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Comparative effects of the toxic dinoflagellate, Karenia brevis, on bivalve molluscs from Florida

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Comparative effects of the toxic dinoflagellate, Karenia brevis, on bivalve molluscs from Florida
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English
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Leverone, James R
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Harmful algae
Molluscs
Larvae
Juveniles
Mortality
Clearance rate
Histopathology
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ABSTRACT: The effects of the toxic dinoflagellate, karenia brevis (Wilson clone), on larval survival and development of the northern quahog (=hard clam, Mercenaria mercenaria), eastern oyster (Crassostrea virginica) and bay scallop (Argopecten irradians) were studied in the laboratory. The effects of K. brevis on feeding activities of juveniles from these species plus the green mussel (Perna viridis) were also examined. Finally, adult bay scallops were exposed to K. brevis for two weeks to investigate possible cytotoxic effects. Survival of 3-day-old larvae was generally > 85% for all shellfish species at Karenia brevis densities of 100 cells . ml-1 or less, and not significantly different between whole and lysed culture. At 1,000 cells . ml-1, survival was significantly less in lysed culture than whole culture for both M. mercenaria and C. virginica. Survival of 7-day-old larvae in all species was not significantly affected at densities up to 1,000 cells . ml-1.^ ^At 5,000 cells . ml-1, however, survival was reduced to 37, 26 and 19% for A. irradians, M. mercenaria and C. virginica, respectively. Development of C. virginica and M. mercenaria larvae was protracted at K. brevis densities of 1,000 cells . ml-1. Clearance rates of juveniles were determined under static and flow-through conditions using whole and lysed cultures of K. brevis. The bay scallop was most sensitive, exhibiting a 79% reduction in clearance rate at 1,000 cells . ml-1 of whole culture. The eastern oyster was least responsive, showing a 38% reduction in clearance rate between the same treatments. The green mussel and the northern quahog displayed intermediate responses. Similar results were observed during longer (2 day) exposures to a continuous supply of K. brevis. Bay scallops showed a significant decline in clearance rate at 100 cells . ml-1 after 24 hr exposure; clearance rate of oysters was not affected by K. brevis at this concentration.^ ^No mortality was observed for any species during these brief exposures. Adult bay scallops exposed to K. brevis for two weeks showed degenerative and inflammatory changes in the digestive gland, including reduced thickness of the epithelium, increased size of digestive tubule lumens and hemocytic infiltration. The prospect for recovery of bay scallop populations in Florida may be hampered by recurring blooms of K. brevis.
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Dissertation (Ph.D.)--University of South Florida, 2007.
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by James R. Leverone.
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Comparative Effects of the Toxic Dinoflagellate, Karenia brevis on Bivalve Molluscs from Florida by James R. Leverone A dissertation submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy Department of Biological Oceanography College of Marine Science University of South Florida Major Professor: Norman J. Blake, Ph.D Sandra E. Shumway, Ph.D, D.Sc. Gabriel A. Vargo, Ph.D. Joseph J. Torres, Ph.D. Richard H. Pierce, Ph.D. Date of Approval: April 2, 2007 Keywords: harmful algae, molluscs, larvae, juveniles, mortality, clearance rate, histopathology Copyright 2007, James R. Leverone

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Dedication This dissertation is wholeheartedly dedi cated to my gracious wife, Barbara. Throughout my graduate studies, she has steadfastly supported me through all my trials and tribulations. Whenever an experimental procedure went awry, especially in the beginning, she was always there to put things in perspective, reminding me that through apparent failures, progress and learning took root. And at times when I was not making even the slightest progress or was not paying proper attention to my work, she would gently push me to keep going. Even at times when I had doubts, she never wavered. She had complete trust and confidence in me at times when I questioned myself. Furthermore, she always maintained a convivial home and family life which provided a welcome respite from my studies. I will be forever grateful to her for her support and hope she may share in the joy of my accomplishment. May this work be an inspiration to any student contemplating a return to graduate school later in life. May you have a partner who will encourage and support you as my wife did me.

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Acknowledgments There are many people to whom I owe a debt of appreciation for their support during the pursuit of this dissertation. I wish to thank Dr. Norman J. Blake and Dr. Sandra E. Shumway for their guidance throughout this process, as well as my remaining committee members, Drs. Gabe Vargo, Jose Torres and Rich Pierce. Candice Way and Noland Elsaesser at USF provided cultured bay scallop larvae and juveniles. Curt Hemmel of Bay Shellfish Company supplied quahog larvae and juveniles, while John Suppan was very gracious in providing oyster larvae. Many personnel from Mote Marine Laboratory were truly remarkable in extending their support and encouragement. Dana Wetzel provided valuable climate-controlled laboratory space. Mike Henry and Trish Blum extracted toxins from numerous algal cultures and performed brevetoxin analyses; Chris Higham and Val Palubok maintained phytoplankton cultures. Jim Gelshleiter helped me acquire a modicum of skill in histological techniques and absorbed the cost laboratory supplies. I am very honored to have had the continual support of Dr. Ernest Estevez, Director of the Center for Coastal Ecology, as I attempted to juggle my dual role of staff biologist at Mote Marine Laborator y and student at the University of South Florida. Dr. Estevez maintained enthusiastic support and encouragement for my academic quest. Finally, this degree could not have been completed without the support and financial assistance of the president, Dr. Kumar Mahadevan, and board of directors of Mote Marine Laboratory. I am deeply appreciative and truly thankful to all of you.

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i Table of Contents List of Tables................................................................................................................. ... iii List of Figures................................................................................................................ .... v Abstract....................................................................................................................... .... viii Chapter One: Introduction.................................................................................................1 Chapter Two: Literature Review.......................................................................................4 Bivalve Mortality Associated with Harmful Algal Species....................................5 Behavioral Responses of Bivalves to Harmful Algal Species..............................10 Feeding Responses of Bivalves Exposed to Harmful Algal Species....................12 Cytotoxic Effects of Harmful Algal Species to Bivalves.....................................20 Chapter Three: Larval Studies........................................................................................ 24 Introduction...........................................................................................................24 Materials and Methods..........................................................................................25 Collection and Maintenance of Bivalves..................................................25 Maintenance of Algal Cultures.................................................................26 Preparation of Lysed Culture....................................................................26 Determination of Cell and Brevetoxin Concentrations.............................27 Three-Day Static Exposure.......................................................................27 Seven-Day Static Exposure......................................................................29 Statistical Analysis....................................................................................30 Results...................................................................................................................30 Toxin Profile of Karenia brevis Cultures.................................................30 Three-Day Static Exposure.......................................................................31 Seven-Day Static Exposure......................................................................32 Discussion.............................................................................................................33 Chapter Four: Juvenile Studies.......................................................................................49 Introduction...........................................................................................................49 Materials and Methods..........................................................................................51 Collection and Maintenance of Juveniles.................................................51 Maintenance of Algal Cultures.................................................................51 Preparation of Lysed Culture.....................................................................52 Determination of Cell and Brevetoxin Concentrations.............................52 Clearance Rate Studies.............................................................................53

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ii Static Exposure Experiments........................................................54 Flow-Through Exposure Experiments..........................................55 Results...................................................................................................................57 Static Exposure Experiments....................................................................57 Bay scallops ( Argopecten irradians )............................................58 Green mussels ( Perna viridis )......................................................58 Northern quahogs ( Mercenaria mercenaria )................................59 Eastern oysters ( Crassostrea virginica )........................................59 Flow-Through Exposure Experiments......................................................60 Discussion.............................................................................................................61 Chapter Five: Histopathology Studies............................................................................ 81 Introduction...........................................................................................................81 Materials and Methods..........................................................................................83 Results...................................................................................................................85 Discussion.............................................................................................................86 Chapter Six: General Discussion..................................................................................... 95 Bivalve Larvae Juvenile Bi valves Conclusion and Significance Impacts from Different Culture Preparations...109 Possible Mechanisms of Toxic Activity...111 Implications for Fisheries Management...114 Literature Cited............................................................................................................... 118 About the Author...................................................................................................End Page

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iii List of Tables Table 1. Cell density, sample matrix and brevetoxin composition of Karenia brevis (Wilson clone) cultures used in larval experiments for each species. (Brevenal is considered a brevetoxin antagonist)............................38 Table 2. Mean (+ SD) number of live and dead larvae, larval stage and percent survival for Argopecten irradians after exposure to Karenia brevis for three days. Treatments consisted of whole and lysed cultures of K. brevis at three concentrations: 10,100 and 1,000 cells ml-1. (n = 5)............39 Table 3. Mean (+ SD) number of live and dead larvae, larval stage and percent survival for Mercenaria mercenaria after exposure to Karenia brevis for three days. Treatments consisted of whole and lysed cultures of K. brevis at three concentrations: 10,100 and 1,000 cells ml-1. (n = 5)............40 Table 4. Mean (+ SD) number of live and dead larvae, larval stage and percent survival for Crassostrea virginica after exposure to Karenia brevis for three days. Treatments consisted of whole and lysed cultures of K. brevis at three concentrations: 10,100 and 1,000 cells ml-1. (n = 5)............41 Table 5. Effect of Karenia brevis concentration and culture preparation on percent survival in three-day-old bivalve larvae. A) Two-way ANOVA ( = 0.05). B) Tukey's ( ) multiple comparison test. Underlined treatments are not signi ficantly different (p > 0.05)....................42 Table 6. Effect of Karenia brevis concentration on percent survival in sevenday-old bivalve larvae. A) One-way ANOVA ( = 0.05). B) Tukeys ( ) multiple comparison test. Underlined treatments are not significantly different (p > 0.05).....................................................................48 Table 7. Experimental conditions, bivalve species, sample matrix, cell and brevetoxin concentration of laboratory cultures of K. brevis (Wilson Clone) used for juvenile feeding experiments. n.d. = not detected................67 Table 8. Decline in Isochrysis galbana cell counts (cells ml-1) for juvenile bivalve molluscs exposed to different concentrations and preparations of Karenia brevis under static conditions. Starting seawater volume in each replicate was 500 ml...............................................................................68 Table 9. Filtration and clearance rates of juvenile bivalve molluscs exposed to

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iv whole and lysed culture of Karenia brevis under static conditions. Starting seawater volume in each replicate was 500 ml.................................69 Table 10. Species, treatment, clearance rate and amount of toxin (g) each species was exposed to during static feeding experiments. Amount of toxin exposure is based on culture cell concentration and amount of toxic (w/v) in each culture...............................................................................75 Table 11. Experimental design for two-week exposure of Argopecten irradians to Karenia brevis ( I. galbana is a common nutritional chrysophyte algae)......89 Table 12. Schedule for the removal of Argopecten irradians from each experimental tank during the two-week exposure to Karenia brevis. Values represent shell height (mm) of individuals removed for fixation (shaded) or dead (unshaded) on that day.........................................................90 Table 13. Scoring of hemocyte inflitration intensity in the digestive diverticula of A irradians exposed to various scenarios of K brevis ...............................91 Table 14. The effects of Karenia brevis on molluscs....................................................116

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v List of Figures Figure 1. Percent survival (mean + SD) of Argopecten irradians larvae after exposure to Karenia brevis for seven days. Treatment with an asterisk was significantly different (p < 0.05). Larvae were seven days old at start of experiment............................................................................................. 43 Figure 2. Percent survival (m ean + SD) of Mercenaria mercenaria larvae after exposure for seven days to Karenia brevis. Treatments with the same letter were not significantly different (p < 0.05). Larvae were seven days old at start of experiment..........................................................................44 Figure 3. Percent of total Mercenaria mercenaria larvae that survived to the umboveliger ( ) and pediveliger ) stages after exposure to Karenia brevis for seven days. Larvae were seven-day-old umboveligers at start of experiment..................................................................................................... 45 Figure 4. Percent survival (m ean + SD) of Crassostrea virginica larvae after exposure to Karenia brevis for seven days. Treatment with an asterisk was significantly different (p < 0.05). Larvae were seven days old at start of experiment............................................................................................. 46 Figure 5. Percent of total Crassostrea virginica larvae that survived to the um boveliger ( ), pediveliger ( ) and spat ( ) stages after exposure to Karenia brevis for seven days. Larvae were seven-day-old umboveligers at start of experiment. ................................................................47 Figure 6. Mean ( S.D.) clearance rate (ml hr-1 mg dry wt-1) for juvenile bay scallops ( Argopecten irradians ) exposed to three concentrations and two preparations of Karenia brevis. Treatments with the same letter are not significantly different (p > 0.05). n = 10. Two-way ANOVA; Tukeys Multiple Comparison Test...................................................................70 Figure 7. Mean ( S.D.) clearance rate (ml hr-1 mg dry wt-1) for juvenile green mussels ( Perna viridis) exposed to three concentrations and two preparations of Karenia brevis Treatments with the same letter are not significantly different (p > 0.05). n = 10. Two-way ANOVA; Tukeys Multiple Comparison Test.................................................................................71 Figure 8. Mean ( S.D.) clearance rate (ml hr-1 mg dry wt-1) for juvenile

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vi northern quahogs ( Mercenaria mercenaria ) exposed to three concentrations and two preparations of Karenia brevis Treatments with the same letter are not significantly different (p > 0.05). n = 10. Two-way ANOVA; Tukeys Multiple Comparison Test..................................72 Figure 9. Mean ( S.D.) clearance rate (ml hr-1 mg dry wt-1) for juvenile eastern oysters ( Crassostrea virginica ) exposed to three concentrations and two preparations of Karenia brevis Treatments with the same letter are not significantly different (p > 0.05). n = 10. Two-way ANOVA; Tukeys Multiple Comparison Test.................................................................................73 Figure 10. Mean ( S.D.) clearance rate (ml hr-1 mg dry wt-1) for juvenile bivalves exposed to (A) whole and (B) lysed cultures of Karenia. brevis under static conditions. = Argopecten irradians ; = Perna viridis ; = Mercenaria mercenaria ; and = Crassostrea virginica (n = 10)............74 Figure 11. Regression of clearance rates for juvenile Argopecten irradians against the amount of brevetoxin exposure under static conditions. Solid line represents regression equation for whole culture of Karenia brevis ; dashed line represents regression equation for lysed culture of K. brevis (n = 5)...............................................................................................76 Figure 12. Regression of clearance rates for juvenile Perna viridis against the amount of brevetoxin exposure under static conditions. Solid line represents regression equation for whole culture of Karenia brevis ; dashed line represents regression equation for lysed culture of K. brevis (n = 5)...................................................................................................77 Figure 13. Regression of clearance rates for juvenile Mercenaria mercenaria against the amount of brevetoxin exposure under static conditions. Solid line represents regression equation for whole culture of Karenia brevis ; dashed line represents regression equation for lysed culture of K. brevis (n = 5)...............................................................................................78 Figure 14. Regression of clearance rates for juvenile Crassostrea virginica against the amount of brevetoxin exposure under static conditions. Solid line represents regression equation for whole culture of Karenia brevis ; dashed line represents regression equation for lysed culture of K. brevis (n = 5)...............................................................................................79 Figure 15. Mean ( S.D.) clearance rate (ml hr-1 mg dry wt-1) of juvenile bivalves exposed to whole (top) and lysed (bottom) cultures of K brevis under flow-through conditions. Species include (A) Argopecten irradians (B) Perna viridis (C) Mercenaria mercenaria and (D) Crassostrea virginica Concentrations of K. brevis in each treatment are: Control ( ), 100 () and 1,000 () cells ml-1. (n = 6). Clearance

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vii rates were measured twice a day (9 A.M. and 5 P.M.) and calculated from inflow and outflow concentrations of a supplemental food algae, Isochrysis galbana .............................................................................................80 Figure 16. (A) The protist, Nematopsis sp? (arrow) and (B) a Rickettsial-like bacterial microcolony in the gill epithelia of A. irradians Magnification equals 400x................................................................................92 Figure 17. An unidentified parasite encysted within the digestive diverticula. Magnification equals 400x................................................................................92 Figure 18. Digestive diverticula from A. irradians exposed to lysed culture of K. brevis and T. Isochrysis (A and B = day 7; C and D = day 9). Hemocyte aggregations (arrows) displaying inflammatory response. Magnification equals 400x................................................................................93 Figure 19. Epithelial layer of the digestive diverticula in A. irradians from different exposure scenarios to K. brevis A) control scallop at 14 days; B) scallop exposed to whole culture of K. brevis at day 12. Magnification equals 400x................................................................................93 Figure 20. Gill tissue from A. irradians showing A) the distal portions of the ordinary filaments (note the lateral cilia) and B) interconnecting vessels of the dorsal expansion......................................................................................94 Figure 21. Mantle tissue from A. irradians showing A) the epithelia of the mantle margin and B) section through an eye on the middle fold of the mantle margin................................................................................................................94

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viii Comparative Effects of the Toxic Dinoflagellate, Karenia brevis on Bivalve Shellfish from Florida James R. Leverone Abstract The effects of the toxic dinoflagellate, Karenia brevis (Wilson clone), on larval survival and development of the northern quahog (= hard clam, Mercenaria mercenaria ), eastern oyster ( Crassostrea virginica ) and bay scallop ( Argopecten irradians ) were studied in the laboratory. The effects of K. brevis on feeding activities of juveniles from these species plus the green mussel ( Perna viridis ) were also examined. Finally, adult bay scallops were exposed to K. brevis for two weeks to investigate possible cytotoxic effects. Survival of 3-day-old larvae was generally > 85% for all shellfish species at Karenia brevis densities of 100 cells ml-1 or less, and not significantly different between whole and lysed culture. At 1,000 cells ml-1, survival was significantly less in lysed culture than whole culture for both M. mercenaria and C. virginica Survival of 7-dayold larvae in all species was not significantly affected at densities up to 1,000 cells ml-1. At 5,000 cells ml-1, however, survival was reduced to 37, 26 and 19% for A. irradians M. mercenaria and C. virginica respectively. Development of C. virginica and M. mercenaria larvae was protracted at K. brevis densities of 1,000 cells ml-1. Clearance rates of juveniles were determined under static and flow-through

