Abstract
Many poikilotherms exhibit adaptive changes in cellular membrane fluidity
(homeoviscous adaptation) to counter potentially disruptive effects of thermal variation in
their environments. The process of homeoviscous adaptation was studied in an intertidal
snail, Littorina keenae, which encounters wide variation in body temperature due to
differences in microhabitat conditions and the effects of tidal rhythms. This study
investigated the effects of thermal variation arising from microhabitat differences
between two geographic sites and from tidal cycle effects within a single site on
membrane fluidity. L. keenae, a species widespread along the North American Pacific
Coast, was studied at two sites on Cabrillo Point at Hopkins Marine Station in Central
California, one site being wind and wave exposed (exposed site), the other relatively
protected by its cove-like orientation (protected site). Protected site individuals exhibited
body temperature fluctuations as great as 20°C within a six-hour period (10 to 30°C).
whereas the exposed population exhibited only a 10°C range in the most extreme case
(10 to 20°C). Fluidities of membranes of gill and mantle tissue were quantified by
measuring fluorescence anisotropy using the membrane probe 1,6-diphenyl-1,3,5-
hexatriene (DPH). Samples from the exposed site population collected prior to and
following midday low tide exhibited no differences in membrane viscosity. However, in
one instance exposed individuals collected at the same time showed two distinct levels of
membrane fluidity, suggesting disparate thermal histories. In addition, comparisons with
previously published data on membrane fluidity for other species may indicate that L.
keenae is compensating toward its upper thermal range.
Introduction
Biological membranes are structurally and functionally essential to all living
organisms. The cell membrane is generally depicted as comprising a dynamic, fluid
phospholipid bilayer, held together by weak hydrophobic interactions among the non¬
polar acyl-chain regions of the phospholipids. These interactions in part allow mobility in
the phospholipids as well as other integral membrane molecules (e.g., glycoproteins,
channels, cholesterols) (Singer and Nicholson, 1972). The fluidity of the membrane
depends on several factors, including composition the lipid acyl chains and temperature.
Membrane fluidity is positively correlated with the degree of lipid acyl chain
desaturation, because the double bonds introduce kinks that prevent efficient packing of
the lipids into the bilayer. More saturated acyl chains favor denser compaction of the
membrane lipids, composing a more ordered membrane (Cossins and Prosser, 1977;
Farkas and Roy, 1989). Increasing temperature also promotes membrane fluidity by
enhancing molecular motion (for review see Hochachka and Somero, 2002). The weak
hydrophobic interactions leave the membrane vulnerable to the possibly deleterious
effects of extreme temperatures. Thermal disruption of the membrane state can critically
impact the essential structure and function of the membrane, indicating a need for a
limited temperature range in which membrane constituents can most effectively function
(for review see Hazel, 1995). For organisms inhabiting different thermal niches.
adjustments to membrane composition may be required to maintain an effective
membrane state in spite of thermal perturbations. One way an organism could respond to
the threat of thermal disruption to physical membrane state is by altering the ratio of
saturated to unsaturated lipid molecules in the bilayer. For example, organisms coping
with extreme heat would need to stabilize their membranes, restructuring lipid
composition to a higher proportion of saturated molecules; whereas an organism
challenged by extremely cold conditions would want to increase the fluidity (lipid
desaturation) of its membranes to counteract the stabilizing effect of the low
temperatures. This adaptive conservation of a biologically optimal range for membrane
fluidity is referred to as homeoviscous adaptation (Sinensky, 1974).
Many studies have investigated the existence and extent of homeoviscous
adaptation through comparisons among poikilothermic microorganisms and across
vertebrate taxa (Sinensky, 1974; Cossins et all, 1984; Behan-Martin et al., 1993; Logue et
al., 2000). Nevertheless, few studies have explored structural compensation in
membranes for thermal stress among invertebrate species (Dahlhoff and Somero, 1993;
Williams and Somero, 1996). Most of the studied vertebrate organisms tend to live in
environments of relative thermal stability. However, for invertebrate poikilotherms
inhabiting marine intertidal habitats, extreme temperature fluctuations can occur on a
daily basis during low tide periods, and these organisms face an intriguing homeostatic
challenge. One study of membrane fluidity in intertidal mussels illustrated seasonal
acclimatization in gill phospholipid structure associated with wide ranges in body
temperature, and alteration of membrane order was also observed during a laboratory
simulation of a hot low tide (Williams and Somero, 1996).
