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Osmotic Response in N. californica
INTRODUCTION
Nuttallina californica (Reeve, 1847), a common intertidal
chiton, has a large vertical range and is found in a variety of
habitats. Though the species is eurytopic, individuals appear
to maintain their particular position in the intertidal for
extended periods (MacGinitie and MacGinitie, 1968). Salinity
variations from 14%, to 45%0 have been measured in pools and
crevices in the intertidal (Boyle, 1969); the range and
duration of osmotic stress would be a function of position in
the intertidal. Chitons are typically isosmotic with seawater,
as are most marine molluscs, but intertidal species subjected
to salinity fluctuations may have developed adaptive responses
to osmotic change, (Prosser, 1973). Previous osmotic studies
on the chiton, Sypharochiton pelliserpentis (Quoy and Gaimard,
1835), in New Zealand (Boyle, 1969 and 1970) showed this species
to be an osmocenformer.
The topic explored in this paper is the response to
osmotic stress in individuals of Nuttallina californica selected
from two micro-habitats differing in vertical height and wave
exposure in a way which has resulted in difference in osmotic
stress histories.
MATERIALS
The study was carried out on individuals taken from Mussel
Point at Hopkins Marine Station, Pacific Grove, California.
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Osmotic Response in N. californica
One group of animals, taken from the lower intertidal, was
from a vertical face of granite normal to the incoming surf.
These organisms thus were not exposed to sitting water or
extensive evaporation. They were found along with the
coralline algae Corallina vancouveriensis (Yendo), Litho¬
phyllum proboscideum (Foslie) and the barnacle, Balanus glandula
(Darwin, 1854). The other set of animals, taken from the
high intertidal, was from an area protected from direct surf
action by offshore rocks. The chitons were collected on
horizontal ledges, making them exposed to salinity fluctuations
from evaporation or precipitation. In the lower part of this
range were Endocladia muricata (Postels & Ruprecht) J. G.
Agardh, Gigagartina paillata (C. A. Agardh) J. G. Agardh,
Tetraclita squamosa (Darwin, 1854) and Pollicipes polymerus
(Sowerby, 1833).
The chitons in the low intertidal were collected at
a tidal range of 2.1 to 3.7 ft. which is almost completely
below the plus 3.5 ft. critical level (Doty, 1957) where
they are subjected to air exposure less than 10.25 hours a
day. Directly above this level there is an almost two¬
fold increase in exposure time and then it gradually increases
until at 5.0 ft. the maximum exposure is twentythree hours.
The high intertidal Nuttallina were collected between 4.0
and 5.2 ft. and lie in the latter range.
Middle sized chitons were chosen, the mean size of the
experimental animals from the low intertidal population was
1.86 g. and 2.10 g. for the high intertidal population.
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Osmotic Response in N. californica
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METHODS AND RESULTS
A. Change in weight following osmotic stress.
In order to test for possible differential response to
osmotic stress in the two populations, a series of laboratory
experiments involving measuring weight changes of stressed
animals was conducted. A method of weighing the chiton
with its substrate was devised to avoid the stress of
constantly detaching the chiton. Plastic petri dishes with
holes drilled in the top to allow free exchange of water were
used as containers and were blotted dry and weighed with the
chitons. Experimental animals were collected and placed in
the dishes, kept in running sea water for forty-eight hours
to allow them to acclimate and partially empty their guts.
The salinity of the ambient and circulating seawater
was determined by a salinometer to be 33.9%.. Using this
information the experimental salinities of 50%, 75%, 90%.
95%, 100%, 105%, 110%, 120%, and 125% of the concentration of
local seawater were mixed from "Instant Ocean Synthetic Sea
Salts" (Aquarium Systems, Inc.).
