standard vs. active metabolism
and its effect on 9,: the 0,s found in this project are
higher than those given by Newell (1970) for standard
metabolism. Finally, once it has been determined that a
population of Tonicella can acclimate, transplantation
experiments would be helpful to test whether animals from
one habitat can acquire the ability to acclimate over a
short period of time.
SUMMARY
1. Two vertically separated populations of Tonicella lineata
pagel2
E. Kincannon
Temperature Response in Tonicella
page 13
(Wood, 1815) were tested for acute and acclimated respiratory
rates at a series of five temperatures.
2. Tonicella show no diurnal or tidal rhythm in respiratory
rate.
seems unaffected by body weight in both populations.
3. 0,
4. Subtidal Tonicella are not acclimated in the field to a
colder temperature than are intertidal Tonicella, but may
be less able to compensate for temperature change.
5. Intertidal Tonicella show a pattern of acclimation after
three weeks in the labotatory; results from subtidal animals
are ambiguous.
ACKNOWLEDGMENT
I wish to thank Dr. Fred Fuhrman and Paul Boothe for
their assistance and advice on this project. I greatly
appreciate the faculty, students, and staff of Hopkins
Marine Station for creating a friendly and stimulating at
atmosphere in which to work. Special thanks are reserved
for Chris Harrold, my advisor, for his guidance and
infinite patience.
E. Kincannon
Temperature Response in Tonicella
page 14
LITERATURE CITED
Barnes, James Ray
1972. Ecology and reproductive biology of Tonicella
lineata (Wood, 1815) (Mollusca--Polyplacophora). Ph.D.
dissertation. Dept. of Zoology, Oregon State University.
161 pp.; 47 figs.
(June 1972)
Bullock, Theodore Holmes
1955. Compensation for temperature in the metabolism
and activity of poikilotherms. Biol. Rev. 30 (3):
311-342; 5 figs.
(1 June 1954)
Bullock, Theodore Holmes, and K. Pampapathi Rao
1954. 010 as a function of size and habitat inpoikilotherms.
Amer. Natur. 88: 33-44; 7 figs.
Demopulos, Peter Andrew
1974. Diet, activity and feeding in Tonicella lineata
(Wood, 1815) with notes on its taxonomy. The Veliger
Edwards, G. A., and Laurence Irving
1943. The influence of temperature and season upon the
oxygen consumption of the sand crab, Emerita talpoida
Say. Journ. cell. comp. Physiol. 21 (2): 169-182; 4 figs.
Kenny,
Ron.
1958. Temperature tolerance of the chiton Clavarizona
hirtosa (Blainville). Journ. Roy. Soc. West. Aust.
41 (4) : 93-101; 6 figs.
(1 January 1958)
Newell, Richard Charles
1970. Factors affecting the rate of oxygen consumptinn.
3/2-450 in R. C. Newell, Biology of intertidal animals
E. Kincannon
Temperature Response in Tonicella
page 15
New York, N.Y. (American Elsevier, Publishing Co., Inc.)
Prosser, Clifford Ladd, ed.
1973. Comparative animal physiology. Vol. I. 3rd
ed. xxii + 456 + xlv pp.; illus. Philadelphia,
Penna. (W. B. Saunders, Co.)
Segal, Earl
1956. Microgeographic variation as thermal acclimation
in an intertidal mollusc. Biol. Bull. 111(1): 129-
152; 8 figs.
Segal, Earl
1961. Acclimation in molluscs. Amer. Zoll. 1: 235-244:
5 figs.
(12 Dêcember 1960)
Sokal, Robert R., and F. James Rohlf.
1969. Biometry. xxi + 776 pp.; illus. San Francisco,
Calif. (W. H. Freeman and Co.)
Umbreit, W. W., R. H. Burris, and J. F. Stauffer
1972. Manometric and biochemical techniques. 5th ed.
v + 387 pp.; 125 figs. Minneapolis, Minn. (Burgess
Publishing co.)
Vernberg, F. John, and Winona B. Vernberg
1970. The animal and the environment. xv + 398 pp.:
illus. New York, N. Y. (Holt, Rinehart and Winston, INC.)
Zeuthen, E.
1970. Rate of living as related to body size in organisms.
Polskie Arch. Hydrobiol. 17(30): 21-30; 7 figs.
E. Kincannon
Temperature Response in Tonicella
TABLE CAPTION
Table 1. Statistics for the weight regression lines shown
in Figures 1 and 2. Values are based on a double
logarithmic transformation. Ninety-five per cent
confidence limits for the y values (rates) are obtained
by constructing parallel lines at a vertical
distance of 2's above and below the regression
line on log scale.
Temperature Response in Tonicella
E. Kincannon
FIGURE CAPTIONS
Figure 1. Relationship between wet, dry, and ash-free dry
weights in selected Tonicella lineata of various sizes.
Upper plot is dry weight as a percentage of wet weight;
lower plot is ash-free dry weight as a percentage of
dry weight.
Figure 2. Respiratory rates of Tonicella lineata measured
over a 24-hour period. Change in manometer reading as
plotted is proportional to oxygen consumption. Weights
are those of the chitons tested.
Figure 3. Oxygen consumption as a function of body weight
for subtidal Tonicella lineata. Rates were measured at
five temperatures 24 hours after collection. Each point
represents one animal.
Figure 4. Oxygen consumption as a function of body weight
for intertidal Tonicella lineata. Rates were measured
at five temperatures 24 hours after collection. Each
point represents one animal..
Figure 5. Rate vs. temperature curves for intertidal ()
and subtidal (0) Tonicella lineata, measured acutely.
Points represent the intersection of a perpendicular,
erected at 0.70 g, with weight regression lines at each
temperature.
E. Kincannon
Temperature Response in Tonicella
Figure 6. Rate vs. temperature curves for intertidal (A)
and subtidal (B) Tonicella lineata acclimated to various
temperatures in the laboratory for three weeks. (8°C -2
13°c -+, 18°c -2). Each point is the mean of two animals,
corrected to a standard weight of 0.70 g as in Figure
5; vertical lines represent the range.
E. Vronne
Orgweight
wet weight
0 +
80 fr. 2. K
47.9 — 17
20
oto+
en-5.
50-
ash-Tree digweght
40 +
drg weight
—
o8a ao 24 28 3.2'30 40
Wer weight (g)
90
F. KinbéANN


