Levin p. 2
ABSTRACT
The marine snail, Littorina planaxis, has the ability
to distinguish between solutions of high and low salinity.
Research indicates that salinity is determined primarily
through the detection of chloride. The region of de¬
teotion seems to be localized in the area, between the
operculum and the foot, known as the operculigerous disc.
Levin p. 3
INTRODUCTION
Littorina planaxis, a gastropod snail, common to
the upper intertidal regions of rocky coastal areas, is
most often found with its operculum closed and attached
to the rocks with a mucus seal. If a snail is dislodged
from the rocks and placed into sea water it begins an
extruding response, moving its operoulum away from the
mouth of the shell and gradually extending its foot and
tentacles. The extruding response consists of two parts.
Stage one is a movement from a closed position to one
where tentacles are exposed. Movement stops momentarily
and the snail may reclose, or may continue with full ex¬
trusion of the foot, head, and tentacles. If a snail is
dislodged and placed into distilled water, no stage one
response occurs. Littorina planaxis must have some mech¬
anism that can determine certain properties of the sur¬
rounding water while the snail is drawn into its shell.
The purpose of this research was to characterize this
response and to localize the region of detection. Results
suggest thatasnail detects primarily chloride concentra¬
tion.
MATERIALS
Littorina planaxis was collected from the rocks at
the Hopkins Marine Station of Stanford University in
Pacific Grove, California. Snails were either placed
into distilled water or left to desiccate in dry wooden
Levin p. 4
aquaria for several hours before each experiment. No
snail was kept for more than 48 hours.
Artificial sea water contained Nacl (450 mM), Mgcl,
(50 mM), Cacl, (10 mM), KCI (10 mM), and Tris(hydroxy¬
methyl)aminoethane Hydrochloride (Tris HCl) buffer at
pH 7.8 (10 mM).
METHODS
Standard testing procedure
Each snail was removed from distilled water or from
desiccation and placed with its operculum up. The oper¬
culum was then covered with 20 microliters of a test solu¬
tion, and the snail's response noted. A stage one response
within two minutes was considered a positive response
whether or not full extrusion occurred. No difference
in response was ever noted between the two groups. No
snail was tested more than once and responses from a group
(4-2) of snails were combined to give a percentage of
positive responses. All snails giving negative responses
were then tested with artificial sea water (ASW) or natu¬
ral sea water (NSW) to make sure that the snails were
alive.
Characterization
To determine the range of salinities over which the
stage one response occurs, test solutions were made from
ASW or from NSW. Test solutions were also made by add¬
Levin p. 5
ing varying amounts of Nacl, MgCl,, Cacl,, or KCl from
1 M stook solutions to a dilution of ASW at 200 mOsm to
characterize the effects of different cations. lo test
the effeots of anions, the following solutions were com¬
pared: 0.5 M sodium chloride, 0.52 M sodium D-gluconate,
0.5 M Tris HCI, 0.5 M and 0.4 M Tris citrate. All solu¬
tions were adjusted to pH 7.8 with either 5 mM Tris HCI
buffer or NaOH. Non-electroyte test solutions con¬
tained sucrose adjusted to pH 7.8 with 10 mM Tris HCI
buffer.
Localization
Mapping of regions sensitive to saline solutions
was carried out by placing small amounts (1 to 5 micro¬
liters) of ASW or NSW on or around the operculum.
To determine the role of the cephalic tentacles in
the deteotion of salinity, it was necessary to remove
these organs from a group of snails. An aqueous solu¬
tion of magnesium chloride isotonic with NSW was used as
an anesthetic and relaxant (Peters, 1964). An immersion
of 15 to 20 minutes was usually adequate to allow for easy
removal of the tentacles. No long-lasting affeots of the
anesthetic were ever apparent, even after immersions of
two hours.
Thirty snails were divided into three groups, an ex¬
perimental group, and two control groups. The experimental
Levin p. 6
group was anesthetized in magnesium chloride, cephalic
tentacles were removed, and the group was placed into
normal sea water for recovery. One control group followed
