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WARNING CONCERNING COPYRIGHT RESTRICTIONS

The copyright law of the United States (Title 17, United States Code)
governs the making of photocopies or other reproduction of
copyrighted material.
Under certain conditions specified in the law, libraries and archives
are authorized to furnish a photocopy or other reproduction. One of the
specified conditions is that the photocopy or other reproduction is not
to be used for any purpose other than private study, scholarship, or
research. If a user later uses a photocopy or reproduction for purpose
in excess of fair use, that user may be liable for copyright
infringement.
17 USC Section 107

RESPONSES OF THE MARSH PERIWINKLE,

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RESPONSES OF THE MARSH PERIWINKLE,

LITTORARIA (LITTORINA) IRRORATA TO
TEMPERATURE, SALINITY AND DESICCATION, AND
THE POTENTIAL PHYSIOLOGICAL RELATIONSHIP
TO CLIMBING BEHAVIOR
RAYMOND P. HENRY*, CHRISTOPHER J. McBRIDE* and
ANN H. WILLIAMS**
* Department of Zoology and Wildlife Science, and the Alabama Agricultural
Experiment Station. 101 Cary Hall. Auburn University, Auburn, Alabama
36849-5414 USA. **Sigma Xi, The Scientific Research Society, 99 Alexander Drive,
Research Triangle Park, North Carolina 27709, USA
October 1992)
Littoraria irrorata climb the stems of Spartina alterniflora in the face of an advancing tide. This
behavior, while allowing escape from predators, may also be correlated with the physiological
responses of the organism to variations in physical factors typically found in an estuarine salt
marsh. To test this hypothesis, snails were exposed to changes in environmental salinity, their
resistance to desiccation was determined, and their ability to take up oxygen from both water and
air was measured at varying temperatures. L. irrorata were found to be tolerant to wide variations
in salinity. The mechanism employed appears to be intracellular volume regulation, which is
common in many other marine and estuarine species. This species is also very tolerant of
desiccation, surviving after losing nearly 70% of its soft tissue weight over a period of weeks.
Upon contact with water, it quickly rehydrates, regaining its initial weight within one hour. At
low temperature (20C) aquatic and aerial oxygen uptake (VO2) are about equal. As temperature
increases to 30C, aquatic VO2) increases, but it declines precipitously between 30 and 40C, the
temperature range in which L. irrorata are stimulated to climb. At high temperatures, when
oxygen solubility in water is low, the failure of aquatic respiration may be the driving
physiological factor behind the emergence of L. irrorata into air where high oxygen availability
is sufficient to maintain oxidative metabolism.
KEY WORDS: Littoraria, respiration, temperature, behaviour

