ABSTRACT
The mussel Mytilus californianus is able to remove a wide array of xenobiotics
(toxins) from its cells using an ATP-driven transport protein. This Multixenobiotic
Resistance (MXR) system, similar to the multidrug transport protein found in human
tumor cells, is thought to be a first line of defense against many natural and
anthropogenic toxins. Three Monterey Bay seaweed species and three Monterey Bay
phytoplankton species were found to contain likely substrates for the MXR protein.
Extracts from all six species showed an increase in intracellular fluorescence using the
rhodamine B accumulation, indicating that these contain substrates for the MXR protein.
Mussels are filter feeders that remove seaweed particulates, phytoplankton, and their
byproducts from the waters that pass over them. Ifthe mussel diet contains compounds
that are substrates for the MXR protein, this would provide insight into the pressures
resulting in selection for this transporter, a mechanism that is a useful defense mechanism
in today’s polluted waters.
INTRODUCTION
A major inhabitant of the rocky intertidal zone of Monterey Bay, the mussel
Mytilus californianus feeds by filtering water through its gills and trapping particulate
matter. These waters contain both natural and anthropogenic toxins (xenobiotics) which
the mussels ingest through their diet or absorb directing from the water. An
understanding of how mussels and other marine organisms deal with xenobiotics, either
by transforming or effluxing them, is now emerging From this research there is
significant evidence that the mussels have a multixenobiotic defense mechanism (MXR)
which may protect them from toxins in their environment (Cornwall et al., 1995: Epel.
1998; Eufemia and Epel, 1998). This defense mechanism, an ATP-utilizing protein
pump, similar to the p-glycoprotein multridrug transporter (MDR) identified in human
tumor cells, provides protection by pumping toxins out of the cell.
The diet of the filter feeder M. californianus consists of particles that are remoyed
from the water and bound to mucus on the gill lamellae and then transported to the mouth
(Bayne, 1976). Mussels mostly consume particles under 1 10um, which includes bacteria.
diatoms, dinoflagellates, inorganic particles and fine organic detritus suspended in the sea
water (Morris et al., 1980; Newell et al., 1989; Hawkins and Bayne, 1992; Duggins and
Eckman, 1997; Raby et al., 1997). In addition to the phytoplankton, the mussel receives
à diet of seaweed particulates, especially during winter months when storms break the
kelp apart (Bayne, 199
Mussels may selectively feed on chlorophyll containing particles (Newell et al.,
1989) and there is also evidence that mussels prefer algal species low in phenolic
compounds, such as the Macrocystis and Egregia species (Steinberg, 1992). However.
thère appears to be no consensus on whether mussels are able to sort particles based on
organic content, size and/or species, and there is certainly no prescribed ideal mussel diet
which many would agree upon (Bayne, 1987; Jorgenson, 1996).
Soluble algal products in Monterey Bay come from seasonal phytoplankton
blooms and from anti-herbivory compounds released from seaweeds as part of their
growth processes (Steinberg, 1985; Paul, 1992). Algal blooms (some types are
commonly known as "red tides") are common during the summer months due to
upwelling, a process that brings more nutrients to the surface allowing for a burst of
phytoplankton growth in the waters (Smayda 1997). Several of the phytoplankton species
in these blooms produce toxins encountered by mussels in significant amounts since
mussels can accumulate the toxins in their tissues (Shumway 1992). Accumulation of
these toxins could come directly from the water or from ingestion of the phytoplankton
and accumulation of their toxic products. In coastal areas around the world, shellfish
poisoning is considered a major problem, as humans who ingest those shellfish during
periods of toxin accumulation can become ill and even die.
My hypothesis is two-fold: first, algae and algal products may be natural
substrates for the MXR protein. This defense mechanism would then prevent the algal
toxins from staying in the mussel or at least decrease accumulation and account for the
eventual removal of the toxins from mussel tissue. My goal was first to identify probable
substrates in seaweeds and phytoplankton that the mussels are exposed to in their natural
environments and secondly, to see if these algal products are inducers of the MXR
protein. To test my hypotheses I focused on identifying algal and phytoplankton
substrates for the MXR transport protein, and then, using the most likely substrates,
carrying out induction studies to see if they affected the level of MXR protein expression.