.259
ol


-- (0 +
-363

§ 20—



a 10 —
.679
0

C

t.429


1e
3.489
3 §791 lan.3 S 7 à 1 10.m.
Time of Oaq
Eigore 2
E. Wesre
10 —
08 +
06 1
0 +
.02
O.01.
oo-
00 7—
o
003
237
130.
.

280.
6
—1—
4681o 10 30 4.0
Wet Weigh+ (g)
Figore 3
.2
E Rionannon
10—

.06-
.041
2
8 -02 +
01
o0-
o07-
05
003 +
.



2
2
230
e
fge.


+
6.810 2.0 3.0 4.0
et wegh+(
ore
0
E Kinore

83
8 0
50151
60
003 -
o


13° (2° 23.
Temperatore (°c.)
Fgore 6
05
03
ol
0161
008 +
00
f. Kincr
8
13

18 23
Temprator (
Fgoret
8
——
13 18 23
B.
e
Temprature Response in Tonicella
. Kincannon
Table 1
-------------------------------------------------—--------
ropulation
Subtidal
Interidal
—----------- —-- ------------ ---- —-----------------

Terperature
38. 10%.
8%.
188
8°
32c.
-------------------------------------------------------
.70.69
.86 .80
.59.68
.80 .57
.01
.81
-------—--------------------L---L----
5 Conl.
mits
.151.085.032 .166.138 .089.121 .050.180 .251.
2.S
------L---------------------------
—
—


—
-