INTRODUCTION
Littoraria (Littorina) irrorata, the marsh periwinkle, is a pulmonate gastropod that inhabits
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intertidal marshes from New York to Texas (Bingham, 1972). This organism displays a pattern of
vertical migration along the stems of the marsh cordgrass, Spartina alterniflora, that appears to
be correlated with the tidal cycle. Snails ascend cordgrass stems in the face the rising tide and
descend at low tide (Bingham. 1972; Hamilton, 1976; Williams, and Appel, 1989). It was initially
proposed by Hamilton (1976) that this climbing behavior could allow the snails to avoid
predation by aquatic predators such as Callinectes sapidus, the blue crab. Recent experimental
evidence, in which predation mortality increased when the cord grass stems were manually
clipped short preventing the snails from climbing to their normal position above the water,
supports this hypothesis (Vaughn and Fisher, 1988).
These animals also experience high mortality in the absence of predators, however, when they are
prevented from climbing off the substratum (Warren, 1985). The author suggested osmotic stress
as a possible cause of mortality, but that idea has never been tested directly. The highest mortality
occurred during the summer months, also suggesting that thermal stress could be a cause of
mortality. McBride et al. (1989) showed that substratum temperatures above 35C stimulated
snails to climb artificial stems in the absence of predators, and that snails preferentially chose
stems that were maintained at cooler temperatures. Furthermore, this behavior was shown to be
effective in allowing the animal to maintain its body temperature below that of the substratum
and below the body temperature that would result if the animal were forced to remain in contact
with the substratum (Williams and Appel, 1989).
In L. irrorata there is a direct relationship between temperature and metabolic rate, a relationship
that is typical of ectothermic organisms. An increase in metabolic rate with temperature is
characterized by an increase in oxygen uptake (V02) e.g.. Newell, 1966, 1969, 1973). A second
factor which would tend to exacerbate thermal stress results from the inverse relationship of 02
solubility in water and temperature (Dejours, 1975); at high temperature there is much less O2
available in water to support the increase in oxidative metabolism. Snails can survive, when
submerged, for extended periods of time entirely on their ability to obtain oxygen from the water
(Bliel and Gunn, 1978), but these studies were done at relatively low temperatures (2123C).
The ability of this organism to respire aquatically in the face of high temperature has never been
tested. Thus, at high temperature, these animals could face both direct thermal stress and a
shortage of oxygen when immersed by the advancing tide.
L. irrorata, a pulmonate, belongs to a genus whose members are known to be bimodal breathers:
organisms that can exchange respiratory gasses with either water or air (see McMahon and
Russell-Hunter, 1977 for a treatment of bimodal respiration in members of the genus Littorina).
As such, climbing behavior which results in emergence during high tide would give the animal
access to the relative high concentration of 02 in the air. This study examined the interaction of
temperature and oxygen uptake in water and air as a potential determinant in the climbing
behavior of this species. The animals ability to tolerate osmotic stress and desiccation are also
directly investigated for the first time.