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ix conditions using whole and lysed cultures of K. brevis The bay scallop was most sensitive, exhibiting a 79% reduction in clearance rate at 1,000 cells ml-1 of whole culture. The eastern oyster was least responsive, showing a 38% reduction in clearance rate between the same treatments. The green mussel and the northern quahog displayed intermediate responses. Similar results were observed during longer (2 day) exposures to a continuous supply of K. brevis Bay scallops showed a significant decline in clearance rate at 100 cells ml-1 after 24 hr exposure; clearance rate of oysters was not affected by K. brevis at this concentration. No mortality was observed for any species during these brief exposures. Adult bay scallops exposed to K. brevis for two weeks showed degenerative and inflammatory changes in the digestive gland, including reduced thickness of the epithelium, increased size of digestive tubule lumens and hemocytic infiltration. The prospect for recovery of bay scallop populations in Florida may be hampered by recurring blooms of K brevis

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1 Chapter One Introduction The dinoflagellate, Karenia brevis (formally Gymnodinium breve Davis) (Daugbjerg et al ., 2001) is responsible for one of the oldest reported harmful algal blooms in North America (Ingersoll, 1882), yet shellfish poisonings in the Gulf of Mexico from this algal species were considered rare and infrequent as late as forty years ago. At that time, it was not known whether shellfish could actually feed upon K. brevis or accumulate the toxins in their tissues. In 1967, laboratory experiments in Texas (Ray and Aldrich, 1967) and field studies from Sarasota, FL (Cummins et al ., 1971) both demonstrated that eastern oysters ( Crassostrea virginica ) could consume K. brevis and become toxic. Partly as a result of these findings, the state of Florida began monitoring shellfish for toxicity from outbreaks of harmful algae in the 1970s. Today, advances are being made in our knowledge and comprehension of the human health impacts from exposure to Florida red tides, which occur either through the consumption of contaminated shellfish or by inhalation of toxin-laden aerosols (Pierce et al ., 1990; Pierce et al ., 2005). We now know that K. brevis produces at least twelve and possibly fourteen potent neurotoxins (=brevet oxins) that are lethal to fish and cause neurotoxic shellfish poisoning (NSP) in human s from the consumption of contaminated shellfish (Baden, 1988; Steidinger et al ., 1998; Bourdelais et al ., 2004). Through these human health related studies, we are learning more about how shellfish accumulate, metabolize and eliminate brevetoxins. Of particular interest is the discovery that eastern

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2 oysters may remain toxic for several months after dissipation of a bloom (Dickey et al ., 1999; Wang, 2004). The literature on the human health effects of Florida red tides has been recently reviewed by Kirkpatrick et al (2004). Not surprisingly, progress in understanding how blooms of K brevis affect the health of individual bivalve species lag considerably behind the human health ramifications of these same blooms; today, however, these red tides have led to greater concerns about the long-term effects these blooms are having on local fisheries, including critical species of bivalve molluscan shellfish (Landsberg, 1996). For instance, we currently do not know whether K. brevis affects critical early life stages, growth and development of juveniles, or reproductive development and fecundity of adults for any species of bivalve mollusc from Florida. Information on possible cytotoxic effects of K brevis on bivalves is also critically lacking. Does exposure to K. brevis affect feeding and behavior in bivalves, or render them more susceptible to predation? What are the effects on population dynamics, particularly larval dispersal and recruitment? We still do not even know if K. brevis causes mortality in any species of bivalve, particularly the bay scallop, Argopecten irradians Any deleterious effect of exposure to blooms of K brevis would potentially threaten Floridas valuable shellfish resources and negatively impact the states growing bivalve aquaculture industry (Blake et al ., 2000; Adams and Sturmer, 2004). The objectives of this research were to investigate the effects of the toxic dinoflagellate, Karenia brevis on four important species of bivalve mollusc from Florida. The bivalve species selected for study were the bay scallop ( Argopecten irradians ), northern quahog (= hard clam, Mercenaria mercenaria ), eastern oyster ( Crassostrea

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3 virginica ) and green mussel ( Perna viridis ). This research was divided into the following separate investigations of the specific effects of K brevis on bivalves: 1) survival and development in larvae, 2) feeding rates in juveniles, and 3) histopathology in adult bay scallops from long-term, sublethal exposure. All studies were conducted under controlled laboratory conditions using a specific culture (Wilson clone) of K. brevis In each instance, experiments were designed to distinguish between effects caused by the toxic dinoflagellate and the effects of its associated toxins (=brevetoxins).

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4 Chapter Two Literature Review The initial motivation for research on toxigenic algae was the potential human health impacts associated with these blooms (Hemmert, 1975; Bicknell and Walsh, 1975; Price et al ., 1991; Todd, 1993; Fremy et al ., 1999; Fernandez, 2000; Garthwaite, 2000). Human health problems generally result from the consumption of bivalve molluscan shellfish rendered toxic by filtering and ingesting harmful microalgae (Shumway, 1995; Bricelj and Shumway, 1998) or by the inhalation of aerosolized brevetoxins incorporated in marine aerosol by bubble-mediated (Pierce et al ., 1990; Pierce et al 2005). Not very long ago, bivalves were thought to accumulate toxins in their tissues without any apparent negative consequences (Prakash et al ., 1971; Quayle, 1969). That impression, however, has since been abandoned as researchers have taken a more thorough look at the chronic, sublethal effects of harmful algae on bivalves and how these interactions affect shellfish populations, mariculture activities and coastal ecosystems (Shumway et al ., 1985; Shumway and Cucci, 1987; Shumway and Cembella, 1993; Bricelj and Shumway, 1998; see reviews by Shumway, 1990 and Landsberg, 2002). Bivalves accumulate microalgal toxins in their tissues through filter-feeding. The way in which they respond to the presence of toxic algae depends upon the species of bivalve as well as the algal species encountered (Shumway and Cucci, 1987; Smolowitz

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5 and Shumway, 1997; Gainey and Shumway, 1988; Bricelj et al ., 1991; Lesser and Shumway, 1993). In turn, behavioral and phys iological differences among bivalves in feeding response also depend upon a number of factors. These factors include, but are not limited to, history of exposure (Shumway and Cucci, 1987; Bricelj et al ., 2000), season (Lesser and Shumway, 1993), algal toxicity (Bricelj et al ., 1996) toxin content (Bricelj et al ., 1991; Li and Wang, 2001), algal cell concentration (Bricelj et al ., 2004), cell selectivity (Shumway et al ., 1985; Shumway et al ., 1990), cell size (Lesser and Shumway, 1993), and differences in digestive function (Wikfors and Smolowitz, 1993). Bivalve Mortality Associated with Harmful Algal Species Global accounts of the lethal, sublethal and chronic effects of harmful algal blooms on shellfish and other molluscs may be found in Shumway and Cucci (1987) and also in two comprehensive reviews (Shumway, 1990; Landsberg, 2002). Reports of massive shellfish mortality linked to harmful dinoflagellate blooms must be interpreted with caution since there can be other unfavorable events or conditions associated with algal blooms. Most often, prolonged peri ods of low dissolved oxygen (=hypoxia) or even the absence of oxygen (=anoxia) in bottom waters will accompany or follow an algal bloom as cells lyse or fish decompose. These conditions generate a high biochemical oxygen demand, which may also be a causative factor in shellfish mortalities associated with toxic algal blooms. In a report on red water organisms (= dinoflagellates) from the Pacific Northwest, Nightengale (1936) listed some of the earliest records of harmful algal blooms and

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6 shellfish that were destroyed (see Table). In 1929, Nightengale (1936) personally Table of historical harmful algal events a nd affected bivalves reported in Nightengale (1936) Year Locality Harmful Alga Shellfish Affected 1891 Pt. Jackson, Australia Glenodinium rubrums Oysters and mussels 1893 Gokasho Bay, Japan Gymnodinium (?) Pearl oysters 1902 Santa Barbara to San Diego, CA Gonyaulax species Fish and bottom fauna 1907 San Pedro to San Diego ,CA Gonyaulax polyedra Pearl oysters, fish and shellfish 1910 Gokasho Bay, Japan Gymnodinium (?) Pearl oysters observed losses of oysters in Oakland Bay, Washington during a bloom of Gymnodinium splendens Although the cause of mortality (toxins or oxygen depletion) in all of these instances was not established, decomposition of organic debris (and concomitant depletion of oxygen?) was suspected as the primary cause. The only reported bivalve mortalities associated with Alexandrium catenella were white mussels ( Donax serra ) and black mussels ( Chloromytilus meridionalis ) off the southern coast of South Africa (Horstman, 1981). Koray (1992) reported unidentified shellfish mortalities due to Alexandrium minutum in Izmir Bay, Turkey. Wardle et al (1975) observed dead surfclams ( Spisula solidissima ) and eastern oysters ( Crassostrea virginica ) among an assemblage of invertebrate and fish fatalities associated with a bloom of Gonyaulax monilata off Galveston, Texas from 1971-72. In this instance, affected species were either sessile, sedentary or weakly motile, suggesting the more motile species were able to avoid the bloom area before accumulating lethal amounts of toxin. Unfortunately, dissolved oxygen was not monitored during these mass mortality events. Species of Alexandrium reportedly toxic to a host of pectinid ( = scallop) species (see Table 1 in

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7 Shumway and Cembella, 1993), have not been linked to scallop mortalities in nature. Mass mortalities of bivalves are occasionally associated with blooms of nontoxic algal species. Mortality in these instances is more often than not due to the subsequent decline in dissolved oxygen that accompanies these blooms. A bloom of the dinoflagellate, Ceratium tripos in New York Bight during the summer of 1976 was followed by mass mortalities of surfclams ( Spisula solidissima ), ocean quahog ( Arctica islandica ), sea scallops ( Placopecten magellanicus ), American lobster ( Homarus americanus ) and fish (Mahoney and Steimle, 1979). Mortalities from this event were attributed to extensive oxygen depletion resulting from degradation of the algal bloom, and not a toxic response to the algal bloom. Several species of Gonyaulax have been implicated in shellfish mortalities worldwide even when other environmental factors, particularly low dissolved oxygen, were at least partially at play during these events. In South Africa, both Gonyaulax grindleyi (= Protoceratium reticulatum ) and G polygramma blooms resulted in massive quantities of dead invertebrates and fish, including a variety of mussels and abalone (Grindley and Nel, 1968; Grindley and Taylor, 1964). Separate G polygramma blooms were associated with mussel mortalities in Venezuela (Ferraz-Reyes et al ., 1979; La Barbera-Sanchez et al ., 1993), Japan (Koizumi et al ., 1996; Schwimmer and Schwimmer, 1968) and Hong Kong (Lam and Yip, 1990). Forbes (1990) reported shellfish mortality in connection with a bloom of G spinifera in 1990. In nearly all of these events, mortality was associated with low dissolved oxygen levels; thus, the cause of death could not be directly attributed to the dinoflagellate. Furthermore, during a PSP event in Venezuela during 1988, HPLC analysis of G. polygramma samples did not reveal any toxins, supporting the idea that

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8 two other species, namely A. tamarensis and G catenatum which were present during that PSP event, were the toxigenic organisms (La Barbera-Sanchez et al ., 1993). Species of Gymnodinium on the other hand, have been implicated in shellfish mortalities, especially the queen scallop ( Pecten maximus ) in European waters. High mortalities of larvae, post-larvae and juveniles (Lassus and Berthome, 1988; ErardLeDenn et al ., 1990) and inhibited growth and reproduction in adults (Erard-LeDenn et al ., 1990) have been documented from France and Ireland in association with Gymnodinium cf aureolum blooms. Abbott and Ballantine (1957) detailed queen scallop mortality in the presence of G. veneficum in the laboratory. The softshell clam, Mya arenaria suffered 30-40% mortality as a result of a Gyrodinium aureolum (= Gymnodinium aureolum ) bloom in Maquoit Bay, Brunswick, Maine in 1988 (Heinig and Campbell, 1992). The blue mussel, Mytilus edulis was also affected. Again, mortality was attributed to both low dissolved oxygen and toxin production by G. aureolum Mortality of (unidentified) shellfish was observed in association with a bloom of Gymnodinium sp. in southern Brazil during April, 1978. Shellfish collected during this episode were found to contain saxitoxins (Machado, 1979). In 1987, North Carolina experienced its first recorded red tide outbreak, caused by the dinoflagellate, Gymnodinium breve (= Karenia brevis ) (Tester et al ., 1991). Strong circumstantial evidence suggested that the red tide caused mortality of both adult and newly recruited bay scallops (Summerson and Peterson, 1990), leading to recruitment failure in subsequent years in the states most productive scallop beds (Peterson and Summerson, 1992). No other information on the effects of K. brevis on bay scallops has been published.

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9 Mass mortality of marine animals due to Karenia brevis red tides was first reported from the west coast of Florida in 1946-47 by Gunter et al (1947). The total number of dead fish from the entire area was estimated to be over 50,000,000. Accounts of dead invertebrates were limited to the statements: Oysters, clams, crabs, shrimp, barnacles, and coquinas were also killed. The clam industry at Marco [did] not appear to be involved. During a red tide outbreak near Sarasota, Florida in 1978, Tiffany and Heyl (1978) reported numerous surf clams ( Spisula solidissima similis ) and coquina ( Donax variabilis ) washed up on local beaches. Peak Gymnodinium breve (= Karenia brevis ) cell counts during the outbreak reached 5 x 106 cells L-1. Sufficient dissolved oxygen was present in offshore bottom waters during the red tide suggesting that mortality was due to the toxic dinoflagellate. Simon and Dauer (1972) observed the near complete destruction of the benthic infaunal community in Old Tampa Bay during a severe (up to 17.7 x 106 cells L-1) K. brevis bloom in 1971. Oxygen depletion accompanying the massive fish kill could not be ruled out as a causative factor. Interestingly, the bivalve Mulinia lateralis was one of the least impacted species in the assemblage, but this may be due to the ability of this species to withstand short periods of anoxia (Shumway et al., 1983). The authors concluded that laboratory studies were needed to confirm the field observations. Under laboratory conditions, Sievers (1969) showed that Crassostrea virginica exhibited no mortality when exposed to K. brevis concentrations up to 9.9 x 106 cells L-1 for 48 hours. Additional laboratory studies are necessary to delineate the direct effects of K. brevis on mortality in Florida shellfish populations.

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10 Behavioral Responses of Bivalves to Harmful Algal Species Measures of behavioral responses of organisms can provide insight into how they cope with potentially harmful conditions; in this case, exposure to harmful or toxic algae. Behavior patterns can provide a useful index of in vivo sensitivity to these algae and their toxins (Bricelj et al ., 1996). Many species of bivalve mollusc exhibit a variety of responses to the presence of toxic dinoflagellates, including changes in shell valve activity, siphon retraction, byssal thread production, burrowing activity, oxygen consumption, and heart rates (Shumway and Cucci, 1987). Shell valve activity in the Pacific oyster ( Crassostrea gigas ) was inhibited when oysters were fed the toxic dinoflagellate Alexandrium minutum for 8 to 15 days (Lassus et al ., 1999), but there was an immediate and significant increase in shell valve activity when the A. minutum diet was followed by a nontoxic food source. This same oyster species, however, displayed vigorous clapping of shell valves when exposed to A. catenella (Dupay and Sparks, 1968), although two other oyster species ( Crassostrea virginica and Ostrea edulis ) remained open and continued to filter in the presence of Alexandrium tamarensis (Shumway and Cucci, 1987), each species exhibited unique behaviors in the presence of this toxic dinoflagellate. In O. edulis there was an initial, partial adduction of the shell valves followed by periodic snaps which continued until clean seawater was introduced. Eastern oysters ( C. virginica ) responded by initially closing their shell valves, followed by a gradual reopening. This pattern repeated itself, although complete closure never occurred. Eastern oysters rarely opened and did not filter when exposed to Gonyaulax monilata (= Alexandrium monilatum ), but opened

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11 frequently in the presence of Karenia brevis (Ray and Aldrich, 1967). It was first suggested by Twarog a nd Yamaguchi (1975), and given further credence by Shumway and Cucci (1987), that a number of molluscan species from locales that periodically experience toxic algal blooms have evolved mechanisms that allow them to exploit the toxic algae as a food source. Mussels ( Mytilus edulis ) from Maine, which have a history of exposure to toxic algal blooms, showed no evidence of shell-valve closure when exposed to Protogonyaulax tamarensis ( = Alexandrium tamarense ) in the laboratory, while the majority of mussels from Rhode Island and Spain, which are not subjected to toxic algal blooms in their native locales, showed initial erratic shell-valve activity followed by complete shell closure (Shumway and Cucci, 1987). In addition, exhalent siphons from many of the mussels from both Rhode Island and Spain were closed, while the mantle edges in otherwise open animals were retracted. Mussels from Rhode Island and Spain also produced copious amounts of a white, mucus-like material in the presence of A. tamerense Many eventually died. Populations of bivalves from the west coast of Florida (northern quahogs and eastern oysters, in particular) have become established in locations which are undoubtedly exposed to a higher frequency of recurring blooms of K. brevis than other populations which rarely, if ever, come in contact with these blooms. An understanding of a species exposure history to harmful algal episodes must be taken into account when attempting to explain the interactions between a toxic alga and a particular bivalve species. The sea scallop ( Placopecten magellanicus ) showed dramatic activity patterns when exposed to Alexandrium tamarense (Shumway and Cucci, 1987). Most individuals