1 found algal substrates and inducers for the MXR protein, suggesting an evolutionary
reason for the existence of the MXR system, a system that presumably evolved when the
mussels primary concern was natural and dietary toxins but may also effectively deal
with anthropogenic pollutants today
MATERIALS AND METHODS
Mussels
Mytilus californianus were collected at the Hopkins Marine Station and kept in
both indoor and outdoor tanks, the former being temperature controlled at approximately
14 degrees C. Both tanks were aerated and received water from the sand filtered intake
pipe 60 ft. below sea surface. All mussels used were between 5-8 cm in length.
Extracts Preparation
The first step in identifying natural substrates for the MXR transporter was
making extracts of various seaweeds and phytoplankton collected from the Hopkins
Marine Station. The following seven prominent alga species were gathered: (1)
Macrocystis pyifera, (2) Egregia menziesii, (3) Phyllospadix scouleri, (4) Endocladia
muricata, (S) Mazzaella flacida, (6) Gelidium robustum. The extracts were made as
follows: the samples were frozen in liquid nitrogen and ground into a powder with a
pestle, 2 grams (wet weight) was placed in a test tube with 5 ml of solvent, after 15
minutes the extract was removed and the extraction was repeated two more times, for a
total of 15 ml of sample extract. The following solvents were initially used: Filtered Sea
Water (FSW), Methanol (100 %, at room temperature and at 70 degrees C), and Ethanol
(95%, at room temperature and at 70 degrees C). We found that the heated MeOH was
produced more extracts in MXR experiments, and thereafter all extractions were done
only with heated MeÖH. Äfter extraction, the solutions were evaporated using a vacuum
evaporator and resuspended in approximately 1 ml of ethanol (95%) and stored at -25° C.
The following phytoplankton species were cultured from Monterey Bay strains
provided by Jason Smith: (1) Alexandrium catenella, (2) Psuedonizchia australis, (3)
Phaeodactylum tricornutum. Extracts were made from approximately 40 ml. of
phytoplankton culture. The culture was centrifuged for 10 minutes at 3500g, the media
was removed, then the pellet was centrifuged for an additional 5 minutes, resuspended in
1 ml of 80% ethanol, sonicated for 20 seconds, and stored at -25° C.
Rhodamine assays:
To determine the levels of MXR activity two different assays were used: a
rhodamine B accumulation assay and a rhodamine B efflux assay. Rhodamine B is a
fluorescent dye that diffuses easily across the cell membrane and then is effluxed via the
MXR protein. Verapamil is a known substrate that blocks rhodamine dye from leaving
the cell via competitive inhibition with the MXR protein.
Rhodamine accumulation assay
For the accumulation assay 1-2 mussels were dissected on ice, the gills were
removed and placed in FSW and the mucus removed with forceps. Small (£ 25 mm)
tissue pieces were cut along the dorsal edge of each lamella and placed in 5ml sea water
+ TuM rhodamine + 20 uM verapamil or 5-10 ul extract. After a one hour exposure
period at 15 degrees C the gill tissues were rinsed in filtered sea water for 30 seconds to
remove excess dye. The tissues were then placed on slides (without a coverslip) and
fluorescence visualized with a fluorescence microscope and images captured by a SIT
camera with an image analyzer.
Rhodamine efflux assay
To verify that the algae contain substrates for MXR, the rhodamine B efflux assay
was used with the extracts yielding positive results in the accumulation assay. In this
assay gill tissue is first loaded with rhodamine dye by incubating the gill fragments in dye
for one hour. The pieces are then removed from the dye, rinsed, and the rate of efflux of
dye into the surrounding media is measured over the next hour. The efflux is rapid in sea
water, but is slowed if a competitive substrate or inhibitor is present.
Six mussels were dissected and their gill tissues were evenly distributed between
petri dishes containing 30 ml FSW + 1 uM rhodamine. Dishes were incubated in the
dark at 15 degrees C for 1 hour. To remove excess dye, the media was removed with a
water aspirator and the tissues were rinsed for 30 seconds with FSW. Then 30 ml FSW +
20 uM verapamil or (30-100 ul ) extract was placed in the dishes, which were then
placed at 15 degrees C in the dark. 450 ul media was removed (in triplicate) and placed
in a 0.5 ml fluorescence cuvette at time points every 10 minutes for one hour and
fluorescence was measured using a fluorometer (EX = 550, EM = 580). To test for
possible fluorescence enhancement or quenching by the extracts, a control experiment
was also run with 1 ul rhodamine in 1 ml FSW + 2 ul each of the 6 extracts and 95% and
80% ethanol. Samples were placed in triplicate in fluorescence cuvettes and, after a 45
minute incubation, were measured in the fluorometer.