the same procedure except cephalic tentacles were not re¬
moved. The second control group was identical to the
other control group except it was not placed into mag-
nesium chloride. All three groups were placed into
distilled water for 30 minutes prior to testing with NSW.
Methylene blue stain (Loefflers) or one percent
usec
OsO, in 0.5 M sodium acetate wasito visualize those
regions exposed to test solutions while the snail
remained drawn into its shell. Snails were exposed to
a stain either by placement of one drop of stain upon
the closed operculum, or by direct immersion of the
snail into the stain. Exposure time was varied. After
exposure, snails were washed with distilled water, placed
into isotonic magnesium chloride, and examined for stain¬
ed areas.
RESULTS AND DISCUSSION
Characterization
The snails response to solutions of varying salin¬
ities is summarized in figure one. The graph is plotted
as the percentage of positive sstage one) responses at a
given osmolality of a test solution. NSW is around 970
Levin p. 7
mOsm. There are three possible methods that Littorina
qualita
planaxis might be using to determine the
surrounding solutions: detection of total osmotic pres¬
sure, deteotion of ionic strength, detection of one
specific ion.
Of the twenty snails tested with sucrose solutions
of 984 mOsm and 826 mOsm, all failed to give positive
responses. The osmolalities of the sucrose solutions
were well within a region that gave 100 percent posi¬
tive response with ASW and NSW. This result dees netsuppert
the possibility that the deteotion of salinity is through
the use of a general osmoreceptor.
Results from additions of specific ions to the
sub-threshold ASW dilution are summarized in figures two
and three. In figure two, the percentage of positive
responses is plotted against the percentage of a specific
cation's concentration relative to the concentration of
that cation found in ASW. A standard dilution curve for
ASW is given as a reference. No positive responses
ocourred with the dilution (arrow). If L. planaxis was
testing for one specific ion, the curve for that cation
should follow the dilution ourve of ASW closely. The
data suggests that the snails are not testing solely for
the calcium, magnesium, or potassium cation. The sodium
Levin p. 8
ion is probably not the only stimulus for the extruding
response either. There may, however, be some sort of
summation of several ions to give a 100 percent positive
response.
No single cation gave 100 percent positive response
except for Ca". As explained previously, Ca
cannot be
preferentially deteoted since its threshold for positive
response is well above that found in NSW. A solution of
Nacl with 10 mM Cacl, gave a curve similar to that of
ASW. From this data either Na' or Cl appear to be pri¬
marily detected. Some concentration of Ca'* (between 2
and 10 mM) is needed for some snails to achieve a stage
one response. At this point it is not entirely clear
what the effeot of the calcium ion is.
The data in figure two can be replotted as the per¬
centage of positive response against the concentration
of the chloride anion (fig. 3). This graph shows a very
steep function where a small increase in the chloride
concentration results in a large increase in the per¬
centage of positive responses. As an example, the Ca
curve rises from no positive responses to 100 percent
positive response over only a 60 mM increase in chloride
concentration. This comes close to resembling an on-off
which
set point,
would be expected if salinity detection
involved chloride only
Levin p. 9
The results of the anion replacement tests also
support the conclusion that Littorina planaxis responds
to the concentration of chloride. Solutions of sodium
chloride (n=8) and Tris HCl (n=6) both gave responses in
over 80 percent of the snails. Test solutions of sodium
D-gluconate (n=9), sodium phosphate (n=6), and Tris
citrate (n=12) gave no positive responses.
Localization
Mapping of the opercular region indicates that the
operculum itself is not sensitive to solutions of high
salinity. The extruding response does not occur unless
the solutions come into contact with the crack between
the operculum and the shell. Further localization was