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12 exhibited an immediate closure of the shell valves followed by either violent swimming activity, partial, sustained shell-valve closure, or a combination of the two. Swimming / clapping activity lasted less than one hour, possibly a result of fatigue. Sea scallops showed a similar response when subjected to starfish extracts, in addition to an accelerated heart rate, increased stroke volume and an enhanced cardiac output (Thompson et al ., 1980). Sea scallops also produced copious amounts of a white mucuslike substance when exposed to bloom conditions of A. tamerense (Shumway and Cucci, 1987). Activity patterns of bay scallops ( Argopecten irradians ) in the presence of toxic dinoflagellates have not been investigated. The northern quahog ( = hard clam) Mercenaria mercenaria showed an initial retraction of the siphons followed by complete closure of the shell valves in the presence of Alexandrium tamarense (Shumway and Cucci, 1987). The animals did not re-open until they were returned to clean sea water. Feeding Responses of Bivalves Exposed to Harmful Algal Species Harmful algae have significant impacts on the feeding activity of bivalve molluscs (Shumway and Cucci, 1987; Bricelj et al ., 1996; Lassus et al ., 1996; Lassus et al ., 1999; Li and Wang, 2001). The exact response depends on the species of bivalve as well as the algal species encountered (Shumway and Cucci, 1987; Gainey and Shumway, 1988; Bricelj et al ., 1996; Lesser and Shumway, 1993; Bardouil et al ., 1993; Bardouil et al ., 1996; Smolowitz and Shumway, 1997; Lassus et al ., 1999). Changes in feeding activity can provide an indication of the sensitivity of bivalves to toxins and thus

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13 potential for toxin uptake of various species (Bricelj et al ., 1996). Feeding rate measurements may also be useful in looking at the response within a species to variation in dinoflagellate cell toxicity (Bricelj and Shumway, 1998). The simplest and most common measure of feeding activity in bivalve molluscs is the clearance rate, which is a measure of the volume of water cleared of particles per unit time. Filtration rate is usually defined as the number of particles removed from suspension per unit time. The clearance rate is a measure of an individuals ability to remove particles from suspension. When no pseudofeces are produced (no captured particles are rejected), the clearance rate equals the ingestion rate. Oysters Early studies on oysters led to erroneous conclusions that they were able to avoid the accumulation of toxic algae and were thus considered to be less prone to becoming toxic than other bivalves (see Shumway et al. 1990 for review). Today, however, there is much more convincing evidence demonstrating that feeding rates in oysters are, indeed, affected by toxic dinoflagellates. These feeding responses, particularly clearance rates, may be quite variable and sometimes contrary (Bardouil et al ., 1993; Shumway and Cucci, 1987). The European oyster, Ostrea edulis exhibited significantly higher clearance rates when fed a mixed nontoxic algal diet plus Alexandrium tamarense when compared to an identical diet without the toxic dinoflagellate (Shumway and Cucci, 1987). The opposite was true for Crassostrea virginica when subjected to identical experimental conditions; the diet containing A. tamarense resulted in lower clearance rates (Shumway and Cucci, 1987). Bardouil et al (1993) offered unialgal cultures of toxic and nontoxic microalgae to Crassostrea gigas under conditions similar in design to Shumway and Cucci (1987).

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14 The same batch of adult C. gigas was exposed successively to non-toxic and toxic Alexandrium tamarense and a different group was exposed to nontoxic Scrippsiella trochoidea and toxic A. minutum Mean algal concentrations were based on field values observed during red tide phenomena. Mean clearance rates showed significant differences between toxic and nontoxic A. tamarense as well as between S. trochoidea and A. minutum Clearance rates of juvenile C. virginica fed separately on unialgal cultures of two toxic dinoflagellates ( A. tamarense and Gyrodinium (= Gymnodinium ) aureolum ) were significantly different from one another and significantly lower than the clearance rates on the nontoxic alga Isochrysis sp. (Lesser and Shumway, 1993). In the same experiment, post-hoc multiple comparison tests showed clearance rates of Ostrea edulis which were fed the same two toxic dinoflagellates grouped together, and were also significantly lower than clearance rates of Isochrysis sp. Bardouil et al (1996) continued their studies on the feeding habits of Crassostrea gigas by investigating the effects of two strain s (toxic and nontoxic) of the dinoflagellate Alexandrium tamarense in combination with the diatom Thalassiosira weissflogii When compared to a unialgal diet of T. weissflogii a diatom/toxic dinoflagellate ratio as low in biomass as 90/10 reduced clearance rates in C. gigas by twenty percent. Clearance rates were slightly, but significantly, lower for a 50/50 diatom/toxic dinoflagellate mixture, while a unialgal toxic dinoflagellate diet resulted in complete inhibition of filtration, ingestion and absorption (Bardouil et al ., 1996). Following the work of Bardouil et al (1996), Lassus et al (1999) evaluated the feeding behavior of Crassostrea gigas on toxic algal diets over a longer time period (up to 30 days) to ascertain whether physiological behavior returned to normal during

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15 detoxification. Oysters fed Alexandrium minutum for 8 to 15 days exhibited significant reductions in clearance rate, filtration rate and biodeposition rate as compared to a nontoxic dinoflagellate, Scrippsiella trochoidea When the A. minutum diet was followed by a diet of either flagellates or diatoms, clearance and filtration rates did not return to their pre-exposure levels (Lassus et al ., 1999). The probability of Eastern oysters becoming exposed to Karenia brevis (= Gymnodinium breve ) is less than other shellfish because oyster beds are typically located in the more oligohaline, upper reaches of an estuary. In the Gulf of Mexico, optimum growth and reproduction occurs in oyster reefs with a salinity of 12 to 30 ppt, but oyster abundance is greatest at salinities between 10 and 20 ppt (Butler, 1954). The salinity preference of K. brevis is > 24 ppt (Tester and Fowler, 1990), which effectively provides a salinity barrier against K. brevis for many oysters. Ray and Aldrich (1965) first noted that the optimum salinity for oysters is lower than that for K. brevis ; however, this does not mean that oysters are never exposed to K. brevis nor are they always free of brevetoxins (Shumway et al ., 1990). Cummins and Hill (1969) found that oysters accumulated 89 95 % of K. brevis in 2-4 hours with an original concentration of 2.4 x 106 cells L-1. Eastern oysters not only survived, but exhibited normal behavior during a 48 hour exposure to K. brevis at cell concentration of 9.9 x 106 cells L-1 (Sievers, 1969). Brevetoxin metabolites have now been isolated and identified in C. virginica from shellfish harvesting areas after a bloom of K. brevis in the Gulf of Mexico in 1996 (Dickey et al ., 1999) and in Sarasota Bay in 2001 (Pierce et al ., 2004). No data have been published on the effects of K. brevis on clearance rates in eastern oysters. Mussels Following a bloom of Gyrodinium (=Gymnodinium) aureolum off

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16 Plymouth, England in 1978, Widdows et al (1979) examined the effects of this dinoflagellate on the physiological and cytological responses of the mussel Mytilus edulis in the laboratory. They showed that the dinoflagellate either produced or contained a substance which was cytotoxic to M edulis It had an acute effect on the clearance rate and caused marked cellular damage to the gut; however, the mussels were capable of rapid recovery when G. aureolum cell concentrations declined. Shumway and Cucci (1987) examined the effects of the toxic dinoflagellate Protogonyaulax tamerensis (= Alexandrium tamarense ) on feeding in several species of commercially important bivalve mollusc. Each species was fed a mixture of nontoxic algae and clearance rates were calculated. The same individuals were purged in filtered seawater overnight and fed an identical algal mixture with the addition of A. tamerense (clone GT 429) the following day. The mussel, Mytilus edulis showed relatively little change in clearance rates when exposed to A. tamerense but this response seemed partly affected by the locality of each mussel population. Mussel populations from Maine showed no selection for or against the toxic dinoflagellates; A. tamerense was readily filtered and appeared in both the pseudofeces and feces. Mussels from other populations showed varying behavioral responses to the presence of A. tamerense (summarized in the previous section), suggesting that feeding rates in these populations were negatively affected by this particular dinoflagellate culture. Clearance rates of juvenile Mytilus edulis fed unialgal cultures of either Alexandrium tamarense or Gyrodinium aureolum were significantly lower than clearance rates on a unialgal diet of Isochrysis sp. (Lesser and Shumway, 1993). Results were similar in experiments conducted during the winter (5o C) and spring (10o C).

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17 In Japan, the clearance rate of the mussel Mytilus galloprovincialis was significantly reduced when exposed to Heterocapsa circularisquama even at a dinoflagellate cell density of only 50 cells ml-1 (Matsuyama et al ., 1997). The reduction in clearance rate did not seem to be caused by size, density, or shape of H. circularisquama cells since no inhibitory effect was observed when mussels were exposed to other morphologically similar dinoflagellates. Repression of clearance rate was not observed when M. galloprovincialis were exposed to a filtrate of H. circularisquama culture or cultures where the cell walls had been removed by centrifugation, indicating that the source of toxicity of H. circularisquama is localized on the cell surface (Matsuyama et al ., 1997). Li and Wang (2001) employed a radiotracer technique to determine the selective feeding behavior of the mussel Perna viridis and the clam Ruditapes philippinarum on an algal mixture containing both toxic and nontoxic Alexandrium tamarense Both bivalves had similar clearance and ingestion rates between the two A. tamarense cultures. No selective ingestion of either algal preparation was observed, indicating that the two bivalves were unable to distinguish the par ticles based on their toxicity. These findings support earlier reports on Mytilus californianus which was observed to ingest both toxic and nontoxic dinoflagellates in natural sea water (Buley, 1936; Fox and Coe, 1943). Scallops Shumway and Cembella (1993) provide a comprehensive review of the impact of toxic algae on scallop culture and fisheries. Less is known about the effects of toxic algae on scallop feeding physiology, particularly clearance and filtration rates, compared to other bivalve molluscs. Li and Wang (2000) suggested that high PSP toxin levels in the scallop Chlamys

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18 nobilis were partly due to its relatively high clearance rate of Alexandrium tamarense Feeding behavior in the king scallop ( Pecten maximus ), after switching from a diet of A. tamarense to a nontoxic diet, changed drastically depending on the algal species used to detoxify the scallops (Bougrier et al ., 2000). A diet based on Tetraselmis suecica appeared to stimulate clearance and filtration rates, whereas one based on Isochrysis galbana had the opposite effect. The sea scallop, Placopecten magellanicus continued to feed normally when exposed to toxic Alexandrium tamarense (Shumway and Cucci, 1987). However, most exhibited striking behavioral responses including violent swimming activity. While this increased activity may provide temporary escape from predators, it does not provide protection from prolonged toxic algal blooms. The associated increase in heart rate, stroke volume and other physiological stresses may actually prove to be detrimental to P. magellanicus Thompson et al ., 1980). Feeding rates for P. magellanicus on A. tamarense and Gyrodinium (= Gymnodinium ) aureolum were significantly different from one another, but still significantly lower than feeding rates on Isochrysis sp. (Lesser and Shumway, 1993).

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19 Juvenile and adult bay scallops ( Argopecten irradians ) did not exhibit feeding inhibition during a two week exposure to the toxic epibenthic dinoflagellate Prorocentrum lima a known producer of diarrhetic shellfish poisoning (DSP) (Bauder et al ., 2001). Clearance rates were similar for bay scallops exposed to equivalent biovolume cell concentrations of P. lima and the non-toxic diatom Thalassiosira weissflogii In contrast, clearance rates of A. irradians fed two species of toxic dinoflagellate ( Alexandrium tamarense and Gyrodinium aureolum ) were significantly higher than bay scallops fed Isochrysis sp. (Lesser and Shumway, 1993). No information is available on the effects of Karenia brevis on feeding behavior in bay scallops (Shumway, personal communication). Clams Northern quahogs ( Mercenaria mercenaria ) retracted their siphons and completely closed their shell valves when exposed to Alexandrium tamarense (Shumway et al ., 1985). The valves remained closed and clams did not feed until the addition of clean seawater. In a separate study, however, M. mercenaria was shown to clear A. tamarense at very low clearance rates during spring and even less so during winter (Lesser and Shumway, 1993). When exposed to a diet composed solely of A. fundyense, M. mercenaria closed their shells and did not resume pumping until a low density of Thallasiosira weissflogii was added (Bricelj et al 1990). Quahogs ingested T. weissflogii and A. fundyense in the same proportion as offered in the algal suspension, thus exhibiting no ingestion selectivity. Quahogs ingested up to 3.4 x 105 toxic cells g-1 day-1 (Bricelj et al 1990). From these studies it appears that a supply of nontoxic algae is necessary to entice northern quahogs to extend their siphons and commence feeding in the presence of certain species of toxic dinoflagellate. The softshell clam, Mya arenaria filtered 7.6 x 103 cells h-1 g dry wt-1 in the

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20 presence of Gonyaulax (= Alexandrium ) tamarense a 47% reduction compared to the same individuals fed the same diet without the toxic dinoflagellate (Shumway and Cucci, 1987). Mya arenaria were not able to clear nontoxic algal cells in the presence of A tamarense During a 1973-74 Karenia brevis bloom in Sarasota, FL, shellfish suspected of being contaminated were processed and analyzed for the presence of toxins (Hemmert, 1975). The surfclam ( Spisula solidissima raveneli ) had a toxicity of 75 MU and the southern hard clam ( Mercenaria campechaenis ) had a toxicity of 96 118 MU, indicating their ability to filter and ingest K. brevis and to accumulate brevetoxins. Cytotoxic Effects of Harmful Algal Species to Bivalves While the role of harmful algal blooms is well documented in human shellfish poisonings and aquatic organism mass mortality events, there is far less information concerning chronic, lethal or sublethal effects on shellfish caused by bioaccumulated or biomagnified algal toxins. This paucity of information also concerns whether such harmful algal blooms render shellfish susceptible to disease (Landsberg, 1996). One of the earlier studies demonstrating the cytotoxic impacts of harmful algae on bivalves was conducted by Widdows et al (1979) who observed cellular damage to the gut of adult Mytilus edulis after a short (< 24 h) exposure to bloom conditions of Gyrodinium (=Gymnodinium) aureolum More comprehensive studies have centered on the pathological effects of bivalve exposure to the toxic dinoflagellate, Prorocentrum minimum (Bardouil et al ., 1993;

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21 Luckenbach et al ., 1993; Wikfors and Smolowitz, 1993; Wikfors and Smolowitz, 1995). Wikfors and Smolowitz (1993) showed that P. minimum when offered as a food source in combination with Isochrysis sp., was poorly ingested by juvenile bay scallops ( A. irradians ) and resulted in 100% mortality after a four-week exposure. After one week, there were appreciable differences in tissues between scallops feeding on Isochrysis sp. and those fed a mixed Isochrysis/P. minimun diet. Affected scallops had poorly developed digestive diverticula, severe attenuation of epithelial cells associated with absorptive-cell necrosis and sloughing of cells into central lumens. Residual cells were more numerous. Large melanized hemocyte clots were present in the open vascular system of the mantle, digestive diverticula, heart, gill, and kidney tissues. Collectively, these findings suggested that P. minimum produced an enterotoxin that gradually affected absorptive cells (Wikfors and Smolowitz, 1993). Juvenile hard clams ( Mercenaria mercenaria ), on the other hand, ingested both P. minimum and the congener P. micans under identical conditions and survived well under all feeding conditions. No histological examinations of hard clam tissues were made (Wikfors and Smolowitz, 1993). Luckenbach et al (1993) observed that juvenile eastern oysters ( C. virginica ) exposed to 100% P. minimum bloom density (8.9 x 103 to 2.5 x 105 cells ml-1)died within 14 days, and 43% exposed to 33% bloom density died within 22 days, but oysters exposed to 5% bloom density had good shell growth and no mortality. Wikfors and Smolowitz (1995) further examined the histopathology of embryos, larvae, spat and juveniles of the eastern oyster ( C. virginica ) fed different diet combinations of P. minimum and Isochrysis sp. Feeding larvae showed poor growth and poor development

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22 of the digestive system on a P. minimum diet only. Growth of oyster larvae fed a 1:1 P. minimum/Isochrysis diet was intermediate, while a 1:2 P. minimum/Isochrysis diet resulted in distinctive changes in the anatomy of the digestive system (Wikfors and Smolowitz, 1995). Oyster spat fed a 1:2 P. minimum/Isochrysis diet showed an abnormal accumulation of lipid in the stomach epithelium. Absorptive cells in the digestive glands of both larvae and spat contained accumulation bodies, often with a laminated, fibrous appearance. Accumulation bodies were periodic acid-Schiff (PAS) positive and may have corresponded to autolysosomal bodies within P. minimum cells. The linkage of accumulation bodies within absorptive cells of oyster digestive diverticula and dinoflagellate autolysosomal bodies suggests a mechanism by which some dinoflagellates interfere with feeding in phytoplankton grazers (Wikfors and Smolowitz, 1995). Flat oysters ( Ostrea rivularis ), which had died following a Prorocentrum bloom in southern China (Yomgjia et al 1995), exhibited a pathology consistent with a systemic toxicosis resulting from the absorption of toxins by the digestive gland (Landsberg, 1996). The most intense lesion was formed by hemocytes that accumulated in and around the hemolymph channels, infiltrated the walls of the blood sinus, and formed intravascular thrombi. Interestingly, this pathology was similar to that found in C virginica by Wikfors and Smolowitz (1993). These studies suggest that Prorocentrum spp. may induce pathological effects in the hematopoietic system of oysters (Landsberg, 1996). Finally, spat of the Pacific oyster ( C. gigas ) showed abnormal histopathologies in the gut and gill when exposed for 21 days to a diet of cultured Prorocentrum rhathymum at 1.2 x 104 cells L-1 (Pearce et al ., 2005). Other toxic algae have caused histopathologies in bivalves. Elevated lysosomal

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23 destabilization rates, which indicate damage to the hepatopancreas, have been reported in eastern oysters ( C. virginica ) exposed to algal blooms dominated by the raphidophyte Heterosigma akashiwo (Keppler et al ., 2005) as well as the dinophyte, Kryptoperidinium foliaceum (Lewitus et al ., 2003). Smolowitz and Shumway (1997) examined gut tissues from juveniles of eight species of bivalve that had been exposed to Gyrodinium aureolum and found the impact to be species-specific. The eastern oyster ( Crassostrea virginica ) and the bay scallop ( Argopecten irradians ) were most severely affected. Several C virginica showed mantle and gill lesions. Bay scallops exhibited decreased height of absorptive cells and increased lumen diameter after exposure, suggesting that G. aureolum was of poor food quality. Evidence of toxic effects was not identified in the digestive gland. Several bay scallops also showed variable amounts of inflammation in the kidney associated with protozoal infestations and variable amounts of predominately rod-shaped bacteria within the urinary space. Another pectinid, the king scallop ( Pecten maximus ) developed obvious STX neoformation in kidneys after exposure to PSP toxins (Bougrier et al ., 2000). Pectinids are apparently unaffected by disseminated neoplasia (Landsberg, 1996), and only one case of germinoma has been reported in bay scallops (Peters et al ., 1996). Brevetoxins produced by Karenia brevis and which are responsible for neurotoxic shellfish poisoning (NSP) in the Gulf of Mexic o, have not yet been shown to play a role in the development of neoplasia in bivalves (Landsberg, 1996). Although brevetoxins are well known for their role in fish kills (Steidinger et al ., 1973; see Landsberg, 2002 for review), their effect in developing hist opathologies in bivalve molluscs is unknown.