For each solution six or more chitons from each site
were blotted and weighed with their containers before being
placed in 250 ml. finger bowls of test solution. The bowls,
covered to reduce evaopration, remained in the sea table at
a constant temperature of 14° + 1°C. At intervals of 1, 2,
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Osmotic response in N. californica
3, 4, 6, 10, 14, and 24 hours the chitons were removed, blotted
dry and weighed.
In order to distinguish between a behavioral and a
physiological difference in the two groups' response to
osmotic stress, eight chitons from each site were put in
small mesh bags and placed on their backs in finger bowls
to prohibit their excluding the external medium. Salinities
of water in the bowls, exposure and weighing was as before.
Results: Low intertidal chitons gained more weight due to
osmotic uptake of water, and at a slightly faster rate than
the high intertidal chitons in dilutions of sea water,
(Figure 1). Weight changes are presented as percent change
of original body weight.
In the 95%, 90% and 75% seawater a small decrease
toward the original weight occurred with time in both groups.
No decrease was seen in the 50% medium where the high intertidal
Nuttallina gained an average of 19.4% of their body weight,
significantly less than the mean gain of 28.4% for the low
intertidal chitons (P(.001, n=30, Student ttest).
Low intertidal chitons initially lost weight at
a slower rate than the high intertidal in hypertonic seawater,
but this was reversed after one to two hours so that they
ultimately evidenced a greater percentage change in weight.
For the 120% medium, after twenty-four hours, the low inter¬
tidal animals lost an average of 8.9% and the high intertidal
chitons lost 6.6% (P.001, n
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Osmotic Response in N. californica
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The high intertidal Nuttallina that were placed in the
mesh bags in 50% seawater gained 19.3% while the low intertidal
chitons gained 26.4%. No significant difference between
weightgain in attached versus unattached animals was found
(student t-test).
B. Freezing Point Depression
The internal body fluid and the external medium of the
chitons were examined for differences in osmotic concentration
using a modification of the method by Gross (Welsh, Smith,
& Kammer, 1968). Nuttallina from both the high and the low
intertidal groups were placed in various salinities as
previously described. At twelve and twenty-four hours one
animal was removed from each salinity, weighed and bled by
inserting a hypodermic needle between the seventh and eighth
plates and extracting with a syringe a small sample of pericardial
fluid, (Boyle, 1969). These samples were placed in capillary
tubes, sealed and frozen along with samples of the solutions.
Results: The results of the freezing point depression
experiments show Nuttallina to be an osmoconformer. The
mean of the osmotic pressures of the internal fluid was within
1.8% of that of the external fluid, which is within the limits
of the definition by Wilbur and Yonge (1964) of osmotic
equilibrium (+ 1 to 2%). Equilibrium had been reached before
the twelfth hour as there was no appreciable difference
between the 12 and the 24 hour samples.
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Osmotic Response in N. californica
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Theoretical Osmometer
C.
To discover how closely Nuttallina follows the predictions
for a gravimetric osmometer, eight chitons, four from each
group, were weighed and dried to constant weight in a 75°c
drying oven. The percent body water was calculated as was
the theoretical weight change that would be predicted to
occur in each salinity if the chiton was a perfect osmometer
with no bound water,
Results: Water constitutes 61% (Range= 58% - 66%) of the
body weight of the low intertidal Nuttallina and 56% (Range-
53 - 60%) of the high intertidal. The predicted weight
gain in 50% seawater is 61% for the low intertidal yet they
gained an average of only 28.4%, significantly less than
predicted (chi-squared = 17.35, PX.01). The prediction
change for the high intertidal is 56% yet the actual average
weight gain was 19.4%, again significantly less (chi-squared
-23.83, P£.005).
DISCUSSION
High intertidal Nuttallina californica, although
osmoconformers, have developed means to cope with the higher
osmotic stresses associated with their environment. Behavioral
changes, such'as partial exclusion of water in osmotic imbalance
by clamping down, do not completely account for their osmotic
response; their equilibrium point for hypotonic solutions
emains the same whether or not they are attached. It may,
however, affect the rate at which equilibrium is reached,
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Osmotic Response in N. californica
page 8
a quantity not measured for unattached animals.