An investigation of membrane fluidity in animals sampled directly from intertidal
microhabitats following high and low tide periods would provide further insight into the
energetic compromises that organisms must make in the face of environmental stressors.
To explore the existence and scope of acclimatory membrane alterations in intertidal
organisms facing myriad physiological challenges in the field, I studied membrane
fluidities in the intertidal snail L. keenae, an invertebrate abundant in the marine intertidal
of the Central Coast of California.
The microhabitats of L. keenae examined in this study expose the animals to a
range of convective heat loss from wind and wave forces, and render them vulnerable to
varying degrees of solar heat radiation, intensified by reflected heat emanating from the
granite boulders inhabited by the snails. Being small poikilotherms, L. keenae are at the
mercy of their thermal environments.
In this study, I examined the membrane fluidity of L. keenae occupying two
specific microhabitats on Cabrillo Point at Hopkins Marine Station on California’s
Central Coast: a protected site and an exposed site. Because of its geographic orientation,
the protected site is sheltered from wind and swell, but faces the sun; whereas the
exposed site is frequently susceptible to wind, spray, and waves. At both sites, L. keenae
are situated in the upper regions of the intertidal zone, about three to four feet above the
mean low water level, and are not regularly submerged (pers. obs.). Both sites are subject
to seawater spray during high tide, and both are air-exposed during low tide (pers. obs.).
Environmental variations between the two sites suggest a possibility for two different
levels of thermal acclimatization, while thermal variations correlated with tidal rhythms
provide a second point of comparison. I hypothesized that snails situated on the exposed
site or snails recently exposed to high tide seawater spray, might have higher membrane
fluidities over a range of temperatures than would snails from the protected site following
air and heat exposure during a warm low tide period.
Materials and Methods
Field Collection
Two variables were considered for this experiment: time and location. To test for
differences in membrane fluidity within one site at different times of day, specimens were
collected from the exposed site just before sunrise following a high tide, and again from
the exposed site in the mid-afternoon immediately following a midday low tide. To test
for variation between the exposed site and the protected site, samples from each location
were collected at the same time of day: mid-afternoon, following a midday low tide.
For both comparisons, specimens were collected from the field using forceps to
minimize thermal conduction that might affect body temperature measurements. Body
temperatures were obtained by inserting a type-K thermocouple probe into the foot tissue
of the organism, and the specimens were then flash-frozen and kept on solid CO2 until
returned to the lab where they were stored at -80°C.
Tissue preparation
Each specimen was dissected in iced seawater for the extraction of gill and mantle
tissues. Following dissection, the sample tissues were stored at -80°C until tested. Frozen
tissue from each snail was homogenized in 3.5mL of 50mM phosphate buffer, using a
ground glass homogenizer. All procedures were performed on ice. I then divided the
homogenized tissue suspension between two 2mL centrifuge tubes and centrifuged both
at 80g (1000 RPM) for three minutes at 4°C. The supernatant from both tubes was
transferred to a 3mL cuvette, and the absorbance was measured at a wavelength of
364nm. The supernatant was diluted to A364-0.15 and placed in a plastic fluorescence
cuvette, into which 2uL of 1.8mM DPH solution was added for a final DPH
concentration of 3.6nM in the cuvette (see below for DPH preparation). Cuvettes were
then sealed with a thin layer of parafilm to prevent dust or lint contamination, and stirred
for thirty minutes in the dark, allowing for the diffusion of the DPH molecule into
membranes.
DPH preparation
I dissolved 2.3 mg 1,6-diphenyl-1,3,5-hexatriene (DPH) crystals into 5 mL N'-N'-
dimethylformamide for a 1.8mM DPH concentration and covered the solution in foil to
prevent the photo-degradation of DPH. The solution was then placed on a stir bar to mix
for one hour prior to being introduced into the tissue preparation.