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24 Chapter Three Larval Studies Introduction Recurring and persistent harmful algal blooms have raised increasing concerns about the long-term effects on local fisher ies, including critical species of bivalve shellfish (Shumway and Cucci, 1987; Shumway, 1990; Landsberg, 1996). The toxic dinoflagellate, Karenia brevis causes periodic and extensive red tides along the southcentral Gulf coast of Florida (Steidinger et al ., 1995) and produces potent neurotoxins (=brevetoxins). These brevetoxins are lethal to fish and cause neurotoxic shellfish poisoning (NSP) in humans from the consumption of contaminated shellfish (Baden, 1988; Steidinger et al ., 1998). At the same time, K brevis blooms pose a potential threat to Floridas shellfish resources and growing bivalve aquaculture industry (Blake et al ., 2000; Adams and Sturmer, 2004). Red tides usually occur in the late summer and fall along the Florida west coast (Kirkpatrick et al ., 2004) at a time when native shellfish species are spawning (Barber and Blake, 1983; Hesselman et al ., 1989). Shellfish populations could thus be exposed to K. brevis blooms at a critical stage in their life history, and the relative success or failure of recruitment could depend on whether there are detrimental effects of exposure to K. brevis on the developmental stages of affected

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25 shellfish species. Therefore, the effects of the NSP-producing alga, K. brevis on survival and development of larvae were examined for three species of bivalve molluscs: the northern quahog (= hard clams) ( Mercenaria mercenaria ), the bay scallop ( Argopecten irradians ) and the Eastern oyster ( Crassostrea virginica ). Experiments were designed to specifically investigate whether differences in survival were due to the dinoflagellate itself or its constituent toxins. Materials and Methods Collection and Maintenance of Bivalves All adult bivalves used for broodstock were collected from the Florida Gulf Coast. Bay scallops were collected from the Anclote estuary while northern quahogs were collected from Tampa Bay. Oyster larvae were provided by John Supan, manager of the Grand Isle (Louisiana) Bivalve Hatchery. Adults were transferred to one of the following facilities for maturation and spawning: University of South Florida, St. Petersburg (bay scallops); Bay Shellfish Co., Palmetto, FL (northern quahogs). Larvae were transferred to Mote Marine Laborator y, Sarasota, FL immediately after spawning for subsequent larval studies. Larvae were maintained in 2L flasks at a density of five larvae ml-1 under gentle aeration and fed Isochrysis galbana daily at a density of 10,000 cells ml-1 prior to experimentation.

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26 Maintenance of Algal Cultures Batch cultures of Karenia brevis (= Gymnodinium breve ) (Wilson clone) were grown in NH15 media without aeration. Cultures of Isochrysis galbana (Tahitian clone) were grown in f/2 media plus Trimsa minus silica with aeration. Seawater was collected locally, filtered through cartridge filters to remove particles > 0.2 m, passed through an activated charcoal filter, sterilized with ultraviolet light and autoclaved. Cultures were maintained at a temperature of 24 to 26o C and a salinity of 33 to 35 ppt (parts per thousand). Lighting was provided by a combin ation of Cool-white and Gro-lite bulbs. Cultures of K. brevis were maintained on a 12:12 hour light: dark cycle, while I. galbana cultures were exposed to constant illumination. All experiments used cultures in stationary growth phase, generally achieved two weeks after inoculation. Preparation of Lysed Culture Larval experiments were conducted using both whole and lysed culture preparations of Karenia brevis Lysed preparations were produced by exposing a sample of K. brevis culture to ultrasonic disruption at 750 W for four minutes using a Sonics Vibracell with 5 mm microtip probe. A small subsample ( < 1 ml) was observed microscopically to verify that the cells had been disintegrated. A 500 ml sample of lysed culture was then subjected to brevetoxin analysis by the same procedure as whole culture samples.

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27 Determination of Cell and Brevetoxin Concentrations Cell concentration for each algal culture was determined prior to each experiment employing a Coulter Multisizer IIE fitted with a 100 m orifice. The number of particles in a 500 l sample of culture was ascertained for Isochrysis galbana (size range: 3.4 8.1 m) and Karenia brevis (size range: 14.2 30.0 m). Three replicate counts were made and the mean value determined. Cell concentrations were verified by microscopic enumeration using a counting chamber (Hausser Scientific Company). Brevetoxin concentration of each culture of K. brevis was determined by high performance liquid chromotography (HPLC). Brevetoxins were extracted by passing a known volume ( 500 ml) of culture through a C-18 extraction disc placed inside a Teflon filtering apparatus under vacuum (20 psi). The C-18 disc was eluted with methanol to recover the brevetoxins and placed in a flask. The flask was then placed on a Labonco rotary evaporator and reduced to dryness. Methanol was added to produce a final volume of 3 ml and the sample injected into a Shimadzu LC-600 HPLC with a Shimadzu SPD-M6A photodiode array UV-VIS detector. Total brevetoxin concentrations were quantified using a C-18 column and an 85:15 methanol: water (1 ml/min) isocratic elution at 215 nm according to the procedure of Pierce et al (2005). Three-Day Static Exposure The first series of experiments consisted of exposing larvae to Karenia brevis for

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28 three days. Bivalve species used for larval studies were bay scallops ( Argopecten irradians ), northern quahogs ( Mercenaria mercenaria ) and eastern oysters ( Crassostrea virginica ). Each experiment employed three concentrations of both whole and lysed culture of K. brevis and a control. K. brevis concentrations were 10, 100 and 1,000 cells ml-1. Each treatment consisted of five replicates. Larvae were three days old at the start of the experiment. For each replicate, approximately 500 larvae were transferred to a petri dish containing 100 ml filtered seawater. Each dish was supplied with 1 x 104 cells ml-1 of Isochrysis galbana K. brevis (whole or lysed culture) was added to each treatment to obtain the desired exposure concentration. After inoculation with K. brevis a small subsample (0.5 ml) was withdrawn and algal cells counted with a hemocytometer under a compound microscope to verify that the desired concentration was obtained. Each dish was covered and left undisturbed. Dishes were neither stirred nor aerated. Each day, the contents of each dish were poured through a 35 m sieve and gently rinsed with filtered seawater. Larvae were transferred to a 100 ml graduated cylinder containing 50 ml of filtered seawater and brought to the appropriate volume prior to reinoculation. Dishes were reinoculated with K. brevis and I. galbana covered, and left undisturbed for 24 hours. The experiment was terminated after seventy-two hours and the larvae preserved in 2% buffered formalin. Straight-hinged larvae which have been dead for as little as one hour have few internal details visible through the transparent shell and are easily separated from larvae preserved while living. Larvae were considered dead if either empty shells or shells with decomposing tissues were found. Larvae were considered

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29 live if tissues were discernable and differentiated under magnification. Developmental stage for each live and dead larva was determined. A two-way analysis of variance with repeated measures was performed for each experiment to determine significant differences in mortality among cell concentration and culture treatment. Seven-Day Static Exposure Seven-day-old larvae were used in this series of experiments to investigate the effects of K. brevis on mortality, development and metamorphosis of each bivalve species. Each experiment consisted of four concentrations of whole K. brevis culture and a control. Each treatment consisted of five replicates. The four K. brevis concentrations were: 10, 100, 1,000, and 5,000 cells ml-1. In each case, approximately 500 larvae were transferred to a 250 ml glass finger bowl (density = 5 larvae ml-1). Each bowl was supplied with 2 x 104 cells ml-1 of Isochrysis galbana as a food source. Whole culture of K. brevis was added to each bowl to obtain the desired exposure concentration. A small subsample (0.5 ml) was withdrawn after inoculation and cells counted with a hemocytometer under a compound microscope to confirm that the desired concentration was achieved. All bowls were covered and le ft undisturbed. Bowls were neither stirred nor aerated. Each day, the contents of each bowl were poured through a 50 m sieve and gently rinsed with filtered seawater. Larvae were transferred to a 250 ml graduated cylinder containing 200 ml of filtered seawater and brought to the appropriate volume prior to reinoculation. Bowls were reinoculated with K. brevis and I. galbana covered,

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30 and left undisturbed for 24 hours. The experiment was terminated after seven days and the larvae preserved in 2% buffered formalin. The number of live and dead larvae (determined by marked morphological disintegration) was determined for each developmental stage. Developmental stage depended upon the bivalve species and included straight-hinged veliger, umbonal veliger, pediveliger, and spat (Sastry, 1965 for A. irradians ; Carriker, 2001 for M. mercenaria ; Waller, 1981 for C. virginica ). Statistical Analyses The square root of the proportion of live and dead larvae was arcsine transformed to satisfy the assumption of normality when dealing with percentages (Zar, 1996). Differences in mortality among cell concentration and culture treatments in the threeday-exposure experiments were determined by two-way ANOVA with repeated measures. Significant differences among treatments were analyzed using Tukeys multiple comparison test. A single factor ANOVA was performed to determine significant differences in mortality among cell concentrations in the seven-day-exposure experiments.

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31 Results Toxin Profile of Karenia brevis Cultures Toxin profiles of K. brevis cultures used in experiments are summarized in Table 1. Two brevetoxins and one antagonist were present in each culture: PbTx-2, PbTx-3, and brevenal, a recently identified brevetoxin antagonist (Bourdelais et al ., 2004). Cell density, brevetoxin composition and total toxin concentration were similar among cultures used for experiments for each shellfish species. Total toxin concentration was higher after cultures had been lysed. Three-Day Static Exposure Survival of bay scallop larvae exposed to K. brevis was > 90% in all treatments and > 80% of surviving larvae reached the umboveliger stage (Table 2). Survival was significantly lower (p < 0.001) at the highest K. brevis concentration in both whole and lysed treatments (Table 5). There was no treatment effect on survival of A. irradians larvae. Survival of M. mercenaria larvae was > 88% in all treatments (Table 3). Most larvae survived to the umboveliger stage. Percent survival was significantly lower (p < 0.001) in the Lysed-1,000 cells ml-1 treatment (Table 5). There was a significant (p < 0.05) concentration and culture effect on larval survival.

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32 Larval survival for C. virginica ranged from 94 to 75%, with < 1% reaching the umboveliger stage (Table 4). Survival decreased with increasing K. brevis concentration; survival was lower in lysed treatments than whole treatments at the same K. brevis concentration. There was a significant (p < 0.05) concentration and culture effect on larval survival (Table 5). Seven-Day Static Exposure Exposure of A. irradians larvae to K. brevis concentrations up to 1,000 cells ml-1 for seven days did not greatly impact survival (Fig. 1). When the quantity was increased to 5,000 cells ml-1, survival declined to 37%. All larvae had reached the pediveliger stage at the termination of the experiment. Survival of M. mercenaria larvae gradually decreased with increasing K. brevis concentration up to 1,000 cells ml-1 (Fig. 2). Larval survival at 5,000 cells ml-1 declined to 26%. A higher percent of larvae from the low dose treatments developed into pediveligers than larvae from higher dose treatments (Fig. 3) Total survival of C. virginica larvae was 88% in the control, 75% in K. brevis concentrations up to 1,000 cells ml-1, and only 19% at 5,000 cells ml-1 (Fig 4). Larval development was similar among exposure doses up to 1,000 cells ml-1 (Fig. 5). Approximately 46% were umboveligers after seven days; 42% developed into pediveligers and 11% had settled as spat. At 5,000 cells ml-1, 67% of surviving larvae were still umboveligers, 32% were pediveligers, and only 1% had settled as spat. Table 6 summarizes the results from one-way ANOVA and Tukeys Multiple

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33 Comparison Test for each species. All three species showed a significant difference in mortality at K. brevis concentrations of 5,000 cells ml-1. Only M. mercenaria showed a significant difference in mortality at K. brevis concentrations of 1,000 cells ml-1. Discussion Studies on the interaction between toxic dinoflagellates and bivalves have focused primarily on juvenile and adult life stages (Shumway, 1990; Bricelj and Shumway, 1998; Landsberg, 1996). Not until more recently has attention begun to focus on the effects of harmful algae on bivalve larvae (Wikfors and Smolowitz, 1995; Matsuyama et al ., 2001; Yan et al ., 2001; Yan et al ., 2003; Jeong et al ., 2004). Bivalve larvae, with their planktonic existence and small size, can be expected to respond in unique ways (compared to their post-larval counterparts) when exposed to harmful algal blooms. In all three species of bivalve ( A. irradians M. mercenaria and C. virginica ), survival of three-day-old larvae in the presence of K. brevis was concentrationdependent. At densities of 100 cells ml-1 or less, survival was generally over 85% and not affected by treatment preparation; i. e., whole or lysed culture. At 1,000 cells ml-1, survival was significantly less in lysed treatments for both M. mercenaria and C. virginica Seven-day-old larvae showed a similar survival response after exposure to K. brevis for seven days. A. irradians and C virginica survival was not significantly reduced at K. brevis concentrations up to 1,000 cells/ml, while survival of M mercenaria larvae was significantly lower at 1,000 cells ml-1. Survival in all three species was significantly reduced at 5,000 cells ml-1. Matsuyama et al (2001) reported lethal effects

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34 of Alexandrium tamarense A taylori Gymnodinium mikimotoi and Heterocapsa circularisquama on larvae of the Pacific oyster, C. gigas at cell densities of 100-1,000 cells ml-1. The process by which K. brevis affects larval survival is not clear, but several possible mechanisms may be involved. Direct cell-to-cell contact with microalgae, either through exposure to toxins present on the cell surface or through mechanical damage to sensitive organs, particularly gills, may negatively affect bivalve larvae (Gallager et al ., 1989; see Landsberg, 2002 for review). Mortality of C. virginica larvae in the presence of the dinoflagellate Cochlodinium heterolobatum was thought to be a result of increased physical contact between larvae and algal cells (Ho and Zubkoff, 1979). Contact with toxic algal cells may also release an unknown inhibitory factor which could negatively affect survival (Yan et al ., 2001). Ultrasonic disruption (=lysing) produces cellular fragments as well as releasing intracellular toxins to the environment, thus making them available for encounters with bivalve larvae. Consumption (or ingestion) of toxic algal cells by bivalve larvae is dependent on a variety of factors, including algal species, cell size and concentration, and larval species and age. Consumption of K. brevis cells may also explain the observed inhibitory effects on larval survival. Larvae of the mussel, Mytilus galloprovincialis readily ingested cells of several species of toxic dinoflagellates w ith mean equivalent spherical diameters of 12-38 m (Jeong et al ., 2004). Eastern oyster ( C. virginica ) larvae ingested P. minimum cells, although algal filtration was depressed in the presence of this toxic algae (Jeong et al ., 2004), and ingestion of this toxic alga resulted in cytological changes in digestive tissues, including the deleterious development of cuboidal and squamous epithelial cells

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35 in the stomach and intestine, reductions in the size of absorptive cells, and the presence of dense inclusions in the cytoplasm, indicating possible phagolytic reactions to dinoflagellate debris (Wikfors and Smolowitz, 1995). Early D-shape larvae of two scallop species ( Argopecten irradians concentricus and Chlamys farreri ) were unable to feed on Alexandrium tamarense cells due to its relatively large size (Yan et al ., 2001; Yan et al ., 2003). During the current study, larvae were fed an optimal ration (Lu and Blake, 1996) of the chrysophyte, I. galbana a common alga used in bivalve culture, in addition to K. brevis Although larval feeding rates were not measured nor K. brevis consumption investigated, ingestion of K. brevis cells was most likely negligible due to the relatively large cell size (ESD = 14-26 m) and low density compared to I. galbana However, the presence of K. brevis especially at higher concentrations, could interfere with bivalve larvae by altering activity patterns (Yan et al ., 2003) and/or feeding rates (Jeong et al ., 2004), resulting in increased mortality and retarded metamorphosis (Matsuyama et al ., 2001). Exposure of seven-day-old larvae to K. brevis had an effect on survival, development and metamorphosis. Even though overall survival was identical in C. virginica larvae exposed to 100 and 1,000 cells ml-1, a higher proportion from 100 cells ml-1 had a) reached the pediveliger stage and b) completed larval development (i.e., settled as spat) than larvae from 1,000 cells ml-1. Almost ninety percent of larvae subjected to 5,000 cells ml-1 did not live beyond the umboveliger stage. Larval development of M. mercenaria was also affected by the presence of K. brevis cells. In this case, progress to the pediveliger stage was inversely related to K. brevis concentration. Similarly, larvae of the Pacific oyster, C. gigas which did not show

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36 significant mortality when exposed to Cochlodinium polykrikoides did suffer retarded metamorphosis to the D-shaped larvae (Matsuyama et al ., 2001). Development of C. virginica larvae was also delayed when exposed to a laboratory clone of the dinoflagellate, P. minimum (Wikfors and Smolowitz, 1995). While the mechanism for increased mortality of bivalve larvae remains unanswered, it is easy to see how the added stress associated with K. brevis and/or its toxins could be reflected in suboptimum development. Sixty percent of brevetoxins in laboratory cultures of K. brevis are extracellular in nature (Pierce et al ., 2001). Ultrasonic disruption, which releases the remaining intracellular toxins, resulted in a 20-24% increase in total brevetoxin in the current study. Two brevetoxins and one antagonist were present in each culture: PbTx-2, PbTx-3, and brevenal, a recently identified brevetoxin antagonist (Bourdelais et al ., 2004). The proportion of each brevetoxin remained unchanged after the cultures were lysed. Except for the absence of PbTx-1, the relative brevetoxin composition of laboratory cultures closely resembled that from water samples collected during a red tide outbreak along the Gulf Coast of Sarasota, FL in 2003 (Pierce et al ., 2005). Larvae of all three bivalve species in this study responded similarly, but with different sensitivities, to cells of K. brevis and its suite of toxins. Mortality was not necessarily dependent on ingestion of algal cells; rather it appears that the toxins were at least partially responsible for increased mortality and delayed larval development. The presence of K. brevis cells at high densities may interfere with larval feeding processes, resulting in suboptimal clearance, inhibited growth and development, and mortality. Blooms of K. brevis may persist in coastal waters for many months (Steidinger et

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37 al ., 1995). Our results clearly indicate that when these blooms and their toxins persist, shellfish larvae are at greater risk of mortality and may continue to be adversely affected even after the disappearance of K. brevis cells. While K. brevis blooms may not directly cause mortality in adult shellfish, they do have the ability to disrupt a critical phase in the life cycle and consequently have important ramifications for recruitment and population stability. The failure of bay scallops to successfully recruit in North Carolina, USA, was attributed to a bloom of Ptychodiscus brevis (= K. brevis ), which interfered with either adult spawning, larval survival and settlement, or survival of newly settled spat (Summerson and Peterson, 1990). Since we demonstrated negative impacts of K. brevis on larvae of northern quahogs (= hard clams) and eastern oysters, we might expect blooms of K. brevis to negatively impact recruitment in these species as well. Thus, there is a clear need for continued research on the relationship between K. brevis and bivalve larvae, ranging from the mechanisms of toxicity to the effects on recruitment and population stability.