achieved through staining experiments. In all cases (n=25)
the only stained region was the area lying between the
foot and the operculum, the operculigerous disc (Fretter &
Graham, 1962). Even after immersion of one hour in
methylene blue, no other areas showed signs of staining.
It should be noted that OsO)--Acetate stained similarly,
did not give a stage one response,did not kill the
snail.
Removal of the cephalic tentacles had no effect upon
the extruding response (n=10). Further operations were
performed by removing the operculigerous disc. After a
Levin p. 10
twenty-four hour recovery period snails were tested with
NSW. No difference was noted in the extrusion response
between experimental (n=14) and control (n=6) groups.
Snails were then allowed to recover for an additional 24
hours, afterwhich half were tested with a dilution of 60
percent ASW and half with sodium D-gluconate. Response
to the dilution was slowed (n-3) or absent (n-4) among
the experimental group. Response was 100 percent among
the controls (n=3) and among snails previosly tested with
dilutions at and slightly below the 60 percent test solu¬
tion (n=13). No difference in response was noted between
experimental (n=7) and control (n=3) groups in response
to sodium D-gluconate. Thus, destruction of this region
does have an effect upon the snail's ability to deteot
salinity. At this point it is unclear why removal of
the dise should have little affect upon the detection
of NSW, but have a large effect upon the detection of
a dilution. Previous research (Kohn, 1961) also suggests
that this region is the primary site of detection in an¬
other species, Nodilittorina granulus (Gray).
In summary, Littorina planaxis has the ability to
determine the concentration of chloride in solution while
the snail is drawn into its shell with its operculum
closed. The calcium cation appears to have some small,
Levin p. 11
unclear effect as well. The region between the foot and
the operculum appears to be the primary region of detec¬
tion as determined by staining and direct removal of the
area.
ACKNOWLEDGEMENT
I wish to thank Dr. William F. Gilly for his tremen-
dous assistance and patience throughout the course of
this research.
Levin p. 12
REFERENCES
Berger, V. Ja., 1978. Comparison of the adaptations of
mollusks to extreme and moderate changes in salinity.
Mal. Rev., Vol. 11, pp. 110-111
Fretter, V. & A. Graham., 1962 British prosobranch
molluses . Ray Society, London, 75 pp.
Kohn, A. J., 1961. Chemoreception in gastropod molluscs.
Am. Zoologist, Vol. 1, pp. 291-308
Peters, R. S., 1964. Function of the cephalic tentacles
in Littorina planaxis Philippi. Veliger., Vol. 7:2,
pp. 143-148
Stephens, P. J., 1978. The sensitivity and control
the scallop mantle edge. J. exp. Biol., Vol. 75,
pp. 203-221
C
Figure One
The percentage of positive (stage one) responses is
plotted against changing osmolalties. Normal sea water
is around 970 mOsm
O
N

9

8
osuoase isod %

9—
t

H




t

H










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O
Figure Two
The percentage of positive responses is plotted
against the percent concentration of a specific cation
in the test solution as comparaed to its normal concen¬
tration in ASW. As an example, a 60 mM Cacl, test
solution yielded 25 percent positive response.
The normal
concentration of Cacl, in ASW is 10 mM. The respective
plot for this point is at (600,25). Activity coefficients
were not considered and are implied to be one at all di¬
litions. The arrow represents the ASW dilution (see also
text).
o



—
5
300
S
S
-
8
kevity
wusdrech and e weplit tu
wettiert wed at
a all diluli









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1
O
O
O
C
a
Figure Ihree
The percentage of positive responses is plotted
against the concetration of chloride in millimolar
units.
he cation indicated refers to the major species
with which the chloride ion was being added to test
solutions.
8
0


—
- —

8
100
75
50
25
N-252
—

0
0——0 Cat
A—Mg
D Na
Na with
—
O mMolar Cacl
9 — — Li

100 200 300 400 500 600
concentration of CI (mM)
..
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N-252
















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