Fluorescence Anisotropy Measurements
All cuvettes were then placed into a water bath at 10°C for approximately 15
minutes for thermal equilibration. I placed each cuvette into the water-jacketed cell of the
spectrofluorometer. Parameters for fluorescence polarization measurements were as
follows: excitation, slit width = 3 nm, A = 364.0 nm; emission, slit width = 10 nm, X -
430.0 nm; response time = 8 s; repetitions = 5; recording range = 0 to 1000. After
checking the temperature of the mixture within the cuvette (using a K-type
thermocouple), I took polarization measurements at the following polarized filter angles:
0°,0°; 90°,0°; 90°,90°; 0°,90°; for excitation, emission angles respectively. This series of
measurements composed one trial. For each sample, two trials were repeated at each
temperature, and the actual temperature within the cuvette was re-measured with the
thermocouple immediately prior to each trial. This procedure was repeated at each of the
following temperatures: 10°C, 15°C, 20°C, 25°C, 30°C, and 35°C.
Values given for the emission of polarized light at each filter angle were recorded
and used to determine the fluorescence polarization of the sample for that trial (Litman
and Barenholz, 1982). Fluorescence polarization values (P) - which are inversely
proportional to membrane fluidity - were calculated using the following equation (Litman
and Barenholz, 1982):
190,0
(0,0)-
10,90
0,90
P-
10,0
(0,0)
10,90
(J0,90
The polarization value was calculated for each replicate and averaged to obtain the
fluorescence polarization value for the sample.
Statistics
Regression lines from the relationship of fluorescence polarization and
temperature were generated for each sample. The slopes of these regression lines were
used for statistical comparisons (i.e., exposed site versus protected site, and exposed site
morning collection versus exposed site afternoon collection) using a single-factor
analysis of variance (ANOVA). The ANOVA was also used to compare estimated
polarization values at common temperature (20°C) for the same comparisons. In the case
of the exposed vs. protected site comparison, a one-tailed student's t-test was also
applied. In all cases, a S 0.05.
Results
Body temperatures for L. keenae fluctuated with tide and time of day at both sites,
but organisms at the protected site were found to experience more extreme fluctuations
than those at the exposed site. Body temperatures (Tp) recorded at the exposed site ranged
from 9°C to 19°C within a six-hour period; whereas measurements at the protected site
exhibited a wider fluctuation, from 10°C to 32°C.
Fluorescence polarization values for every sample fell between 0.22 and 0.31
across the tested temperature range. As the temperature increased, polarization values
declined (enhanced membrane fluidity).
Exposed site samples collected in the morning and afternoon showed similar
membrane fluidities. Specimens collected before sunrise from the exposed site following
high tide had a mean Tp of 13°C, and membrane fluidities were similar for all snails
collected. The second group of exposed specimens collected in the afternoon following a
midday low tide had a mean Tp of 18°C and resulted in two pairs of differing membrane
fluidities. One pair closely matched the fluidities of the morning collection, while the
other pair displayed lower polarization values across the temperature range. (Fig. 1). The
effect of temperature on fluidity was significantly different between the morning and
afternoon samples (ANÖVA, p-0.02), but this difference disappeared when I excluded
the two individuals having considerably lower polarization values (ANÖVA, p-0.07).
(Fig. 2). Estimated fluorescence polarization values at 20°C (disregarding the two
individuals having low polarization values) of morning and afternoon samples were not
significantly different (ANOVA, p-0.92).
Although the comparison between morning and afternoon collections at the
exposed site yielded no significant variation, membrane fluidities between exposed and
protected snails were different. Fluorescence polarization values for samples from the
exposed site following a midday tide (average Tp =16°C) were lower than those collected
from the protected site at the same time on the same day (average Tp -25°C). (Fig.3). The
effect of temperature on fluidity was not significantly different between sites (ANOVA).
However, the membrane fluidities of protected and exposed snails were statistically
different across the temperature range: using 20°C as a point of comparison, estimated
fluorescence polarization values for that temperature were compared using two statistical
methods. The one-tailed t-test showed a significant difference between the exposed site
and protected site fluidity measurements, p-0.014, while the more conservative ANOVA
gave a barely non-significant p of 0.075.
Discussion
Littorina keenae living in the intertidal microhabitats of Cabrillo Point on
California’s Central Coast regularly experience changes in ambient temperature as large
as 20°C in a six hour period. I recorded body temperature fluctuations spanning a range
of 10°C for L. keenae inhabiting the exposed site, while L. keenae at the protected site
experienced a range of 20°C for the same time period. This pattern makes intuitive sense,
as the exposed site is more consistently vulnerable to heat loss through wind convection
and to seawater spray, even during hot low tides. Conversely, organisms inhabiting the
protected site are sheltered from both wind and swell. On hot days, these animals would
be unable to cool themselves, either convectively to wind or conductively to water.