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MATERIALS AND METHODS

Littoraria irrorara were collected from a Spartina alterniflora saltmarsh on the north side of
Dauphin Island, Alabama. Animals were transported to the laboratory in plastic coolers filled
with damp vegetation. The snails were maintained in the laboratory in 20 L aquaria filled with
approximately 4 L of water (10 ppt salinity) and covered with fiberglass mesh to prevent escape.
Animals were allowed to acclimate to laboratory conditions for one week before being used in an
experiment; during that time they were not fed.
In order to assess the ability of L. irrorata to withstand changes in ambient salinity, snails were
divided into two group: one group was acclimated to 5 ppt and the other to 20 ppt. The animals
were forced to remain submerged in the aquarium by a plastic grid that was fixed slightly below
the water level. The salinity in the aquarium was changed at a rate of 3 ppt per day by the
addition of either distilled water or concentrated brine until the desired value was reached;
animals were then allowed to remain at that salinity for a period of two weeks. After that time the
snails acclimated to 5 ppt were transferred to water of 20 ppt, and vice versa. During the
subsequent two week period the following variables were measured: wet weight, hemolymph
osmolarity, and whole-animal ammonia excretion.
The ability of L. irrorata to regulate/adjust cell volume was determined by following changes in
tissue wet weight after a salinity change. Wet weight was determined by weighing the intact
animal to the nearest milligram on an analytical balance (Sartorius). Tactile stimulation of the
foot was used to cause maximal (and presumably uniform) withdrawal of the animal into the shell
before weighing to minimize variation in extracellular water. At the end of the experiment the
shell was removed, dried, and weighed: that weight was then subtracted from the previous values
for total weight to give the wet tissue weight of the animal. Changes in wet weight were reported
as percent changes in the pre-transfer value which was normalized to 100%.
Hemolymph was obtained the method of Pierce (1970). The foot was slashed with a scalpel
blade, the animal was placed in a funnel, and hemolymph was drained into a 1.5 ml
microcentrifuge tube (Eppendorf). The sample was centrifuged for 1 min (Fisher microfuge), and
osmotic concentration was determined on a 10 l sample using a vapor pressure osmometer
(Wescor 5100C).
Ammonia excretion was measured by the phenol-hvpochlorite method (Sol orzano. 1969). Snails
were placed in 10 ml of water of the appropriate salinity: 5 ml were immediately withdrawn to
determine background ammonia concentrations. The animals were left in the remaining water
volume, forcibly submerged by fiberglass mesh fixed at the water surface, for 30 min; at that time
they were removed, and the ammonia concentration was determined in the remaining 5 ml. The
differences between the background concentration and that at 30 min was taken to be the amount
of ammonia excreted by the animal. At the end of the experiment the shell was removed, the soft
tissue was dried at 60C to constant weight, and ammonia excretion was reported as mol NH3
per gm dry weight per hour. A separate group of animals was used for the determination of each
of the three variables (volume regulation, hemolymph osmolarity, and ammonia excretion).
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Tolerance to desiccation was also determined by measuring changes in soft tissue in plastic
beakers with fiberglass mesh secured to the top to prevent escape. The beakers containing the
snails were weighed to the nearest 1 mg on an analytical balance (Sartorius): they were then
placed in a desiccator at room temperature (23C) and weighed periodically over the following
two weeks. At that time, animals were removed from the beakers and allowed free contact with
water (10 ppt salinity): they were weighed periodically until they had regained their original
weight. Ultimately, the shells were removed and weighed, and the weights of the beakers, mesh,
and shell were subtracted from the total weights where appropriate to give the weight of the soft
tissue. Changes in weight were expressed as a percent of the initial wet tissue weight (set at
100%).
Oxygen uptake (V02) of L. irrorata in water and air at 20, 30 and 40C was measured by
following the decline in O2 tension (P02) in a closed chamber. The chamber consisted of a 30 ml
Erlenmeyer flask that was partially filled with glass beads to reduce the internal volume to 10 ml.
The mouth of the flask was fitted with a rubber stopper. Two needles (22 ga), each attached to a
three-way stopcock, were inserted through the stopper to allow air or water samples to be taken
from the chamber. Samples were drawn with a 1 ml syringe (after flushing three times to
eliminate the dead air space in the syringe) and analysed for PO2 on a blood gas analyser
(Radiometer PHM 73 and E5046/D616 electrode thermostatted to the experimental temperature).
PO2 values in water (10 ppt) were converted to molar concentrations using the solubility
coefficients of Dejours 1975); final values for VO2 were reported as ml O2 per gram dry tissue
weight per hour.
The mean VO2 in air vs water was determined as paired observations on individuals at a given
temperature. All chambers were equilibrated to the experimental temperature in a water bath prior
to the addition of the snails. All animals were acclimated to 20C for one week: thus the values
for VO2 at the higher temperatures were acute measurements. For each trial, an individual snail
was placed in a chamber, the stopper was inserted, and gas (21% O2, balance N2) was circulated
through the chamber for 2 min. At that time a 1 ml sample of either air or water was taken, and
the initial P02 determined. The volume of air water was replaced, and the chamber was sealed by
closing the stopcocks. The duration of the experiments ranged from 30 min to 15 hr, depending
on the temperature and the medium being measured. This was a result of the varying rates of
VO2 at different temperatures in air vs water. At the end of the experiment, a second 1 ml sample
was taken from the chamber and analysed as above; the P02 inside the chamber was never
allowed to fall below 60 torr. The snail was then removed from the chamber, and its soft tissue
was removed from the shell and dried at 60C to a constant weight. Values for VO2 in air and
water at the three temperatures were compared using two-way analysis of variance (ANOVA)
and Duncans Multiple Range test (SAS}.