Induction studies
Four separate induction studies (see table 2) were conducted, each using different
substrates, methods of exposure (injection or water exposure), and lengths of time (from
24 hrs to 16 days). At the end of each study a gill tissue rhodamine accumulation assay
was carried out using half of the gills and the other half was frozen in liquid nitrogen and
saved at -80 degrees F. for the Western Blot analysis.
Increased protein activity and concentration was assessed by rhodamine B
accumulation assay and Western blotting, respectively. The rhodamine accumulation
assay was done as described above, with rhodamine + verapamil tested with each group.
Western blot analysis was carried out according to Eufemia and Epel 1998. Proteins
were resolved on 7.5 % polyacrylamide gels and proteins were transferred to
nitrocellulose membranes via semi-dry eletrophoretic transfer. Membranes were
incubated with MXR specific monoclonal antibody C219 and goat anti-mouse HRP
conjugated secondary antibody. Bands were detected by electrochemiluninescence
(ECL) Protein concentrations were determined using a modified Bradford Assay
(BIORAD) assay.
RESULTS
Rhodamine accumulation assay
I found fluorescence levels significantly (p = 0.05) lower than the rhodamine
control in: Gelidium (15% less) and Mazzaella (40% less), and not significantly different
from controls in Endocladia (2% higher), indicating these algal extracts did not contain
MXR substrates(figure 2). I found fluorescence levels significantly above the controls in
Macrocystis (50% higher), and Egregia (30% higher), and not significantly different but
above controls in Phyllospadix (7% higher), indicating that these extracts do contain
MXR substrates. I found fluorescence levels significantly higher than controls in the
phytoplankton species A. catenella ()and P. australis Q, and not significantly different
but above controls in P. tricornutum (), indicating that these extracts do contain MXR
substrates (figures 1 and 3, table 1). Ethanol controls were run with every experiment
and fluorescence levels were not significantly different than the rhodamine controls (data
not shown).
Rhodamine efflux assay
The rhodamine efflux assay showed evidence of substrates in the phytoplankton
extracts A. catenella, P. australis, P. tricornutum and the algae Egregia. (figures 4 and 5)
These four exhibited fluorescence efflux rate that was less than rhodamine, at
approximately the same rate as verapamil, indicating the extracts contained compounds
interfering with rhodamine efflux. Two algal species (Macrocystis and Phyllospadix)
showed fluorescence efflux at or above rhodamine control levels (figure 6) To eliminate
the possibility of enhanced fluorescence or fluorescence quenching caused by the extracts
a control experiment was run, and showed no significant effects on rhodamine
fluorescence intensity by any of the algae or phytoplankton extracts.(Figure 7)
Induction studies
In the long term (10 - 16 day) induction study 6 out of 29 mussels died, with the
probable cause of death wbeingas that too much blood was being withdrawn. Two
induction experiments were done in which Macrocystis induced increased levels of MXR
proteins in the western blot. The results of the Western blot for Psuedonizchia and
Alexandrium showed no difference in band darkness between control and extract-injected
mussels. (figure 8) Half of the gill tissue was used for a rhodamine accumulation assay
in every induction experiment. In these I found no significant difference between control
groups and extract groups, though all showed very high levels of MXR activity (figure 9).
DISCUSSION
Mussels, as filter feeders, are continuously exposed to algae and algal products,
whether it is from winter storms breaking up giant kelp into fine particulates or summer
phytoplankton blooms releasing toxic byproducts into the water. In examining the
mussel diet to find any natural substrates, I approached the question from an
environmental perspective, hoping the natural environment would provide clues to how
mussels deal with their natural environment as well as a polluted one. Mussels have
higher MXR titer in polluted environments versus pristine ones (Kurelec 1995), and T am
interested in understanding if a natural event, such as a phytoplankton bloom causes a
similar response in MXR protein levels.
My rhodamine accumulation experiments indicate six likely candidates for MXR
substrates (table 1). This data supports the idea that natural substrates are commonly
found in the mussel diet. Mussels are known consumers of all 3 of the phytoplankton
species that contain substrates, and given the abundance of the 3 seaweeds that contain
substrates, they would therefore be exposed to their products on a regular basis (Gosling
1992).