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38 Table 1 Cell density, sample matrix and brevetoxin composition of Karenia brevis (Wilson clone) cultures used in larval experiments for each species. (Brevenal is considered a brevetoxin antagonist). K. brevis Culture Brevetoxin Amount (ug L-1) Species (Cells/ml) Matrix PbTx-2 PbTx-3 TOTALBrevenal Whole 20.03 5.07 25.10 30.23 Bay scallop ( Argopecten irradians ) 12,000 Lysed 26.31 8.11 34.42 34.47 Whole 23.36 2.46 25.82 29.17 Northern quahog ( Mercenaria mercenaria) 12,800 Lysed 32.33 2.42 35.32 35.32 Whole 22.19 4.10 26.29 24.93 Eastern oyster ( Crassostrea virginica ) 10,000 Lysed 32.94 0.97 33.91 33.90

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39 Table 2 Mean ( + SD) number of live and dead larvae, larval stage and percent survival for Argopecten irradians after exposure for three days to Karenia brevis Treatments consisted of whole and lysed cultures of K. brevis at three concentrations: 10, 100 and 1,000 cells ml-1. (n = 5). Straight-Hinged Veliger Umboveliger Larvae Survival (%) Treatment Live Dead Live Dead Total Total Umboveliger Control 41.0 (24.7) 3.6 (3.0)395.8 (125.6)4.0 (3.1)444.4 (128.3)98.3 (0.8)89.1 (5.9) Whole-10 45.8 (19.4) 7.2 (2.9)410.8 (140.9)3.2 (2.0)467.0 (125.0)97.8 (1.4)88.0 (7.8) Lysed-10 47.6 (7.1) 3.6 (3.6)350.0 (70.8)3.6 (0.9)404.8 (74.9)98.2 (0.6)86.5 (2.5) Whole-100 61.0 (33.8) 4.6 (2.3)475.6 (92.5)1.2 (1.1)542.4 (115.5)98.9 (0.2)87.7 (4.3) Lysed-100 57.6 (5.5) 10.2 (2.8)384.2 (95.9)1.8 (2.7)453.8 (101.5)97.4 (0.7)84.7 (2.4) Whole-1,000 39.4 (16.4) 22.8 (10.4)430.0 (76.4)3.8 (2.8)496.0 (77.9)94.6 (2.3)86.7 (4.5) Lysed-1,000 63.2 (33.6) 21.8 (11.6)402.2 (123.0)5.4 (2.3)492.6 (111.9)94.5 (2.5)81.6 (10.2)

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40 Table 3 Mean ( + SD) number of live and dead larvae, larval stage and percent survival for Mercenaria mercenaria after exposure for three days to Karenia brevis Treatments consisted of whole and lysed cultures of K. brevis at three concentrations: 10, 100 and 1,000 cells ml-1. (n = 5). Straight-Hinged Veliger Umboveliger Larvae Survival (%) Treatment Live Dead Live Dead Total Total Umboveliger Control 8.4 (2.0) 4.0 (2.9)448.4 (41.6)46.2 (17.0)507.0 (53.8)90.1 (2.1)88.4 (0.7) Whole-10 7.0 (4.2) 3.2 (2.3)474.4 (90.0)36.4 (6.9)521.0 (91.5)92.4 (1.3)91.1 (0.5) Lysed-10 4.8 (3.7) 0.4 (0.6)428.4 (138.9)48.8 (10.7)482.4 (150.9)89.8 (2.0)88.8 (0.5) Whole-100 5.2 (2.7) 1.0 (1.0)454.0 (155.7)41.0 (14.9)501.2 (171.6)91.6 (0.8)90.6 (0.5) Lysed-100 5.4 (2.7) 3.4 (1.3)463.8 (158.9)47.2 (19.0)519.8 (177.9)90.3 (1.0)89.2 (0.8) Whole-1,000 10.6 (4.0) 4.6 (2.9)512.4 (129.5)49.4 (9.1)577.0 (142.6)90.6 (0.8)88.8 (0.6) Lysed-1,000 7.4 (2.0) 5.0 (4.0)539.6 (166.1)68.6 (21.8)620.6 (191.3)88.1 (0.9)86.9 (0.2)

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41 Table 4 Mean ( + SD) number of live and dead larvae, larval stage and percent survival for Crassostrea virginica after exposure for three days to Karenia brevis Treatments consisted of whole and lysed cultures of K. brevis at three concentrations: 10, 100 and 1,000 cells ml-1. (n = 5). Straight-Hinged Veliger Umboveliger Larvae Survival (%) Treatment Live Dead Live Dead Total Total Umboveliger Control 241.6 (56.7) 20.4 (6.9)1.6 (3.0)10.4 (9.2)274.0 (59.2)88.8 (3.1)0.6 (1.1) Whole-10 304.8 (74.1) 17.0 (7.9)1.2 (0.8)1.2 (1.1)324.2 (81.1)94.4 (1.8)0.4 (0.3) Lysed-10 259.4 (21.5) 15.8 (8.3)0.4 (0.5)0.2 (0.4)275.8 (27.1)94.2 (2.9)0.1 (0.2) Whole-100 299.0 (19.4) 42.0 (5.5)0.00.4 (0.5)341.4 (19.5)87.6 (1.8)0.0 Lysed-100 226.4 (27.0) 48.4 10.7)0.01.6 (1.1)276.4 (34.6)81.9 (2.3)0.0 Whole-1,000 252.6 (38.1) 46.4 (14.0)0.6 (1.3)1.4 (0.9)301.0 (43.5)84.1 (4.0)0.2 (0.4) Lysed-1,000 199.8 (16.8) 65.2 (9.5)0.00.8 (0.8)265.8 (12.4)75.2 (4.1)0.0

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42 Table 5 Effect of Karenia brevis concentration and culture preparation on percent surviv al in three-day-old bivalve larvae. A) Two-way ANOVA ( = 0.05). B) Tukeys ( ) multiple comparison test. Underlined treatme nts are not significantly different (p > 0.05). Argopecten irradians Source of Variation SS df MS F P-value F crit k = 7 q(alpha)=4.541 Concentration 0.06320.031523.200.00003.40 v = 28 Sy = 0.0161 Treatment 0.00210.00151.140.29714.26 0.05 0.0731 Interaction 0.00920.00453.280.05493.40 Within 0.033240.0014 L-1,000 W-1,000 L-100 W-10 Control L-10 W-100 Total 0.10629 Mercenaria mercenaria Source of Variation SS df MS F P-value F crit k = 7q(alpha)=4.464 Concentration 17.05328.52664.750.01833.40 v = 28 Sy = 0.6822 Treatment 34.810134.810119.390.00024.26 0.05 3.0455 Interaction 3.61421.80721.010.38043.40 Within 43.086241.7952 L-1,000 L-10 Control L-100 W-1,000W-100 W-10 Total 98.56329 Crassostrea virginica Source of Variation SS df MS F P-value F crit k = 7 q(alpha)=4.541 Concentration 0.28420.141869.350.00003.40 v = 28 Sy = 0.0205 Treatment 0.03110.030815.070.00074.26 a = 0.05 0.0929 Interaction 0.01620.00824.020.03123.40 Within 0.049240.0020 L-1,000 L-100 W-1,000W-100 Control L-10 W-10 Total 0.38029

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43 Karenia brevis (cells ml -1 ) Control1010010005000 Survival (%) 0 20 40 60 80 100 120 Figure 1. Percent survival (mean + SD) of Argopecten irradians larvae after exposure to Karenia brevis for seven days. Treatment with an asterisk was si gnificantly different (p < 0.05). Larvae were seven days old at start of experiment.*

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44 Karenia brevis (cells ml-1) Control1010010005000 Survival (%) 0 20 40 60 80 100 Figure 2. Percent survival (mean + SD) of Mercenaria mercenaria larvae after exposure to Karenia brevis for seven days. Treatments with the same letter were not significantly different (p < 0.05). Larvae were seven days old at start of experiment.a a a b c

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45 Karenia brevis (cells ml -1 ) Control1010010005000 Percent of Total Larvae 0 20 40 60 80 Figure 3. Percent of total Mercenaria mercenaria larvae that survived to the umboveliger ( ) and pediveliger ( ) stages after exposure to Karenia brevis for seven days. Larvae were seven-day-old umboveligers at beginning of experiment.

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46 Karenia brevis (cells ml -1 ) Control1010010005000 Survival (%) 0 20 40 60 80 100 120 Figure 4. Percent survival (mean + SD) of Crassostrea virginica larvae after exposure to Karenia brevis for seven days. Treatment with an asterisk was si gnificantly different (p < 0.05). Larvae were seven days old at start of experiment.*

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47 Karenia brevis (cells ml -1 ) Control1010010005000 Percent of Total Larvae 0 10 20 30 40 50 60 Figure 5. Percent of total Crassostrea virginica larvae that survived to the umboveliger ( ), pdiveliger ( and spat ( ) stages after exposure to Karenia brevis for seven days. Larvae were seven-day-old umboveligers at start of experiment.

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48 Table 6 Effect of Karenia brevis concentration and culture preparation on percent surviv al in seven-day-old shellfish larvae. A) Two-way ANOVA ( = 0.05). B) Tukeys ( ) multiple comparison test. Underlined treatments are not significantly different (p > 0.05). A) Two-Way ANOVA B) Tukeys Multiple Comparison Test Argopecten irradians k = 5 q(alpha)= 4.232 Source of Vari ation SS df MS F P-value F crit v = 20 Sy = 0.0567 Between Groups 1.8261 .4565 25.35E-082.87 a = 0.05 w = 0.2399 Within Groups 0.3213200.0161 Total 2.147524 5000 100010 100 Control Mercenaria mercenaria k = 5 q(alpha) = 4.232 Source of Variation SS df MS F P-value F crit v = 20 Sy = 0.0366 Between Groups 0.8554 0.2139 22.00E-082.87 a = 0.05 w = 0.1549 Within Groups 0.1340200.0067 Total 0.989424 5000 1000100 10 Control Crassostrea virginica k = 5 q(alpha) = 4.303 Source of Variation SS df MS F P-value F crit v = 17 Sy = 0.0679 Between Groups 1.61 0.40 .547.26652.96 a = 0.05 w = 0.2922 Within Groups 0.3920170.0231 5000 1000100 10 Control Total 2.009621

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49 Chapter Four Juvenile Studies Introduction The effects of diets that include toxic dinoflagellates on feeding in bivalve molluscs have received increased attention in the past twenty years (Shumway and Cucci, 1987; Gainey and Shumway, 1988; Bricelj et al ., 1996; Lassus et al ., 1996; Lassus et al ., 1999; Li and Wang, 2001; Lesser and Shumway, 1993; Bricelj and Shumway, 1998). The recurring conclusion is that bivalve responses are species-specific (Shumway and Cucci, 1987; Gainey and Shumway, 1988; Shumway, 1990; Lesser and Shumway, 1993; Smolowitz and Shumway, 1997), and depend upon a variety of factors, including the algal species encountered (Shumway and Cucci, 1987; Gainey and Shumway, 1988; Shumway, 1990; Lesser and Shumway, 1993), algal toxicity (Bricelj et al ., 1991; Bardouil et al ., 1993; Bricelj et al ., 1996; Lassus et al ., 1996; Li and Wang, 2001), algal concentration (Li et al ., 2002), cell size and selectivity (Shumway et al ., 1985; Shumway et al ., 1990; Lesser and Shumway, 1993; Matsuyama et al ., 1997), history of exposure (Shumway and Cucci, 1987; Chebib et al ., 1993; Bricelj et al ., 1996), season (Lesser and Shumway, 1993) and differences in digestive function (Wikfors and Smolowitz, 1993).

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50 Blooms of K. brevis may be especially harmful to bay scallops ( Argopecten irradians ) (Summerson and Peterson, 1990), and could jeopardize efforts to restore Floridas dwindling bay scallop populati ons (Geiger and Arnold, 2003; Leverone et al ., 2005) and the potential for a successful aquaculture program (Blake et al ., 2000). The burgeoning hard clam (= quahog) aquaculture industry in Florida (Adams and Sturmer, 2004) has many lease sites in Pine Island Sound (Lee County), an estuary with a history of repeated red tide outbreaks (Tester and Steidinger, 1997). The nonindigenous green mussel, Perna viridis became established in Tampa Bay in 1999 (Ingrao et al ., 2001), and has since spread south along the Florida Gulf Coast (Benson et al ., 2001), the same geographic area where blooms of K. brevis are most frequent (Tester and Steidinger, 1997). Lastly, restoration and creation of oyster habitats ( Crassostrea virginica ) is receiving increased attention within this same region (Savarese et al ., 2004). The effects of K brevis on oyster populations in Florida have not yet been examined. This study was undertaken to determine the effects of the toxic dinoflagellate, Karenia brevis on the clearance rate of juveniles of four species of common bivalve molluscs from Florida: the bay scallop ( Argopecten irradians ), northern quahog (= hard clam Mercenaria mercenaria ), eastern oyster ( Crassostrea virginica ) and green mussel ( Perna viridis ). Both short-term (one hour) and long-term (two day) effects were investigated. We also examined the effects of whole culture (intact cells) and lysed culture (disrupted cells) of K brevis on clearance rate to distinguish between the effects of the dinoflagellate and its toxins.

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51 Materials and Methods Collection and Maintenance of Juveniles Juveniles of four species of bivalve were used in these experiments: the bay scallop ( Argopecten irradians ), eastern oyster ( Crassostrea virginica ), northern quahog ( Mercenaria mercenaria ) and green mussel ( Perna viridis ). Bay scallops and northern quahogs were obtained directly from the hatchery. Eastern oysters and green mussels were collected from upper Tampa Bay, which had not experienced blooms of K brevis for decades. All bivalves were maintained in aerated aquaria at 25o C and fed Isochrysis galbana daily at a density of 2 x 104 cells ml-1 prior to experimentation. Maintenance of Algal Cultures Batch cultures of Karenia brevis (= Gymnodinium breve ) (Wilson clone) were grown in NH15 media without aeration. Cultures of Isochrysis galbana (Tahitian clone) were grown in f/2 media plus Trimsa minus silica with aeration. Seawater was collected locally, filtered through cartridge filters to remove particles > 0.2 m, passed through an activated charcoal filter, sterilized with ultraviolet light and autoclaved. Cultures were maintained at a temperature of 24 to 26o C and a salinity of 33 to 35 ppt (parts per thousand). Lighting was provided by a combin ation of Cool-white and Gro-lite bulbs. Cultures of K. brevis were maintained on a 12:12 hour light: dark cycle, while I. galbana cultures were exposed to constant illumination. All experiments used cultures in

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52 stationary growth phase, generally achieved two weeks after inoculation. Preparation of Lysed Culture Larval and juvenile bivalve experiments were conducted using both whole and lysed culture preparations of Karenia brevis Lysed preparations were produced by exposing a sample of K. brevis culture to ultrasonic disruption at 750 W for four minutes using a Sonics Vibracell with 5 mm microtip probe. A small subsample ( < 1 ml) was observed microscopically to verify that the cells had been disintegrated. A 500 ml sample of lysed culture was then subjected to brevetoxin analysis by the same procedure as whole culture samples. Determination of Cell and Brevetoxin Concentrations Cell concentration for each algal culture was determined prior to each experiment employing a Coulter Multisizer IIE fitted with a 100 m orifice. The number of particles in a 500 l sample of culture was ascertained for Isochrysis galbana (size range: 3.4 8.1 m) and Karenia brevis (size range: 14.2 30.0 m). Three replicate counts were made and the mean value was determined. Cell concentrations were verified by microscopic enumeration using a counting chamber (Hausser Scientific Company). Brevetoxin concentration of each culture of K. brevis was determined by high performance liquid chromotography (HPLC). Brevetoxins were extracted by passing a known volume ( 500 ml) of culture through a C-18 extraction disc placed inside a

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53 Teflon filtering apparatus under vacuum (20 psi). The C-18 disc was eluted with methanol to recover the brevetoxins and placed in a flask. The flask was then placed on a Labonco rotary evaporator and reduced to dryness. Methanol was added to produce a final volume of 3 ml and the sample injected into a Shimadzu LC-600 HPLC with a Shimadzu SPD-M6A photodiode array UV-VIS detector. Total brevetoxin concentrations were quantified using a C-18 column and an 85:15 methanol: water (1 ml min-1) isocratic elution at 215 nm according to the procedure of Pierce et al ., 2005). Clearance Rate Studies All feeding experiments were carried out at a temperature of 25o C and a salinity of 30 o/oo. Two separate sets of feeding experiments were conducted for each bivalve species. The first set consisted of short-term experiments under static conditions. Individuals were exposed to an initial set of conditions and left undisturbed for one hour. Feeding rates were calculated at the end of the hour. The second set consisted of longterm experiments under flow-through conditions. Individuals were subjected to experimental conditions that were continuously replenished over a 48 hour period. Feeding rates were calculated twice each day. Complete details for each set of experiments are provided below.