RESULTS

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L. irrorata appears to be very tolerant of large changes in ambient salinity. There was no
mortality among groups of 10 snails acclimated to 20 ppt and abruptly transferred to 5 ppt or vice
versa. The snails reacted to the salinity changes as typical euryhaline osmoconforming volume
regulators. For animals transferred from 20 to 5 ppt, there was an initial gain in soft tissue weight
of about 8% followed by a 2 hr period in which the original weight was restored (Figure 1). The
hemolymph osmotic concentration of the snails decreased by about one-third during the first 3 hr
after transfer, indicating that the gain in weight was the result of an osmotic water load which
subsequently resulted in an increase in cell volume (i.e.. cell swelling). During both the periods of
swelling and volume readjustment ammonia excretion increased approximately two-fold (Figure
1). While restoration of the initial values of soft tissue weight was complete within 2 hours,
hemolymph osmotic values continued to decline through 24 hr, and ammonia excretion in 5 ppt
remained elevated through 7 days (Figure 1).
For animals acclimated to 5 ppt and transferred to 20 ppr a similar general pattern of response
was seen but with a different time course. There was an initial weight loss

of about 8% that in this case took place over a period of two hours (Figure 2): the process during
which the initial weight was restored then took 14 days. The restoration was never perfect with
animals recovering to about 9899% of the initial weight values (Figure 2). The initial weight
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loss was accompanied by an increase in hemolymph osmotic concentration of about 350 mOsm
and a decrease in the rate of ammonia excretion.
L. irrorata also appeared to be highly tolerant of desiccation. Snails suffered no mortality over
the initial 9 days of desiccation during which they lost 50% of their wet weight (Figure 3).
Throughout the duration of the experiment (16 days) there was only 10% mortality at a maximum
of 60% loss of wet weight. Upon reintroduction

to water, the snails rapidly rehydrated their tissues. Animals restored their weights to
approximately 95% of the initial value within 1 hr. and 100% by 2 hr (Figure 3).
The pattern of bimodal breathing in response to increasing temperature was the only indicator of
physiological stress induced by a physical factor that could be correlated with emergent climbing
behavior. At low temperature (20C) aquatic and aerial VO2 were not significantly different
(P>0.05, Duncans multiple range test).

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At 30C aquatic VO2 increased four-fold, while there was a doubling of in aerial VO2 (Figure 4).
When the temperature was increased to 40C, aerial V02 was maintained, but aquatic VO2
dropped precipitously, decreasing to about one-third the value for V02 in air (Figure 4).

DISCUSSION
Thermal stress, independent of predation pressure, has been shown to initiate climbing behavior
in L. irrorata (McBride et al., 1989; Williams and Appel, 1989). The results of this study indicate
that the failure to obtain adequate amounts of oxygen from 02-poor water at high temperature to
meet the metabolic demands of the animal is one of the principle physiological determinants of
climbing behavior. This may be a result of lower O2 content in water of high temperatures, or a
failure of the snails ability to adequately ventilate and/or perfuse the respiratory surface at high
temperature.
Many intertidal gastropods exhibit an increase in oxidative metabolism with temperature, with
Q10 values near 2.0 (McMahon and Russell-Hunter, 1977). The most common examples are
from northern, temperate regions which rarely exceed 30C. For L. irrorata, which routinely
experiences summer temperatures in excess of 30C, total VO2(aquatic plus aerial) increases 3.5
fold from 20 to 30C. At 40C total VO2 decreases, but it is still roughly twice the value for 20
C, indicating an approximate doubling of metabolic rate between 20 and 40C. While metabolic
rate is increasing with temperature, the concentration of oxygen in the ambient water is
decreasing. Oxygen solubility in water decreases with increasing temperature (Dejours, 1974)
such that in a liter of water at 40C there is one-third less O2 available than at 20C (8.8 vs 12 ml
O2 assuming air-saturation in both cases). In comparison, one liter of air contains about 200 ml
of O2 regardless of temperature. Temperature