The major problem with the rhodamine accumulation assay was large variability
with controls. I found varying levels of fluorescence in tissues from the same mussel that
had been incubated with rhodamine, the only difference being the incubations started 20
minutes apart. I was able to eliminate the possibility of variations in the camera or
microscope with a fluorescent rhodamine light standard. This problem may be caused by
localization of MXR within the gill tissue, tissue deterioration, or damage during the
experiment. In future experiments I will add more rhodamine controls to account for this
variation. Since I pooled results of multiple experiments my results account for this
variation, but larger sample sizes would be preferred and will be used in future studies.
In the rhodamine efflux assay I found evidence for substrates in four of the six
likely candidates, three of which were phytoplankton. The two algal extracts
(Macrocystis and Phyllospadix) that indicated they did not contain substrates in the efflux
assay need to be retested with varying amounts, as their pigment may be interfering with
the fluorometer results and the fluorometer controls were only conducted for one
concentration of extract.
I did find that Macrocystis is an inducer for the MXR protein based on western
blot results. In both induction experiments where Macrocystis extract was injected into
the posterior adductor muscle, Western blotting showed significantly stronger staining of
a band at 170 KD then the controls exhibited, indicating induction of MXR protein took
place. This induction could be part of a “stress response" to a natural event, such as
when Macrocystis is releasing high amounts of byproducts (as seen with heavy
foam/mucus levels in the water), in which the mussel responds by elevating MXR
expression. Öther physical stressors, such as heat shock and or exposure to cadmium, are
thought to cause a general stress response; the question of whether exposure to a range of
chemical stresses could cause induction of MXR in mussels needs further research
(Eufemia and Epel 1998).
Phytoplankton are also important in the mussel diet, and if the phytoplankton
release toxic products, it would seem logical that the mussel has a defense against these
toxins. Currently (June 1998), there is a large phytoplankton bloom occurring in
Monterey Bay (species analysis is still occurring), and the mussels are exposed to these
blooms just about every year. In order to survive, the mussels must be able to deal with
extreme situations like this, especially if the phytoplankton produce toxins that could
harm the mussels. Mussels accumulate some of these toxins, but the accumulated toxins
are later released, perhaps effluxed via the MXR proteins. Understanding how a mussel
deals with a phytoplankton bloom is one of the future goals of my research.
The multidrug resistance (MDR) protein of mammalian tumor cells is
immunologically similar to MXR. Current research suggests the marine algae Caulerpa
taxifolia may contain substrates for MDR, making it a new candidate for reversal of
MDR in cancer treatment (Smital et al. 1996). In the future I would like to test algal
substrates I have identified for the MXR system in mussels with the mammalian MDR
protein.
Tobserved large natural variation in the amount of MXR protein in the mussels.
If natural products are substrates and inducers of the MXR transporter, the diet of the
mussels becomes as important a factor as pollution, temperature, and other stressors on
MXR. One of the major problems I encountered was that some mussels expressed either
very high or very little MXR. This might reflect the environment of the tanks in which
they were stored or the rocks on which they live. In future experiments it would be
interesting to control natural factors (using temperature controlled tanks, artificial sea
water, etc.) as well as anthropogenic ones.
The results of this study raise several important questions. What is the identity of
the compounds that are substrates for the MXR protein? The extracts presumably contain
not only the plant/phytoplankton tissue but also chemical products the algae released as
byproducts or chemical defenses. Isolating the compounds would help identify the class
of substrates that are used with the MXR transporter.
As marine waters become more polluted it is extremely relevant to understand the
mechanisms that marine organisms have to deal with this problem. As development on
coastal area increases, with agriculture alongside it, the amount of nutrient run-off into
the ocean has increased dramatically. The increase in nutrients leads to higher plant
productivity and a greater number of algal blooms as well (Anderson and Garrison.
1997). The MXR system is not fully understood; its substrates and limitations are not vet
known. Algae and algal products are not only a possible explanation for the presence of
the MXR system, as they are a major part of the diet, but are also a current stressor on the
system. This ability of mussels to efflux natural toxins is critical in understanding their
ecology.
ACKNOWLEDGMENTS
This work is really the brain child of the fabulous Nancy Eufemia, the academic guidance
of Dave Epel, and technical support of Chris Patton. Thank you's to Jason Smith (for
phytoplankton), Richard Kuo, and Melissa Kaufman.
LITERATURE CITED
Anderson, D. and Garrison, D. 1997 Preface in The Ecology and Oceanography of
Harmful Algal Blooms. Limnology and Oceanography. 42 (5, part 2) preface.
Bayne, B. et al. 1976. Physiology: 1. Pp. 121-140 in Bayne, B. ed. Marine Mussels: their
ecology and physiology. Cambridge University Press, Cambridge.