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54 Static Exposure Experiments Separate static feeding experiments were carried out for each bivalve species under identical conditions. Individuals were placed in separate beakers containing 500 ml filtered seawater and allowed to acclimate for one hour. Each beaker was lightly aerated. Experimental treatments included three concentrations and two culture preparations of K. brevis and a control (no K. brevis added). Each treatment consisted of five replicates. Cell densities of K. brevis were 10, 100, and 1,000 cells ml-1 and culture preparations included lysed and whole cultures. Isochrysis galbana a common food alga, was added to each beaker at an initial concentration of 2-4 x 104 cells ml-1. Beakers were left undisturbed and the reduction in cell concentration of both I. galbana and K. brevis was determined after one hour. At the end of each experiment, tissue dry weight (mg) was determined by drying soft tissues in preweighed aluminum pans at 103o C for 24 hours. Weight specific clearance rates were calculated as: CRdw (ml/hr) = (volume in ml) (ln C1 ln C2) / t, where C1 and C2 are the cell concentrations at the beginning and end of each time increment. Weight-specific filtration ra te (if no pseudofeces production was observed) was calculated as: FRdw (cells/l/hr) = CRdw (C1 + C2)/2, where C1 and C2 are the cell concentrations at the beginning and end of each time increment. A two-way analysis of variance with equal replication was performed to determine significant differences in weight-s pecific filtration and ingestion rates among cell concentrations and culture treatments. Multiple comparison analyses were

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55 performed using Tukeys procedure (designed to prevent Type I errors) and Dunnetts (designed to compare all means with a control). The amounts of brevetoxin to which individual bivalves were exposed were determined by multiplying the total toxin concentration of the culture (Table 10) by the amount (volume) of culture added to each treatment. In this way, the relationship between clearance rate and toxin exposure could be determined for whole and lysed cultures of K brevis for each species. Significant differences between regression lines (whole vs. lysed cultures) were tested by a simple F test (for both residual variance and slope). Flow-Through Exposure Experiments Continuous-flow feeding experiments were conducted using a test system developed by Singer et al (1990). This system operated by pumping the algal solution for each treatment through an enclosed 290-ml exposure vessel. There were eighteen separate vessels available for any given experiment. Each test vessel consisted of two halves which are sealed using a silicon O-ring and a full-circumference clamp. The top half had two ports. One port was used as the inflow line and the second port was available for feeding or treatment injection if required. When not in use, the second port was sealed with a Teflon-lined cap. The bottom half had a single port, which served as the outflow port. The treatment solution flowed through the inlet into the top half of the vessel, through a fritted glass disk (40-60 m pore size) which was permanently fastened within the lower half of the vessel and finally through the outflow port.

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56 Treatment solutions of K. brevis were prepared in 20L polypropylene carboys and allowed to mix by gentle aeration. Treatment solutions were introduced into each chamber using an ISMATIC MCP Pumpsystem Model 78002 multihead pump at a flow rate approximately two to three times the clearance rate of the control bivalves (i.e., the cell concentration in the outflow fell between 50 and 80% of the inflow concentration). All tubing used throughout the pump system was made of platinum-cured silicon. The outflow (waste) solution was treated with bleach before disposal. Each experiment consisted of two treatments (six replicate chambers), a control (five replicate chambers) and a blank (one chamber). Treatment concentrations were 100 and 1,000 cells ml-1 of K. brevis Isochrysis galbana a common food alga, was added to each beaker at an initial concentration of 2-4 x 104 cells ml-1. Since there were a limited number of chambers available for a given experiment, consecutive experiments were run for each bivalve species, the first using whole cultures of K. brevis and the second using lysed cultures. Each experiment ran for two days and feeding rates were calculated twice a day (9 AM and 5 PM). At the end of each experiment, tissue dry weight (mg) was determined. Weight-specific clearance rates were calculated as: CRdw (ml hr-1) = flow rate (ml hr-1) (Ci Co) / Ci, where Ci is the inflow concentration of I. galbana and Co is the outflow concentration from each experimental chamber. Weight-specific filtration rates were calculated as: FRdw (cells hr-1) = CRdw (Ci + Co)/2, where Ci and Co are the inflow and outflow I. galbana cell concentrations during each feeding rate determination. A single factor analysis of variance was performed to determine significant differences in weight-specific clearance and filtration rates among cell concentrations.

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57 Results Cell concentration of K. brevis cultures ranged from 2.1 2.2 x 104 cells ml-1 for static experiments and from 2.0 2.5 x 104 cells ml-1 for flow through experiments (Table 7). Static experiments (run simultaneously for each species) used the same culture while flow-through experiments (run consecutively for each species) required separate cultures. Total brevetoxin concentration ranged from 23.1 80.3 g L-1 for static experiments and 29.7 75.1 g L-1 for flow-through experiments. PbTx -2 and PbTx-3 were the most abundant brevetoxins in cultures of K. brevis for all experiments. PbTx-1, which was detected only cultures used in the static experiments, was present in concentrations < 8 g L-1. Brevenal, a putative inhibitor of brevetoxin action, was not identified prior to the flow-through experiments; however, it is possible, even likely, that it was present, yet undetected, in cultures of K. brevis used in the static experiments. Total brevetoxin was typically higher after a culture was lysed. Static Exposure Experiments Table 8 summarizes the decline in I. galbana for each bivalve species exposed to different concentrations and preparations of K brevis under static conditions. Table 9 summarizes filtration and clearance rates for each species. No pseudofeces production was observed for any species under any treatment condition. Results for each species are discussed separately.

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58 Bay scallops ( Argopecten irradians ) Mean dry weight for juvenile bay sca llops ranged from 16.9 19.5 mg dry wt. Clearance rate was highest in the control (11.19 ml hr-1 mg dry wt-1) and lowest in the Whole-1,000 treatment (2.33 ml hr-1 mg dry wt-1) (Fig. 6). This equals a 79% reduction in clearance rate between the two treatments. There was a significant difference in clearance rate among treatments (ANOVA; p < 0.001). A two-factor ANOVA showed a concentration effect (p < 0.001), a treatment e ffect (p < 0.001), and an interaction effect (p < 0.001). Bay scallops filtered 3% of K. brevis over one hour at 1,000 cells ml-1 (calculated from Table 8). Green mussels ( Perna viridis ) Mean dry weight for juvenile green mussels ranged from 40.3 46.5 mg dry wt. Mean clearance rate was highest in the control (16.39 ml hr-1 mg dry wt-1) and lowest in the Whole-1,000 treatment (4.37 ml hr-1 mg dry wt-1) (Fig. 7), a 73% reduction in clearance rate between the two treatments. There was a significant difference in clearance rate among treatments (ANOVA; p < 0.001). A two-factor ANOVA showed a concentration effect (p < 0.001), a treatment e ffect (p < 0.001), and an interaction effect (p < 0.001). Green mussels filtered 32% of K. brevis over one hour at 1,000 cells ml-1 (calculated from Table 8).

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59 Northern quahogs ( Mercenaria mercenaria ) Mean dry weight for juvenile northern quahogs ranged from 13.8 16.3 mg dry wt. Clearance rate was highest in the control (12.91 ml hr-1 mg dry wt-1) and lowest in Whole-1,000 (4.28 ml hr-1 mg dry wt-1), or a 73% reduction in clearance rate (Fig. 8). There was a significant difference in clearance rate among treatments (ANOVA; p < 0.001). A two-factor ANOVA showed a concen tration effect (p < 0.001), a treatment effect (p < 0.001), and an interaction effect (p < 0.001). Northern quahogs filtered 9% of K. brevis over one hour at 1,000 cells ml-1 (calculated from Table 8). Eastern oysters ( Crassostrea virginica ) Mean dry weight for juvenile oysters ranged from 40.6 50.6 mg dry wt. Clearance rate was highest in the control (13.57 ml hr-1 mg dry wt-1) and lowest in the Whole-1,000 treatment (8.42 ml hr-1 mg dry wt-1) (Fig. 9). This equals a 38% reduction in clearance rate between the two treatments. There was a significant difference in clearance rate among treatments (ANOVA; p < 0.001). A two-factor ANOVA showed a concentration effect (p < 0.001) but no treatme nt effect (p = 0.73). Oysters filtered 54% of K. brevis over one hour at 1,000 cells ml-1 (calculated from Table 8). Differences in mean clearance rate among the four bivalve species is summarized in Fig. 10A for whole cultures and Fig. 10B for lysed cultures. Significant differences were found among species, K. brevis concentration and culture (p < 0.001). There were also significant interaction differences (p < 0.001) among all factors (Multifactor

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60 ANOVA; univariate test of significance for clearance rate). Relationships between clearance rate and brevetoxin concentration for each bivalve species are summarized in Figures 11 through 14. Bay scallops showed a significant decrease in clearance rate with increasing brevetoxin concentration for both whole and lysed cultures of K. brevis (Fig. 11). There was no significant difference (p > 0.05) between the two culture treatments. Green mussels (Fig. 12) and northern quahogs (Fig. 12) showed a decline in clearance rate with increasing brevetoxin concentration only for whole cultures. There was a significant difference (p < 0.05) between the two cultures for both species. Finally, eastern oysters showed a slight decline in clearance rate for both whole and lysed cultures of K. brevis (Fig. 14). There was no significant difference (p > 0.05) between the two culture treatments. Flow-Through Exposure Experiments Figure 15 summarizes clearance rates for all species under continuous flowthrough exposure to whole (top) and lysed (bottom) cultures of K. brevis. Mean clearance rate of juvenile A. irradians was significantly reduced (p < 0.05) at K. brevis concentrations of 100 cells ml-1 and higher in both whole (Fig 15A) and lysed (Fig 15B) experiments. The bay scallop was the only bivalve species to show a concentration effect of lysed K. brevis culture on clearance rate. This effect was delayed until day two, when there was a significant decrease in clearance rate at 100 cells ml-1 and higher. Mean clearance rate of P. viridis exposed to whole K. brevis culture (Fig 15B)

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61 was significantly lower (p < 0.05) at 1,000 cells ml-1. There was no significant difference (p > 0.05) in clearance rate with lysed K. brevis over time, although rates increased slightly during the two-day exposure. Mean clearance rate of M. mercenaria exposed to whole culture was significantly lower (p < 0.05) at 1,000 cells ml-1 (Fig 15C). There were no significant differences (p > 0.05) in clearance rate when M. mercenaria was exposed to lysed (Fig 15C) K. brevis There was no significant difference (p > 0.05) in clearance rate of juvenile C. virginica exposed to different concentrations of lysed (Fig 15D) or whole (Fig 15D) K. brevis over time. Discussion The species-specific response of bivalve molluscs to the presence of toxic or noxious algae in their diet (Shumway and Cucci, 1987; Shumway, 1990) is supported in the current laboratory study. Each of the four species responded differently when exposed to K. brevis at different concentrations and culture preparations. Furthermore, each species responded similarly under two very different exposure regimes: short-term (1 hr) exposure to a non-replenished supply of K. brevis and long-term (2 day) exposure to a continuous supply of K. brevis In the present study, the bay scallop ( A. irradians ) was the most sensitive to the presence of K. brevis in terms of clearance rate. This was the only species that showed a significant reduction in clearance rate when fed K. brevis at a concentration of 100 cells ml-1 independent of culture preparation. The response was immediate when exposed to

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62 intact cells, but took 24 hr to be manifested with lysed cells. Poor growth, histopathologies and mortality of A. irradians exposed to other toxic dinoflagellates suggest a systemic toxic effect (Wikfors and Smolowitz, 1993; Smolowitz and Shumway, 1997; Lesser and Shumway, 1993). The delayed feeding response to lysed K. brevis in our study was not related to any observed behavioral changes (e.g., shell valve closure Shumway and Cucci, 1987), but likely indicat es an unknown cytotoxic or neurotoxic effect. Green mussels ( Perna viridis ) and northern quahogs ( M. mercenaria ) were intermediate in their feeding responses when exposed to K. brevis Both species showed significantly reduced clearance rates at 1,000 cells ml-1 whole culture while neither species was affected by lysed culture. In fact, the clearance rate of P. viridis increased gradually during the two-day exposure to lysed culture, regardless of concentration. Clearance rate in juvenile P. viridis was also unaffected by another toxic dinoflagellate, Alexandrium tamarense (Li et al ., 2002); however, the congener, P. canaliculus was able to clear, ingest and absorb laboratory cultures (EPA-JR strain) of K. brevis (Ishida et al ., 2004). The effects of toxic algae on feeding activity in the northern quahog ( M. mercenaria ) are more species-specific. While M mercenaria can ingest and survive exposure to potentially toxic strains of Prorocentrum (Wikfors and Smolowitz, 1993), ingestion of Alexandrium fundyense was low and could only be induced by the addition of a nontoxic diatom (Bricelj et al ., 1990). Additionally, feeding rates of M. mercenaria fed A. tamarense and Gyrodinium aureolum were low compared to rates when fed I. galbana, and exposure to G aureolum resulted in significant mortalities (Lesser and

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63 Shumway, 1993). Eastern oysters ( C. virginica ) were the least responsive bivalve when exposed to K. brevis with respect to clearance rate, although there was a significant concentration effect in the static experiment. Of the four species of bivalves tested, oysters removed the highest percentage of K. brevis cells from the surrounding media. Sievers (1969) showed that Eastern oysters maintained normal shell valve activity at high densities of K. brevis in the laboratory. During red tides in the Gulf of Mexico, oysters became toxic (Cummins et al ., 1971), easily accumulating (Dickey et al ., 1999) and metabolizing (Poli et al ., 2000) brevetoxins. Oysters were more toxic than clams taken at the same time from the same location during a red tide outbreak in North Carolina (Tester and Fowler, 1990). Our results support the view that eastern oysters are relatively unharmed by exposure to bloom concentrations of K. brevis (Shumway et al ., 1990). Overall, whole cultures of K. brevis (intact cells) had a greater effect than lysed cultures (disrupted cells) on clearance rate in all species except C. virginica even though the amount of total brevetoxin was similar between the two preparations, suggesting that encounters with the dinoflagellate interfered with filtering capability. The New Zealand cockle ( Austrovenus stutchbury ) and the greenshell mussel ( P. viridis ) were shown to assimilate brevetoxins from K. brevis culture as well as from the supernatant from disrupted culture (Ishida et al ., 2004), but the effects of these preparations on feeding was not investigated. Additional studies using recently isolated strains of K. brevis including a non-toxic Wilson clone and two new isolates from Sarasota Bay (Florida, USA), could further elucidate these differences in bivalve feeding behavior. There was close within-species agreement in clearance rates between static and

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64 flow-through systems; however, the effects of K. brevis on A. irradians was shown to be significantly affected by exposure time, whereby clearance rates at both medium (100 cells ml-1) and high (1,000 cells ml-1) densities declined only after 24 hr exposure. For this reason, continuous flow-through systems are generally preferred over static systems when measuring physiological performance. With static systems, conditions are not held constant and therefore clearance rates may be affected if algal concentrations fall below a critical level (Widdows and Salkeld, 1993). Conditions in flow-through systems can be held constant (i.e., algal concentration), thus enabling continuous monitoring of clearance rate over extended time periods which more closely reflect environmental conditions during algal blooms. Additionally, flow-through systems allow for the monitoring of possible behavioral or physiological changes a ssociated with long term exposure to toxic algae (Lassus et al ., 1999). Bardouil et al (1996) suggested that longer exposure times are necessary to assess the effects of toxic algae on algal ingestion and toxin absorption in bivalve shellfish. Recurring blooms (= red tides) of K. brevis are common along the Florida west coast (Tester and Steidinger, 1997; Kirkpatrick et al ., 2004). Our results showed that the effects of laboratory cultures of K. brevis on clearance rates of juveniles of four important bivalves were species-specific, suggesting th at the ecological and fisheries impacts from these algal blooms could be quite different depending upon bivalve species, bloom concentration and duration. The most sensitive species in the present study was the bay scallop, A. irradians A rare bloom of K. brevis in North Carolina during 1987-88 was implicated in the massive mortality and subsequent recruitment failure of local bay scallop populations (Summerson and Peterson, 1990). Recently, bay scallops have been

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65 the focus of restoration activities in several southwest Florida estuaries (Geiger and Arnold, 2003; Wilbur et al ., 2005; Leverone et al ., 2005). In 2001, a restoration project was irrevocably compromised when a dense (105 107 cells L-1) bloom of K. brevis infiltrated Sarasota Bay, FL, resulting in complete mortality of captive scallops (Leverone, unpublished). While more precise studies are necessary to resolve the relationship between red tide intensity and duration on bay scallop mortality, prediction and monitoring of algal blooms would be beneficial in identifying potential restorations sites that are less prone to chronic K. brevis blooms. Floridas hard clam ( M. mercenaria ) aquaculture industry would also benefit from improved red tide prediction and monitoring. Relocating lease sites to areas less susceptible to red tides would benefit the industry twofold: reduce the deleterious effects of high K. brevis concentrations on feeding rates which, in turn, would affect growth rates, and 2) reduce the probability that cultured clams will be prevented from reaching the market due to harvest closures (Shumway, 1990). Locating aquaculture sites in lower salinity waters might reduce the frequency and duration of exposure to red tides, which typically initiate in more saline offshore waters. If a red tide does penetrate the estuary, the lower salinity further into the bay could serve as a potentially effective salinity barrier to a bloom of K. brevis. Similarly, reduced feeding rates in the green mussel ( P. viridis ) at high K. brevis concentrations should theoretically make it more difficult for mussel populations to remain established in estuaries where red tides are more frequent and/or severe. Emperical observations, however, suggest a different outcome. An intense red tide during 2005-06 resulted in high mortality of green mussels attached to pilings and other structures in lower Tampa Bay (personal observation). Intense recolonization by juvenile

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66 green mussels, however, was observed in late 2006, several months after the bloom had dissipated. The prolific and dynamic recruitment rates of green mussels and their ability to rapidly recolonize a previously inhabited space after a red tide has disappeared suggests populations of this exotic species have no difficulty overcoming the temporary effects of exposure to K, brevis. Finally, the relative insensitivity of C. virginica feeding rates to K. brevis suggests that the structure and function of Eastern oyster habitats in southwest Florida should not suffer serious negative impacts from K. brevis blooms.