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measurements in the salt marsh confirm this situation. While air temperature was 31C, substrate
temperatures ranged from 3436C (X = 35.7 1.1). The temperature of standing water in the
marsh (x = 35.1 = 0.99) was not significantly different from that of the substrate (McBride,
1987). Therefore, with the stems of S. alterniflora serving as a refuge from high substratum and
water temperatures and as a route to access high atmospheric O2 concentrations, it is plausible to
suggest the combination of thermal and respiratory stress as the physical physiological driving
forces behind the vertical migration of L. irrorata.
Recent field studies appear to support this idea. Mark-recapture data for L. irrorata from a Texas
marsh show that small size snails remain significantly closer to Spartina stems than do larger
ones (Vaughn and Fisher. 1992). Smaller individuals should have a greater susceptibility to
thermal/hypoxic stress, and therefore be more reliant on rapid vertical migration, for the
following reasons. First, because they are small (and therefore have less mass), their internal
temperature would change more rapidly than would that for larger snails. Also, smaller animals
have a much higher mass-specific metabolic rate (i.e.. higher oxygen uptake) and would therefore
be more compromised by water of low O2 content. Interestingly, for periods of low temperature
during winter, vertical migration ceased (Vaughn and Fisher, 1992). This occurred when water
oxygen content would be higher (due to higher solubility at low temperature), and when the
snails metabolic rate would also be depressed. Thus, in winter, both the physical and
physiological factors that tend to induce climbing are at a minimum, and the snails do not climb.
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The temperature-dependent pattern of bimodal breathing observed in L. irrorata is also found in

other closely related species of intertidal gastropods as well. In L. littorea, L. obtusata, and L.
saxatilis, measured between 5 and 40C, aquatic VO2 predominates at the lower temperatures,
but above 30C aerial VO2 is the primary route for obtaining oxygen (McMahon and Russell]Hunter, 1977). The change to the reliance on aerial VO2 is most striking in L. irrorata, but its
apparent widespread presence in other intertidal species supports the idea that thermal stress and
concomitant low aquatic O2 concentrations could have been important general factors in the
development of aerial respiration in the gastropods.
Predation, on the other hand, may be more important in determining the position above the water
level to which the snail climbs. L. irrorata has been shown to climb to a height of at least 15 cm
above the waler (Bingham, 1972; Hamilton. 1976), high enough to avoid most natant predators
(Vaughn and Fisher, 1988). Position on a cordgrass stem, other than the fact that it is off the
substratum and above the water level, would make little difference in behavioral
thermoregulation or oxygen uptake.
L. irrorata appears to be quite tolerant of desiccation, the major physiological constraint on
emergence behavior. The snails lose water slowly and rehydrate rapidly. They survive a 50% loss
of soft tissue weight over 14 days, but during a typical tidal cycle a snail would lose at most 10%
of its wet weight while emerged. Other terrestrial gastropods have been shown to initiate drinking
to rehydrate their tissues after a loss of about 8% of initial wet weight (Prior, 1984). It is,
therefore, probable that any water loss in L. irrorata through dehydration would be quickly
regained via contact rehydration when the animal returned to the substratum.
Osmotic stress does not appear to play any role in emergence behavior of intertidal gastropods. L.
irrorata is euryhaline, surviving well from 5 to 20 ppt salinity. It can also survive large, stepwise
reductions in salinity (e.g., 15 ppt) which simulate field conditions of catastrophic dilutions
brought about by storms and/or freshwater runoff. Animals responded to salinity changes as
typical osmoconforming, cell volume rcgulators. They initially gained or lost weight when
transferred to low or high salinity, respectively, but they were able to restore soft tissue weight to
near the original values. The increase in ammonia excretion after low salinity transfer is
indicative of cell volume regulation via reduction of the intracellular free amino acid (FAA) pool
via deamination and excretion of the resultant amino group. Another species, L. littorea, has also
been shown to be a euryhaline osmoconformer that tolerates salinity changes via cell volume
regulation and adjustment of the intracellular FAA pool (Taylor and Andrews, 1988). This
mechanism is common in many euryhaline marine and estuarine invertebrates: its presence in
gastropods obviates the need for vertical migration to avoid salinity stress.
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