Cornwall, R., Toomey, B.H., et al. 1995. Characterization of multixenobiotic/multidrug
transport in the gills of the mussel Mytilus californianus and identification of
environmental substrates. Aquatic Toxicology. 31:277-296
Duggins, D. and Eckman, J. 1997. Is kelp detritus a good food for suspension feeders?
Effects of kelp species, age and secondary metabolites. Marine Biology. 128: 489-495
Epel, David. 1998, in press. Use of multidrug transporters as first lines of defense against
toxins in aquatic organisms. Comp. Biochem and Physiology. Part A.
Eufemia, Nancy, and Epel, David. 1998, in press. The multixenobiotic defence
mechanism in mussles is induced by substrates and non-substrates: Implications for a
general stress response. Marine Environmental Research
Gainey, Louis, and Shumway, Sandra. 1988. A compendium of the responses of bivalve
mulluscs to toxic dinoflagellates. Journal of Shellfish Research. 7 (4): 623-628.
Gosling, Elizabeth, ed. 1992. The Mussel Mytilus: Ecology, Physiology, Genetics, and
Culture. Elsevier, Amsterdam.
Hawkins, Anthony and Bayne, Brian. 1992. Physiological Interrelations, and the
regulation of Production. pp. 171-176. in Gosling, Elizabeth, ed. The Mussel Mytilus.
Ecology, Physiology, Genetics, and Culture. Elsevier, Amsterdam.
Jorgensen, Barker. 1996. Bivalve filter feeding revisited. Marine Ecology Progress
Series. 142: 287-302.
Kurelec, Branko. 1995 Inhibition of multixenobiotic resistance mechanism in aquatic
organisms: ecotoxic consequences. The Science of the Total Enviornment 171 (1995);
197-204.
Minier, C. and Moore, M.N.1996. Induction of Multixenobiotic Resistance in Mussel
Blood Cells. Marine Environmental Research. 42 (1-4): 389-392.
Morris, R., Abbott, D. and Haderlie, E. 1980. Intertidal Invertebrates of California.
Stanford University Press, Stanford, CA.
Newell, Carter et al. 1989. The Effects of Natural Seston Particle Size and Type on
Feeding Rates, Feeding Selectivity and Food Resource Availability for the Mussel
Mytilus Edulis Linnaeus, 1758 at Bottom Culture Sites in Maine. Journal of Shellfish
Research. 8 (1): 187-196.
Raby et al. 1997. Food-particle size and selection by bivalve larvae in a temperate
embayment. Marine Biology. 127: 665-672.
Shumway, S. 1992. Mussels and Public Health. p. 520 in Gosling, Elizabeth, ed. The
Mussel Mytilus: Ecology, Physiology, Genetics, and Culture. Elsevier, Amsterdam.
Smayda, T. 1997. What is a bloom? A commentary. Limnology and Oceanography.
42 (5, part 2): 1132-1136.
Smital, T. et al. 1996. Reversal of multridrug resistance by extract from the marine alga
Caulerpa taxifolia. Periodicum Biologorum. 98(2): 197-203.
Steinberg, Peter. 1985. Feeding Preferences of Tegula funebralis and Chemical Defenses
of Marine Brown Algae. Ecological Monographs. 55(3): 33-349
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Ecological Roles of Marine Natural Products. Cornell University Press, Ithaca.
FIGURE LEGEND
Error Bars always represent standard deviation. ** = significant, p £0.01,
Fig. 1. Rhodamine Accumulation Assay 1. The following were tested in 4 separate
experiments and the results were pooled- 1. Rhodamine alone, 2. Verapamil, 3.
Macrocystis pyifera, 4. Phyllospadix scouleri, 5. Egregia menziesii.
Fig. 2. Rhodamine Accumulation Assay 2. The following were tested in 1 experiment.
1. Rhodamine alone, 2. Verapamil, 3. Gelidium robustum, 4. Mazzaella flacida, 5.
Endocladia muricata
Fig. 3. Rhodamine Accumulation Assay 3. The following were tested in 3 experiments
and the results were pooled- 1. Rhodamine alone, 2. Verapamil, 3. Alexandrium
catenella, 4. Phaeodactylum tricornutum. 5. Psuedonizchia australis.
Fig. 4. Rhodamine Efflux Assay 1. The following were tested in 1 experiment- 1.
Rhodamine alone, 2. Verapamil, 3. Alexandrium catenella.