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67 Table 7 Experimental conditions, bivalve species, sample matrix, cell and brevetoxin concentration of laboratory cultures of K. brevis (Wilson Clone) used for juvenile feeding experiments. n.d. = not detected. EXPERIMENT K. brevis Culture Brevetoxin Amount (ug L-1) Static Matrix (cells ml-1) PbTx-1 PbTx-2 PbTx-3 Brevenal TOTAL Bay scallops Whole n.d. 32.9 1.0 33.9 67.8 ( Argopecten irradians ) Lysed 22,000 n.d. 12.2 4.1 24.9 41.2 Green mussel Whole 1.9 17.6 3.6 ----23.1 ( Perna viridis ) Lysed 21,650 1.9 20.0 6.5 ----28.4 Northern quahog Whole 0.7 17.4 22.2 ----40.3 ( Mercenaria mercenaria ) Lysed 22,000 5.9 36.2 18.4 ----60.5 Eastern oyster Whole 5.9 36.6 21.1 ----63.5 ( Crassostrea virginica ) Lysed 21,300 7.6 52.7 20.1 ----80.3 Flow-through Bay scallops Whole 19,600 n.d. 30.4 4.7 31.9 67.0 ( Argopecten irradians ) Lysed 21,800 n.d. 32.9 1.0 33.9 67.8 Green mussel Whole 21,400 n.d. 10.7 9.2 9.7 29.7 ( Perna viridis ) Lysed 23,800 n.d. 34.4 5.5 13.9 53.8 Northern quahog Whole 21,500 n.d. 32.9 1.0 33.9 67.8 ( Mercenaria mercenaria ) Lysed 23,100 n.d. 43.2 12.1 19.8 75.1 Eastern oyster Whole 24,600 n.d. 24.8 5.3 31.8 61.9 ( Crassostrea virginica ) Lysed 23,300 n.d. 36.2 5.9 18.4 60.5

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68 Table 8 Decline in Isochrysis galbana cell counts (cells ml-1) for juvenile bivalve molluscs exposed to different concentrations and preparations of Karenia brevis under static conditions. Starting seawater volume in each replicate was 500 ml. Argopecten irradians Cell concentration (t=0 hr) (cells ml-1) Cell concentration (t=1 hr) (cells ml-1) Treatment Mean Dry Wt (mg) (SD) T. iso K. brevis T. iso K. brevis Reduction in cell concentration mg dry wt-1 hr-1 (cells ml-1) Control 19.5 (0.92) 25,758-----16,671----466 Whole-10 18.8 (1.34) 25,5743217,35522 437 Lysed-10 16.9 (1.95) 25,334-----17,751----449 Whole-100 18.2 (1.75) 25,34314222,181115 174 Lysed-100 18.4 (1.50) 25,728-----21,520----229 Whole-1,000 17.7 (1.28) 25,2611,06823,2601,036 113 Lysed-1,000 18.4 (1.02) 25,651-----21,414----230 Perna viridis Control 43.5 (3.87) 20,736-----5,005----362 Whole-10 44.9 (4.38) 21,547275,58914 355 Lysed-10 43.3 (3.49) 20,928-----5,388----359 Whole-100 43.4 (6.18) 21,52411710,023111 265 Lysed-100 44.4 (1.90) 21,285-----6,704----328 Whole-1,000 46.5 (7.21) 22,2981,13214,891770 153 Lysed-1,000 40.3 (3.16) 20,944-----7,092----344 Mercenaria mercenaria Control 14.9 (1.43) 22,988-----15,727----487 Whole-10 16.3 (2.41) 23,6001316,60113 429 Lysed-10 16.0 (1.81) 24,098-----17,610----406 Whole-100 15.3 (1.01) 23,2079917,46755 375 Lysed-100 13.8 (0.98) 24,154-----17,094----512 Whole-1,000 16.1 (1.43) 22,82097919,897889 182 Lysed-1,000 15.2 (0.79) 23,787-----17,370----422 Crassostrea virginica Control 43.9 (5.88) 19,210-----2,637----378 Whole-10 40.6 (8.99) 18,98693,1938 389 Lysed-10 47.1 (5.84) 18,800-----3,355----328 Whole-100 47.1 (5.43) 19,2511083,98733 324 Lysed-100 50.6 (6.54) 20,364-----4,439----315 Whole-1,000 48.3 (8.14) 20,4471,0355,557560 308 Lysed-1,000 45.7 (4.75) 20,493-----5,053----338

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69 Table 9 Filtration and clearance rates of juvenile bivalve molluscs exposed to whole and lysed culture of Karenia brevis under static conditions. Starting seawater volume in each replicate was 500 ml. Argopecten irradians Treatment Dry tissue (mg) Mean (SD) Filtration Rate (cells hr-1) Clearance Rate (ml hr-1) Weight-Specific Clearance Rate (ml hr-1 mg dry wt-1) Control 19.5 (0.92) 9,087 218 11.19 Whole-10 18.8 (1.34) 8,219 194 10.40 Lysed-10 16.9 (1.95) 7,583 178 10.54 Whole-100 18.2 (1.75) 3,161 67 3.69 Lysed-100 18.4 (1.50) 4,207 89 4.89 Whole-1,000 17.7 (1.28) 2,001 41 2.33 Lysed-1,000 18.4 (1.02) 4,236 90 4.93 Perna viridis Control 43.5 (3.87) 15,731 714 16.39 Whole-10 44.9 (4.38) 15,958 679 15.13 Lysed-10 43.3 (3.49) 15,540 682 15.80 Whole-100 43.4 (6.18) 11,501 385 9.01 Lysed-100 44.4 (1.90) 14,581 580 13.06 Whole-1,000 46.5 (7.21) 7,407 205 4.37 Lysed-1,000 40.3 (3.16) 13,852 545 13.60 Mercenaria mercenaria Control 14.9 (1.43) 7,261 191 12.91 Whole-10 16.3 (2.41) 6,999 176 10.99 Lysed-10 16.0 (1.81) 6,488 157 9.93 Whole-100 15.3 (1.01) 5,739 142 9.31 Lysed-100 13.8 (0.98) 7,060 173 12.54 Whole-1,000 16.1 (1.43) 2,923 69 4.28 Lysed-1,000 15.2 (0.79) 6,417 157 10.37 Crassostrea virginica Control 43.9 (5.88) 16,573 613 13.57 Whole-10 40.6 (8.99) 15,794 530 11.91 Lysed-10 47.1 (5.84) 15,444 497 11.76 Whole-100 47.1 (5.43) 15,265 755 10.74 Lysed-100 50.6 (6.54) 15,924 612 10.02 Whole-1,000 48.3 (8.14) 14,889 245 8.42 Lysed-1,000 45.7 (4.75) 15,440 548 8.95

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70 Treatment ControlWhole-10Lysed-10Whole-100Lysed-100Whole-1,000Lysed-1,000 Clearance Rate (ml hr-1 mg dry wt-1) 0 2 4 6 8 10 12 14 Figure 6. Mean ( S.D.) clearance rate (ml hr-1 mg dry wt-1) for juvenile bay scallops ( Argopecten irradians ) exposed to three concentrations and two preparations of Karenia brevis Treatments with the same letter are not significantly different (p > 0.05). n = 10. Tw o-way ANOVA; Tukeys Multiple Comparison Test. a a a b, c b c b

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71 Treatment ControlWhole-10Lysed-10Whole-100Lysed-100Whole-1,000Lysed-1,000 Clearance Rate (ml hr-1 mg dry wt-1) 0 2 4 6 8 10 12 14 16 18 20 Figure 7. Mean ( S.D.) clearance rate (ml hr-1 mg dry wt-1) for juvenile green mussels ( Perna viridis ) exposed to three concentrations and two preparations of Karenia brevis Treatments with the same letter are not significantly different (p > 0.05). n = 10. Two-way ANOVA; Tukeys Multiple Comparison Test. a a a,b d c e b, c

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72 Treatment ControlWhole-10Lysed-10Whole-100Lysed-100Whole-1,000Lysed-1,000 Clearance Rate (ml hr-1 mg dry wt-1) 0 2 4 6 8 10 12 14 16 18 Figure 8. Mean ( S.D.) clearance rate (ml hr-1 mg dry wt-1) for juvenile northern quahogs ( Mercenaria mercenaria ) exposed to three concentrations and two preparations of Karenia brevis Treatments with the same letter are not significantly different (p > 0.05). n = 10. Two-way ANOVA; Tukeys Multiple Comparison Test. a a b c d c, a e b,

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73 Treatment ControlWhole-10Lysed-10Whole-100L ysed-100Whole-1,000Lysed-1,000 Clearance Rate (ml hr-1 mg dry wt-1) 0 2 4 6 8 10 12 14 16 Figure 9. Mean ( S.D.) clearance rate (ml hr-1 mg dry wt-1) for juvenile eastern oysters ( Crassostrea virginica ) exposed to three concentrations and two preparations of Karenia brevis Treatments with the same letter are not significantly different (p > 0.05). n = 10. Tw o-way ANOVA; Tukeys Multiple Comparison Test. a a b d, e c,d,e b ,c, d a b

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74 Figure 10. Mean ( S.D.) clearance rate (ml hr-1 mg dry wt-1) for juvenile bivalves exposed to (A) whole and (B) lysed cultures of Karenia brevis under static conditions. = Argopecten irradians ; = Perna viridis ; = Mercenaria mercenaria ; and = Crassostrea virginica (n = 10).

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75 Table 10 Species, treatment, clearance rate and amount of toxin (g) each species was exposed to during static feeding experiments. Amount of toxin exposure is based on culture cell concentration and amount of toxic (w/v) in each culture. Clearance Rate Culture Toxins ( g L-1) Species Treatment ( ml min-1 mg dry wt-1)67.8 41.2 Bay scallop (cells ml-1) Whole Lysed Whole Lysed ( Argopecten irradians ) 1010.410.540.015 0.009 1003.694.890.154 0.094 1,0002.334.931.541 0.936 Clearance Rate Culture Toxins ( g L-1) Treatment ( ml min-1 mg dry wt-1) 23.1 28.4 Green mussel (cells ml-1)Whole Lysed Whole Lysed ( Perna viridis ) 1015.1315.80.005 0.007 1009.0113.060.053 0.066 1,0004.3713.60.533 0.656 Clearance Rate Culture Toxins ( g L-1) Treatment ( ml min-1 mg dry wt-1)40.3 60.5 Northern quahog (cells ml-1)Whole Lysed Whole Lysed ( Mercenaria mercenaria ) 1010.999.930.009 0.014 1009.3112.540.092 0.138 1,0004.2810.370.916 1.375 Clearance Rate Culture Toxins ( g L-1) Treatment ( ml min-1 mg dry wt-1)63.5 80.5 Eastern oyster (cells ml-1)Whole Lysed Whole Lysed ( Crassostrea virginica ) 1011.9111.760.015 0.019 10010.7410.020.149 0.189 1,0008.428.951.491 1.890

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76 y (whole) = -1.7524Ln(x) + 2.196 R2 = 0.8722 y (lysed) = -1.2182Ln(x) + 3.9016 R2 = 0.7447 0 2 4 6 8 10 12 0.0010.0100.1001.00010.000 Brevetoxin Amount (ug)Clearance Rate ( ml hr-1 mg dry wt 1 ) Figure 11. Regression of clearance rates for juvenile Argopecten irradians against the amount of brevetoxin exposure under static conditions. Solid line represents regression for whole culture of Karenia brevis ( ); dashed line represents regression for lysed culture of K. brevis ( ). Regression equation shown for each line. (n = 5).

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77 y (whole) = -2.3365Ln(x) + 2.6553 R2 = 0.9937 y (lysed) = -0.4777Ln(x) + 12.852 R2 = 0.5744 0 3 6 9 12 15 18 0.0010.0100.1001.000 Brevetoxin Amount (ug)Clearance Rate ( ml hr-1 mg dry wt -1 ) Figure 12. Regression of clearance rates for juvenile Perna viridis against the amount of brevetoxin exposure under static conditions. Solid line represents regression for whole culture of Karenia brevis ( ); dashed line represents regression for lysed culture of K. brevis ( ). Regression equation shown for each line (n = 5).

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78 y (whole) = -1.4571Ln(x) + 4.7103 R2 = 0.9233 y (lysed) = 0.0955Ln(x) + 11.136 R2 = 0.0248 0 2 4 6 8 10 12 14 0.0010.0100.1001.00010.000 Brevetoxin Amount (ug)Clearance Rate ( ml hr-1 mg dry wt -1 ) Figure 13. Regression of clearance rates for juvenile Mercenaria mercenaria against the amount of brevetoxin exposure under static conditions. Solid line represents regression for whole culture of Karenia brevis ( ); dashed line represents regression for lysed culture of K. brevis ( ). Regression equation shown for each line (n = 5).

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79 y (whole) = -0.7578Ln(x) + 8.9142 R2 = 0.9651 y (lysed) = -0.6102Ln(x) + 9.2267 R2 = 0.9814 0 2 4 6 8 10 12 14 0.0010.0100.1001.00010.000 Brevetoxin Amount (ug)Clearance Rate ( ml hr -1 mg dry wt -1 ) Figure 14. Regression of clearance rates for juvenile Crassostrea virginica against the amount of brevetoxin exposure under static conditions. Solid line represents regression for whole culture of Karenia brevis ( ); dashed line represents regression for lysed culture of K brevis ( ). Regression equation shown for each line (n = 5).