During high tides, both sites are subject to seawater spray, so one might expect both sites
to experience similar minimal body temperatures. The Tp values recorded over the course
of this study precisely mirror this expectation. The recorded Tp values for the exposed
site ranged from 10°C at high tide to 19°C at low tide; the Tp values for the protected site
varied between 11°C and 32°
For the exposed and protected samples that were collected following a midday
low tide period, one might expect the protected site collection to have had a warmer
thermal history than the exposed site collection, and indeed the average body
temperatures for individuals in that collection from the protected site were 25°C, while
the average Tp for exposed site individuals was nine degrees cooler at 16°0. Thus I
predicted that the protected snails, with a relatively warm thermal history, would
compensate for the heat-induced increase in membrane fluidity by stabilizing its tissue
membranes.
The membrane fluidities of the exposed site collections taken at different times of
day reflect the relatively narrow range of thermal fluctuation observed at the exposed site.
Although inclusion of all of the afternoon exposed site samples in statistical comparisons
returned significant differences between morning and afternoon exposed-site collections,
when the two afternoon samples displaying lower polarization values were removed, the
differences disappeared. The rationale for the removal of these data follows.
In evaluating the slopes of each individual tested over the course of the study,
every tissue preparation demonstrated a decrease in fluorescence polarization as
temperature increased. The low polarization values for the two individuals in the
afternoon collection group not only were inconsistent in their raw polarization values, but
the slopes for both individuals were also nearly zero (-0.0004 and -0.0007 fluorescence
polarization per °C, respectively), indicating the physically unlikely lack of change in
membrane fluidity with respect to temperature. This insensitivity to temperature led me
to suspect that the preparation of the sample was flawed. Therefore, I included two
graphical and statistical analyses for the same comparison, one including and one
excluding the inconsistent data. (Figs. 1 and 2). However, for the purposes of discussion,
1 will refer to the data that exclude the anomalous samples.
No significant differences in membrane fluidity could be seen between the
exposed group collected in the early morning following high tide and the exposed group
collected later that afternoon following low tide (ANÖVA, p-0.07). Based on the
assumption that any adjustments in membrane composition would occur in response to
differing thermal histories, a lack of alteration may indicate a lack of thermal disparity
between the two groups. The Tp values for the morning collection averaged 13°C and the
mean Ty for the collection was 18°C, so the average body temperature range remained
relatively narrow. Moderate changes in body temperature in such a short period may
present less of a cost to the organism than would an energy expenditure to restructure its
membranes during that time.
The disparity between the two thermal environments (exposed and protected) is
reflected in the membrane orders of organisms taken from each site. (Fig.3). Over a range
of temperatures, samples collected from the protected site exhibit higher membrane
stability than do those individuals from the exposed site. To quantify this difference, I
used both a single-factor ANÖVA and a student's t-test for the estimated polarization
values at 20°C. Because 20°C lies in the middle of the data set, using this x-value would
11
show the least amount of error in the estimation of the slope, giving the most accurate
prediction of a common polarization value. The one-tailed t-test showed a significant
difference between the two estimated polarization values (p-0.014), but the conservative
ANOVA gave p-0.07, marginally insignificant.
Variation in membrane order between the exposed and protected sites, though
apparent and consistent in graphical representation, can be considered at best marginally
significant, with only four individuals from each site for replication, and only the least
conservative statistical test giving significant results. However, the subtle implication of
one significant result suggests the possibility that organisms inhabiting more protected
geographical regions of Cabrillo Point may have adjusted the composition of their tissue
membranes to compensate for the often extreme changes in thermal stress. The
polarization values imply that at a given temperature within the tested range, protected-
site L. keenae may have more stable membranes than do L. keenae at the exposed-site.
A comparison between the estimated polarization values for the two groups at
their respective average Th values (14°C and 24°C) reveals a striking similarity. The
predicted value for the exposed site is 0.276, and 0.270 for the protected site, suggesting
conservation of a specific, target membrane state even between differing thermal
microhabitats.