Fig. 5. Rhodamine Efflux Assay 2. The following were tested in 1 experiment- 1.
Rhodamine alone, 2. Verapamil, 3. PCP, 4. Egregia menziesii, 5.Psuedonizchia australis
Fig. 6. Rhodamine Efflux Assay 3. The following were tested in 1 experiment- 1.
Rhodamine alone, 2. Verapamil, 3. Macrocystis pyifera. 4. Phyllospadix scouleri
Fig. 7. Fluorescence controls, Rhodamine efflux assay. The following were tested in
one experiment- 1. Rhodamine alone. 2. Macrocystis pyifera. 3. Egregia menziesii,
4. Psuedonizchia australisa 5. Alexandrium catenella, 6. Phaeodactylum tricornutum
Phyllospadix scouleri. 8. 80% Ethanol. 9. 95% Ethanol,
Fig. 8. Induction studies. Western blot. This represents two separate induction
experiments, each one shows a control band versus a Macrocystis pyifira band.
Fig. 9 Induction studies. Rhodamine accumulation assay. The following were tested
in 1 24 hr. induction experiment- 1. Control (mussel physiological saline solution). 2.
Macrocystis pyifira 3. Psuedonizchia australis.
Table 1: Summary of experimental results
Rhodamine
Extract
Accumulation Assay¬
Possible Substrate?
Macrocystis
Yes
pyifera
Endocladia
muricata
No
Gelidium
robustum
No
Egregia
menziesii
Yes
Mazzaella
flacida
No
Phyllospadix
scouleri
Yes
Alexandrium
catenella
Yes
Phaeodactylum
tricornutum
Yes
Psuedonizchia
Yes
australis
Rhodamine Efflux
Assay-
Possible Substrate?
No
Yes
No
Yes
—
Yes
Yes
Induction
Injection
Experiment
Extract
amount
Saline
100 ul
Solution
Macrocystis
100 ul
A. catenella
100 ul
Saline
100 ul
Solution
(3 times)
PCP
100 ul
(7 times)
A. catenella
100 ul
(4 times)
Saline
100 ul
solution
3
P.australis
100 ul
Macrocystis
100 ul
Artificial
Water
Sea water
exposure
only
Unfiltered
Water
Sea Water
exposure
4
only
Table 2: Induction Experiment summary
Time of
Exposure
48 hr.
48 hr.
48 hr.
8 days
16 days
10 days
24 hours
24 hours
24 hours
72 hours
Western
Blot¬
induction?
Yes
No
Invalid
comparison
Unsure
Yes
Not shown
Not shown
Gill Rho.
Acc. Assay¬
Substrate?
—
Not shown
Not shown
—
——
Likely
Not shown
Not shown
Not shown
250
200
100
50
Rhodamine Accumulation Assay: Algae 1
Rhodamine Verapamil Macro. Phyllo. Egregia
Fig. 2
160
140
120
100
40
20 -
Rhodamine Accumulation Assay: Algae 2
Rho.
Ver. Gelidium Mazz.
Endo.
Fig. 3
Rhodamine Accumulation Assay: Phytoplankton
200
180
160
140
120
100
60 -
40
20
0 -
Rho.
Ver.
A.cat. P. aus. P. tric.
Figure 3: pooled results from 3 experiments
60
50
40
30 -
20
10 -
fflux Assay
—

—

50 60 70
10 20
30 40
Time (minutes)
—°— Rhodamine
Verapamil
v— A. catenella
Fig. 4
50
5 40
3 30
20
10
Efflux Assay




10 20 30 40 50 60 70
Time in Minutes
—•— Rhodamine
-O
Verapamil
v— Egregia
V PCP
—— Psuedonizchia
Fig. 5
70 —
50
3 40
1
F 20
10 -
0
Efflux Assay


1

20 30 40 50 60 70
Time in minutes
Fig. 6.
—e— Rhodamine
Verapamil
Macrocystis
V Phyllospadix
Fig. 7
40
15
5
o
Fluorescence Control Experiment
cat. P. aust. Egregia Philo. P. tric. Etoh(95) Etoh(80)
Fig. 8. Induction Studies. Western Blot.
Exp. 2
Exp. 1
250 KD
160 KD
Con Mac Con Moc
Fig. 9
180
160 -
140 -
120 -
100
80
60 -
40 -
20
Induction experiment + 4
rhodamine accumulation assay
Rho. Ver. Rho. Ver.
Rho. Ver.
Control Macrocystis
Psuedonizchia