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 Steinberg, Peter. 1992. Geographical Variation in the Interaction between Marine Herbivores and Brown Algal Secondary Metabolites. pp. 51-62 in Paul, Valerie, ed. 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