In hypotonic solutions the chitons from the high intertidal
area gained less than the low intertidal chitons. Both
groups, however, showed a decrease in body weight after
reaching an extreme, except in 50% seawater; thus volume
regulation does occur. The difference between the two
groups must be due either to the efficiency of volume regulation
or to differences in the threshold of stress required to
initiate regulation. By having a lower threshold volume
regulation of the high intertidal chitons would start sooner,
thereby reducing the weight gained. Since the chiton is not
an osmoregulator it never returns to its original weight
while still in the hypotonic solution.
The theoretical weight changes deviate from the observed
changes for the 50% and 75% solutions. This can partly be
explained by assuming that 10 - 15% of the body water is
bound by protein and osmotically inactive (Prosser, 1973),
but some type of mechanism working on a switch basis, such
as ionic excretion or the making of monosaccharides from
stored polysaccharides, must also be present to account for
the difference between the two populations,
Since, in hypertonic solutions there is no appreciable
return of body weight toward the original weight it is
unclear if any volume regulation especially since the actual
weight changes come close to the theoretical weight changes.
The greater ability for high intertidal Nuttallina
californica to withstand osmotic stress could have a
genetic basis as in selective larval settling or differential
Simonsen
Osmotic Response in N. californica
page 9
survival. This ability seems more likely, though, to be
ontogenetic by development of differences in tissues or
metabolism as a result of exposure to the stress. Further
work needs to be done before this can be answered.
SUMMARY
1) High intertidal Nuttallina californica can cope with
osmotic stress better than the low intertidal chitons as shown
by their smaller weight change.
2) The low intertidal chitons have a slower initial rate
of weight loss in hypertonic seawater.
3) Attached and unattached animals reach the same equilibrium
point so behavior alone can't explain the differences in
osmotic response.
4) There is a large deviation from predicted weight changes
in 50% and 75% seawater which shows that both populations
do exhibit some regulation, but with a difference in
effectiveness.
ACKNOWLEDGMENTS
I am indebted to the faculty and staff of Hopkins Marine
Station, expecially to Mr. Chuck Baxter and Dr. Robin Burnett
for their advice and encouragement during the course of this
experiment.
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Osmotic Response in N. californica
page 10
LITERATURE CITED
Boyle, P. R.
1969. The survival of osmotic stress by Sypharochiton
pelliserpentis (Mollusca: Polyplacophora). Biol. Bull.
136: 154-166.
1970. Aspects of the ecology of a littoral chiton,
Sypharochiton pelliserpentis (Mollusca: Polyplacophora).
New Zealand Journ. of Marine & Freshwater Research
4 (4) : 364-384.
Doty, Maxwell S.
1957. Rocky intertidal surfaces, pp. 558-559.
Hedgepath, Joel, W. (ed.), Treatise on marine ecology
and paleoecology, Vol. 1-Ecology; New York, Waverly
Press; viii + 1296 pp.; illust.
MacGinitie, George E. and Nettie MacGinitie
1968. Natural History of Marine Animals. 2nd ed.; New
York, N. Y., McGraw Hill. xii + 523 pp.; 286 figs.
Prosser, C. Ladd (ed.)
1973. Comparitive animal physiology Vol. 1 3rd ed.
W. B. Saunders, 456 pp.; illust.
Wilbur, Karl, M. and C. M. Yonge, (eds.)
1964. Physiology of mollusca Vol. 1; New York & London
Press, 473 pp.; illust.
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Osmotic Response in N. californica
FIGURE CAPTION
1.) Mean weight changes (as 8 of original body weight)
of N. californica in experimental solutions of
various salinities. Test salinities in percent
of ambient seawater and number of animals tested
for each condition is indicated. The standan
errors of the means were all less than 38 of the
percent change indicated and thus too small to
indicate graphically.
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