In context of previous interspecific vertebrate studies, the snails' conservation of a
fluorescence polarization value of 0.27 at average Tp is much higher than other conserved
fluidity ranges observed in vertebrates. In a 1993 study by Behan-Martin et al.,
membrane fluidities for several vertebrate species were compared by plotting the
temperature at which they displayed a polarization value of 0.22 against their accustomed
12
body temperature. The resulting line is exceptionally conserved. (Fig. 4). The equivalent
value for L. keenae was included as an *X" and is clearly divergent from the data of
Behan-Martin (1993). (Fig. 4). The disparity between membrane fluidity conservation in
L.keenae and fluidity conservation among the vertebrate data might be due to several
factors, the most parsimonious of which would be to assume error in my own data
collection. The illustrated vertebrate polarization values were obtained using purified
lipid vesicles, while my data were obtained using crude homegenates of both gill and
mantle tissues. The inconsistency could also be a result of phylogenetic differences
between vertebrates and invertebrates, though more data concerning invertebrate
membrane fluidities would be required to make that distinction.
Nevertheless, membrane fluidities of L. keenae are not as divergent from other
published data as suggested by the above analysis. Inclusion of my data on a plot of
previously published polarization values as a function of temperature (Logue, et al.,
2000) indicates that L. keenae seems to be conserving its membranes in a more ordered
state than the given vertebrates. (Fig.5). The range for L. keenae’s polarization values is
comparable to the illustrated endothermic vertebrates, suggesting that L. keenae is
acclimatized to higher body temperatures. However, the slopes for the snail polarization
values are less steep than the most similar vertebrate slopes. One explanation might be
that L. keenae is routinely exposed to the upper thermal range included in this figure and
would be adjusting membrane fluidity to these warm temperatures. Both the similarity of
L. keenae to warm-adapted animals and the presence of a more gradual slope in the snail
data suggest the possibility that keenae is compensating toward its upper thermal limits.
Conclusions
L. keenae inhabiting the marine intertidal environment at Cabrillo Point
experience rapid and dramatic fluctuations in ambient and body temperatures. Snails in
the microhabitat of the protected site see warmer, more extensive fluctuations than do the
exposed site snails. Samples collected from the exposed site in the morning and later that
afternoon showed no significant differences in the membrane states between the two
groups. Conversely, the comparison between exposed and protected sites following a
midday low tide revealed slightly significant differences that may imply acclimatory
alterations in membrane compostion. At the very least, this subtle dissimilarity demands
closer inspection.
Acknowledgements
I would like to thank my advisors George Somero and Jonathon Stillman for their
extensive guidance and communication in working on this project. In addition I would
like to express my gratitude to the Somero Lab group for their support and assistance, as
well as to express my appreciation for the support and generosity of the faculty and staff
of the Stanford Hopkins Marine Station.
Literature Cited
Behan-Martin, M.K. and G.R. Jones, K. Bowler, A.R. Cossins. 1993. A near perfect
temperature adaptation of bilater order in vertebrate brain membranes. Biochim.
Biophys. Acta. 1151: 216-222.
Cossins, A. R. and C. L. Prosser. 1977. Evolutionary adaptations of membranes to
temperature. Proc. Natl. Acad. Sci. USA. 75: 2040-2043.
Dahlhoff, E., and G. N. Somero. 1993b. Effects of temperature on mitochondria from
abalone (genus Haliotis): adaptive plasticity and its limits. J. Exp. Biol. 185: 151-
168.
Farkas, T. and R. Roy. 1989. Temperature-mediated restructuring of
phosphatidylethanolamines in livers of freshwater fishes. Comp. Biochem.
Physiol. 93B: 217-222.
Hazel, J.R. 1995. Thermal Adaptation in Biological Membranes: Is Homeoviscous
Adaptation the Explanation? Annu. Rev. Physiol. 57: 19-42.
Hazel, J.R. and E. Williams. 1990. The Role of Alterations in Membrane Lipid
Composition in Enabling Physiological Adaptation of Organisms to Their
Physical Environment. Prog. Lipid. Res. 29: 167-227.
Hochachka, P.W. and G. Somero. 2002. Temperature. pp. 290-379 in Biochemical
Adaptation: Mechanism and Process in Physiological Evolution, Oxford
University Press, New York, NY.
Litman, B.J. and Y. Barenholz. 1982. Fluorescent probe: diphenylhexatriene. Meth.
Enzymol. 81: 678-685.
Logue, J. A. and A.L. De Vries, E. Fodor, A.R. Cossins. 2000. Lipid Compositional
Correlates of Temperature-Adaptive Interspecific Differences in Membrane
Physical Structure. J. Exp. Biol. 203: 2105-2115.
Sinensky, M. 1974. Homeoviscous Adaptation - A Homeostatic Process that
Regulates the Viscosity of Membrane Lipids in Escherichia coli. Proc. Nat. Acad.
Sci. USA. 71: 522-525.
Singer, S. J. and G. L. Nicholson. 1972. The Fluid Mosaic Model of the Structure of Cell
Membranes: cell membranes are viewed as 2-dimensional solutions of globular
proteins and lipids. Science. 175: 720-731.
Figure Legends
Fig. 1. Fluorescence polarization (inversely proportional to membrane fluidity) of DPH in
lipid membranes of gill and mantle tissue extracted from two groups of L. keenae
individuals from the exposed site, one group collected before sunrise following an early-
morning tide (closed triangles, N=4), the other collected in the afternoon following a
midday low tide (open circles, N=4). Single factor analysis of variance shows a
significant difference between the two slopes (morning collection, y--0.0022x+0.3166,
=0.9973; afternoon collection, y—-0.001 1x+0.2569, R=-0.8655), p-0.02. Data points
depict individual samples.
Fig. 2. Fluorescence polarization of DPH in lipid membranes of gill and mantle tissue of
two groups of L. keenae individuals collected from the exposed site: one group collected
prior to sunrise following an early-morning high tide (closed triangles, N-4), and the
other collected in the afternoon following a midday low tide (open circles, N-2).
Samples here are the same as those illustrated in Fig. 3, minus the two samples showing
low polarization values. A single-variable analysis of variance shows no significant
difference between the slopes for the two groups (morning collection, y-
0.0022x+0.3166, R==0.9973; afternoon collection, y—-0.0017x+0.3053, R°=0.9405),
p-0.92. Data points depict means + 1 S.D.
Figure 3. Fluorescence polarization of DPH probe (inversely proportional to membrane
fluidity) in lipid membranes of gill and mantle tissue excised from Littorina keenae
specimens collected from both the exposed site (open circles, N=4) and the protected site
(closed triangles, N=4) in the afternoon immediately following a midday low tide. A
single-factor analysis of variance showed no significant difference between the slopes
(exposed, y—-0.0021x+0.3057, R°-0.9923; protected, y--0.0023x+0.3256, R°-0.9589)
p=0.6. At 20°C, estimated polarization values show significant difference using a one-
tailed student's t-test, p-0.014. Data points depict means + 1 S.D.
Fig 4. Conservation of membrane fluidity across several vertebrate species (adapted from
Behan-Martin, et al., 1993). Temperature at which the animal shows a polarization of
0.22 is plotted against the animal’s accustomed body temperature. Snail value is the“
symbol.
Fig. 5. Fluorescence polarization (membrane fluidity) values shown as a function of
temperature for vertebrates adapted from Logue et al., 2000, including exposed site and
protected site data for L. keenae (from Fig. 1 for comparison) shown as patterned lines.
Fig. 1
0.32
0.3
0.28
§ 0.26
g 0.24
5 0.22
0.2
0.18
0.16




S

4
8
O
8
O
O
O
5 10 15 20 25 30 35 40
Temperature °0
Fig. 2
50.32
0.3
g 0.28
0.26
3 0.24
E 0.22


S

5 10 15 20 25 30 35 40
Temperature °0
Fig. 3
0.32
0.3
0.28
0.26
0.24
0.22
5 10 15 20 25 30 35 40
Temperature °0
Fig. 4
50
L. keenae
40
FPigeon
Mouse Rat
1 Astarling
MousenI
30
mC
M
o
20
4.

arch
Sia an
G Gerbil
H Hamster
10 Ta
Co Cam

Gi Goldfish
Notothenia
Tr Trout
Ci Cichlid
40
10
30
20
Body temperature (°0)
50
Fig. 5
0.34
0.28
0.22
30.16
0.10
urkey
L keenae Protected Site

Rat
24°0
.

Toadfish (24°C)
Tilapia (25°c)

Blue grunt (24°c)
40
20
Temperature (C)
L. Keenae
Exposed Site
(14°c
— Striped bass (17°C
—Trout (8°0)
Notothenia (0°0
Pagothenia (5 100