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80 Figure 15. Mean ( S.D.) clearance rate (ml hr-1 mg dry wt-1) of juvenile bivalves exposed to whole (top) and lysed (bottom) cultures of Karenia brevis under flowthrough conditions. Species include (A) Argopecten irradians (B) Perna viridis (C) Mercenaria mercenaria and (D) Crassostrea virginica Concentrations of K. brevis in each treatment are: Control ( ), 100 () and 1,000 () cells ml-1. (n = 6). Clearance rates were measured twice a day (9 A.M. and 5 P.M.) and calculated from inflow and outflow concentrations of a supplemental food algae, Isochrysis galbana

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81 Chapter Five Histopathology Studies Introduction Studies on bivalve exposure to harmful microalgae have mostly focused on shortterm acute and lethal effects. The consequences of more long-term, chronic or sublethal contact with toxic algae and/or bioaccumulated toxins have received less attention (Shumway and Cucci, 1987; Shumway, 1990; La ndsberg, 1996). Chronic exposure to biotoxins typically leads to impaired f eeding, avoidance behaviors, physiological dysfunction, weakened immune function and reduced growth and reproduction (Shumway, 1990; Wikfors and Smolowitz, 1993), which in turn may lead to an increased susceptibility to disease, abnormal development, histopathologies and the induction of neoplasia (Landsberg, 1996). Types of histopa thologies that have been observed include mantle and gill lesions (Nielsen and Strmgren, 1991; Smolowitz and Shumway, 1997), cellular changes and increased lumen diameter within the digestive diverticula (Wikfors and Smolowitz, 1993), reproductive abnormalities, protozoan and/or bacterial infections (Smolowitz and Shumway, 1997) and disseminated neoplasia and germinomas (see Landsberg, 1996 for review; Barber, 2004). The impact of Gyrodinium aureolum on the histology of gut tissue from eight species of juvenile bivalve was species-specific (Smolowitz and Shumway, 1997). The

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82 eastern oyster ( Crassostrea virginica ) and the bay scallop ( Argopecten irradians ) were the most severely affected species. Several C virginica showed mantle and gill lesions. Bay scallops exhibited a decrease in the height of absorptive cells and an increase in lumen diameter after exposure, suggesting G aureolum is of poor food quality. Evidence of toxic effects was not identified in the digestive gland. Several bay scallops also showed variable amounts of inflammation in the kidney associated with protozoal infestations and variable amounts of predominately rod-shaped bacteria within the urinary space. Bay scallops exposed to a Prorocentrum isolate also showed tissue abnormalities (Wikfors and Smolowitz, 1993). Bay scallops fed mixed diet of P. minimum and Isochrysis galbana showed distinctive lesions. Control scallops showed normal, welldeveloped digestive diverticula while experimental scallops exhibited an assortment of abnormalities in this organ, including contracted absorptive cells, abnormal vacuolation, necrosis of absorptive cells and their exfoliation into the lumen. All other organs (gills, muscle, kidney, foot and heart) in the experimental group appeared moderately to well developed (Wikfors and Smolowitz, 1993). Another pectinid, the king scallop ( Pecten maximus ) developed obvious saxitoxin neoformation in kidneys after exposure to paralytic shellfish poisoning toxins (Bougrier et al ., 2000). The mussel Mytilus edulis was shown to be cytotoxic in the presence of the dinoflagellate, Gyrodinium aureolum which had an acute effect on the clearance rate and caused marked cellular damage to the gut (Widdows et al ., 1979). Likewise, exposure of juvenile hard clams ( Mercenaria mercenaria ) and blue mussels ( Mytilus edulis ) to a toxic isolate of the picoplankter Aureococcus anophagefferens (which causes brown tides in

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83 coastal bays of the mid-Atlantic USA) caused reduction in digestive epithelium height and overall appearance of absorptive cells (Bricelj et al 2004). These observations are similar to those in bivalves that have undergone starvation. The effects of long-term exposure of bivalves to Karenia brevis have not yet been studied. Consequently, we do not know if the brevetoxins produced by K. brevis which are responsible for neurotoxic shellfish poisoning (NSP) in the Gulf of Mexico, have a role in the initiation of any specific pathologies in bivalve tissues (Landsberg, 1996). Although brevetoxins have been well known for their role in fish kills (Steidinger et al ., 1973), their role in developing histopathologies in bivalve molluscs is unknown. Of the four bivalves studied in the present work, the bay scallop ( Argopecten irradians ) has been the most sensitive to K. brevis exposure. Therefore, this chapter focuses on the histological effects of long-term exposure to K. brevis in adult bay scallops. Furthermore, the effects of whole and lysed cultures of K brevis in unialgal and mixed suspensions, are examined in the following tissues: digestive diverticulum, mantle and gill. Materials and Methods Adult bay scallops ( Argopecten irradians ) were collected from the Anclote Anchorage (28o 17N; 82o 45W) and Hommosassa Springs (28o 43N; 82o 43W) on June 30, 2006 and transferred to Mote Marine Laboratory. Scallops were gently scrubbed to remove any attached fauna or debris and equally divided into five separate 25-liter aquaria. Aquaria were mildly aerated and maintained at 32 ppt salinity and 27o C

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84 in a temperature-controlled exposure room. Scallops were suspended above the aquaria bottom by a mesh partition to allow for the settlement of feces. Scallops were allowed to acclimate without food for two days prior to the start of the experiment. Each aquarium held twenty-five scallops at the start of the experimental exposure. For those treatments receiving Isochrysis galbana and Karenia brevis algal concentrations were set at 1 x 105 cells ml-1 and 5 x 102 cells ml-1, respectively. Experimental conditions are summarized in Table 11. A water exchange (ca. ninety percent) was made each day and algal concentrations adjusted to maintain the desired concentrations. A sample (n = 4) of individuals was removed from each aquaria on days 2, 7 and 14 and fixed for histology. Scallops were observed daily and any individual showing signs of stress or abnormal behavior (i.e., shell gaping or mantle retraction) was immediately removed and fixed. Shell height was measured before dissection. Scallops were dissected and the mantle, gill and digestive gland were fixed in Davidsons fixative (Howard and Smith, 1983). Tissues remained in fixative for 48 hours before being transferred to 70% ethanol where they remained until embedding. Each tissue was processed in paraffin (Tissue Prep ), five m sections prepared and stained with hematoxylin and eosin (Howard and Smith, 1983). Sections were observed under magnification to determine if any abnormalities had developed after a two week exposure to K. brevis Results are descriptive and qualitative in nature. Photomicrographs accompany descriptive pathologies.

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85 Results Table 12 summarizes the sampling schedule, withdrawals and mortality of A. irradians during the two-week exposure to K. brevis Mean shell height was approximately 50 mm in all treatments. All scallops in every treatment survived the first week of exposure. On day nine, several scall ops began to show signs of stress, indicated by slight gaping of the shell valves and partial retraction of the mantle edges. These included one scallop from Tank 3 ( K. brevis only) and two from Tank 5 (lysed K. brevis and T. iso ). At the end of day twelve, only scallops from Tank 1 (Control) and Tank 4 (whole K. brevis and T. iso ) were still alive. Scallops from both of these treatments were still alive on day fourteen when the experiment was terminated. Various parasitic infections were observed in gill and digestive tissue from scallops in all treatments. The most common infections were ciliates ( Nematopsis sp?) and Rickettsias-like bacterial infections (Fig. 16). An unidentified parasite within the digestive diverticula is pictured in Fig. 17. On the other hand, several histomorphologies, particularly in the digestive diverticula, were observed that appear to be associated with several of the exposure scenarios to K. brevis The most noticeable and pervasive of these pathologies was the presence of hemocyte aggregations and infiltrations in the digestive diverticulum. This particular pathology, which is indicative of an inflammatory response, was found to some degree in all treatments, but was particularly associated with scallops that were either starved or exposed to lysed K. brevis and Isochrysis (Tank 5; Fig. 18). This pathology first appeared on day 2 in starved scallops; in the other treatment it appeared on day 7

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86 and was present through the remainder of the experiment. The relative intensity of this inflammatory response was scored on a scale of 0 3+ and summarized in Table 13. The appearance of the epithelial layer in the digestive diverticula showed a range of atrophic degradation, including variously reduced thickness of the epithelial layer and reduced sizes of the digestive tubules. These changes were pervasive throughout all treatments except the control and were noticeable from as early as day two in starved scallops. The degree of modification of the epithelial layer is shown in Fig. 19. Gill and mantle tissue from A. irradians exposed to K. brevis did not show any obvious or noticeable histopathologies. The epithelia of the ordinary filaments in the gill appeared normal, as did the supporting structures, septa and cilliary tracts (Fig 20A). The variously-shaped interconnecting vessels of the dorsal expansion of the gill also appeared normal and healthy (Fig 20B). Finally, the free edge of the mantle, which is divided into three folds and two grooves, showed no deformities or abnormalities (Fig 21). Discussion Bivalve parasites, notably the Rickettsiales and Protista, are commonly found in the epithelial cells of the gills and digestive diverticula of many species, including scallops (Chang et al ., 1980). Most infections appear benign, despite relatively dense colonization. Light to moderate rickettsial-like infections of the gill have been previously found in wild, captive and cultured adult bay and sea scallops by Leibovitz et al. (1984). The coccideans, Nematopsis ostrearum and N. duorari have been found in

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87 bay scallops, but no pathogenicity has been described for these infections (Kruse, 1966; Sprague, 1970). In the present study, bacterial infections were not intense, nor were they predominant in any particular treatment or related to time of exposure to K. brevis The presence of bacterial and protist parasites did not appear to be positively associated with any of the observed cytological histopathologies in this study. This study showed distinctive and pervasive hemocytic infiltrations in the digestive diverticula of A. irradians particularly in individuals that had either been starved or exposed to lysed culture of K. brevis Hemocytes are known to recruit from circulation to sites of inflammation and tissue damage (Cheng, 1967). The fact that a higher incidence of inflammation occurred from exposure to lysed cultures suggests a cytotoxic response rather than a reaction to the actual dinoflagellate. Damage to adsorptive cells in the digestive diverticula and systemic pathologies characteristic of toxin effects has previously been observed in juvenile bay scallops by Wikfors and Smolowitz (1993) after exposure to a diet which included Prorocentrum minimum These scallops suffered rapid mortality. Similar changes in the digestive cells of the mussel Mytilus edulis were noted during a bloom of Gyrodinium aureolum and were attributed to a toxic response rather than a result of starvation (Widdows et al., 1979). Additional studies are necessary to elucidate the mechanism by which toxicity from harmful algae leads to such rapid mortality in the bay scallop (as opposed to other bivalves). Decreased height of absorptive cells and increased lumen diameter suggest that K brevis is, at best, a poor quality food (Smolowitz and Shumway, 1997). Starved bivalves display similar changes in epithelia of the absorptive cells (Wikfors and Smolowitz,

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88 1995). Smolowitz and Shumway (1997), however, did not observe sloughing of digestive epithelial cells in juvenile A. irradians exposed to Gyrodinium aureolum leading them to conclude that there was pr obably no toxic effect. In this study, there were signs of epithelial sloughing which lends support to an unknown toxic mechanism (in addition to poor nutritional processes) in the digestive diverticula of A. irradians exposed to K. brevis The use of a nontoxic dinoflagellate in addition to K. brevis in future feeding studies might help elucid ate the histological differences between nutritional and toxic responses in bay scallop digestive tissues.

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89 Table 11. Experimental design for two-week exposure of Argopecten irradians to Karenia brevis ( Isochrysis galbana is a common nutritional chrysophyte algae). Treatment Tank 1 Tank 2 Tank 3 Tank 4 Tank 5 Isochrysis galbana (1 x 105 cells ml-1) Yes No No Yes Yes Karenia brevis (5 x 102 cells ml-1) No No Yes Yes Yes Whole or lysed Karenia brevis None None Whole Whole Lysed

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90 Table 12. Schedule for the removal of Argopecten irradians from each experimental tank during the two-week exposure to Karenia brevis Values represent shell height (mm) of individuals removed for fixation (sha ded) or dead (unshaded) on that day. Day Tank 1 Tank 2 Tank 3 Tank 4 Tank 5 Control ( T. iso only) Starved (no algae) Whole K.b only Whole K.b + T. iso Lysed K.b + T. iso 0 2 54.9 60.8 59.8 56.5 52.5 52.4 58.9 56.0 57.1 55.6 53.1 56.1 55.6 53.2 52.0 46.4 50.1 46.8 50.1 46.5 3 4 5 6 7 31.3 40.9 44.1 41.4 40.2 42.1 44.6 53.2 43.9 42.6 49.7 48.7 48.8 46.1 48.6 49.3 54.2 47.7 54.1 52.2 8 59.0 43.0 46.2 9 44.9 53.9 57.3 45.2 10 52.9 52.2 11 12 53.7 52.7 45.2 43.5 55.6 13 14 53.4 56.4 52.3 55.1 49.3 48.3 Mean 49.7 51.551.451.5 50.8 S.D. 6.49 5.804.965.65 5.49

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91 Table 13. Scoring of hemocyte infiltration intensity in the digestive diverticula of A irradians exposed to various scenarios of K brevis Tank 1 Tank 2 Tank 3 Tank 4 Ta nk 5 Day Control Starved Whole K brevis only Whole K brevis & T. Iso Lysed K brevis & T. Iso 2 0 1 2+ 0 0 0 0 2 3+ 0 0 0 1+ 0 2+ 0 1+ 0 1+ 0 1+ 1+ 1+ 2+ 0 7 0 1 2+ 0 0 1+ 2+ 1+ 1+ 0 1+ 1+ 2+ 1+ 2+ 0 1+ 0 1 + 3+ 0 2+ 0 3 + 2+ 9 0 2+ 3+ 10 0 12 0 1+ 0 14 0 1+ 0 0 0 1+ 1+ 0 0

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92 Figure 16. (A) The protist, Nematopsis sp? (arrow) and (B) a Rickettsial-like bacterial microcolony in the gill epithelia of A. irradians Magnification equals 400x. Fig. 17. An unidentified parasite encysted within the digestive diverticula. Magnification equals 400x. A B

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93 Figure 18. Digestive diverticula from A. irradians exposed to lysed culture of K. brevis and T. Isochrysis (A and B = day 7; C and D = day 9). Hemocyte aggregations (arrows) displaying inflammatory response. Magnification equals 400x. Figure 19. Epithelial layer of the digestive diverticula in A. irradians from different exposure scenarios to K. brevis A) control scallop at 14 days; B) scallop exposed to whole culture of K. brevis at day 12. Magnification equals 400x. BA C D A BA

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94 Figure 20. Gill tissue from A. irradians showing A) the distal portions of the ordinary filaments (note the lateral cilia) and B) interconnecting vessels of the dorsal expansion. Figure 21. Mantle tissue from A. irradians showing A) the epithelia of the mantle margin and B) section through an eye on the middle fold of the mantle margin.A A B B

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95 Chapter Six General Discussion Florida red tides caused by the dinoflagellate Karenia brevis are some of the oldest reported harmful algal blooms, with fish kills being reported since the middle of the nineteenth century (Ingersoll, 1882). These massive fish mortalities are the most consistent observation from reports of red tides, and much has since been learned regarding the toxic mechanisms involved in fish mortality (see Landsberg, 2002 for review). Filter feeding bivalve molluscs also become contaminated with these toxins during blooms of K. brevis and cause a disease called neurotoxic shellfish poisoning in humans who consume contaminated shellfish. The focus of previous research on K. brevis and bivalves has been on the human health consequences of eating shellfish contaminated with these toxins, called brevetoxins, or their analogs (Hemmert, 1975). Studies of K. brevis and its effects on bivalves themselves are much more limited and can be divided into three categories: 1) observations of bivalve mortalities from natural blooms (Simon and Dauer, 1972; Tiffany and Heyl, 1978); 2) toxicity of shellfish exposed to laboratory cultures (Ray and Al drich, 1967; Sievers, 1969; Hemmert, 1975) or blooms in the field (Cummins et al ., 1971; Tester and Fowler, 1990; Wang et al ., 2004); and 3) the dynamics of toxin uptake, metabolism and elimination after controlled exposure to K. brevis cultures and/or pure toxins (Fletcher et al ., 1998; Plakas et al ., 2002; Ishida et al ., 2004). These studies and their findings are summarized in Table 14.

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96 Field observations of mortality from natural blooms demonstrate the ecological consequence of these perturbations on resident fauna, but rarely do they unequivocally relate mortality to the intensity and duration of exposure to K. brevis Invariably, there are other concommitant and complicating conditions that contribute to mortality, most notably severe and prolonged depressions in dissolved oxygen, which may or may not be monitored. Monitoring of shellfish toxicity during red tides has provided valuable information on the toxicity of exposed bivalves, but no consideration has ever been given to the health of the shellfish. Recent la boratory studies focusing on the identity of toxins and their derivatives have contributed greatly to our knowledge of how bivalves process brevetoxins; however, these studies did not investigate the behavioral or physiological responses, such as changes in feeding rate, that bivalves undergo when confronted with these toxic dinoflagellates. Not until the early work of Shumway and colleagues was attention given to the impacts of harmful and toxic algae on specific shellfish or to the potential long-term impacts to bivalve fisheries and culture (Shumway and Cucci, 1987; Shumway, 1990; Shumway et al., 1990). These studies reveal ed no universal effects on bivalves from exposure to toxic algae; rather, the response depends on the interaction between specific alga and bivalve species. It was also demonstrated that bivalve populations which are periodically exposed to toxic algal blooms may have evolved mechanisms permitting them to exploit the toxic organisms as food with no ill effects. Recurring blooms of K. brevis have become common along the Florida west coast (Tester and Steidinger, 1997). These blooms occur almost annually, usually in the late summer and autumn (see Kirkpatrick et al ., 2004 for review). At the same time,

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97 populations of important bivalve species in Florida are under increasing threat from these persistent algal blooms. For example, projects aimed at restoring bay scallops within several southwest Florida estuaries have increased in recent years (Geiger and Arnold, 2003; Wilbur et al ., 2005; Leverone et al 2005). The green mussel, Perna viridis which is commercially exploited in its native New Zealand, became established in Tampa Bay in 1999 (Ingrao, 2001), and has since spread throughout the state and has been found as far as South Carolina along the Atlantic coast. The state of Florida has developed a burgeoning clam ( M. mercenaria ) aquaculture industry in the past decade and is responsible for managing leases in coastal areas that coincide with the most frequent episodes of red tide. Specific knowledge regarding the ecological consequences of prolonged or repeated exposure to K brevis in Florida bivalve populations would help tremendously in developing responsible management plans for each of these valuable shellfish species. This dissertation research was undertaken to add to the knowledge of how K. brevis affects the different life stages of important bivalve molluscs from Florida. Bivalve Larvae Most studies on the interaction between toxic dinoflagellates and bivalves have focused on juvenile and adult life stages (see reviews by Shumway, 1990; Bricelj and Shumway, 1998; Landsberg, 2002). Recently, attention has been given to the effects of harmful algal on bivalve larvae (Wikfors and Smolowitz, 1995; Matsuyama et al ., 2001; Yan et al ., 2001; Yan et al ., 2003; Jeong et al ., 2004). The veliger is an important and delicate stage in the early development of bivalve shellfish and is generally considered

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98 more sensitive than its post-larval counterparts to perturbations and stressors, including exposure to harmful algal blooms (Yan et al., 2003; Wang et al, 2006). The research presented in this thesis demonstrated that survival of veliger larvae for all three bivalve species ( Argopecten irradians Mercenaria mercenaria and Crassostrea virginica ) was dependent upon the cell concentration of Karenia brevis Overall survival was quite high (85%) at K. brevis cell concentrations less than bloom strength (100 cells ml-1 and less), but decreased to roughly 25% at high bloom concentrations (5,000 cells ml-1). Larval development was also protracted in surviving larvae of C. virginica and M. mercenaria at K. brevis densities of 1,000 cells ml-1. Larval survival was generally higher when exposed to whole cultures of K. brevis compared to lysed cultures. Matsuyama et al (2001) reported that certain species of harmful algae were lethal to larvae of the Pacific oyster, C. gigas at cell densities (100-1,000 cells ml-1) similar to those in the present study. While certain dinoflagellates ( Alexandrium tamarense A taylori Gymnodinium mikimotoi and Heterocapsa circularisquama ) were shown to be lethal, four other species ( Chattonella antiqua Gymnodinium catenatum Heterosigma akashiwo and Scrippsiella trochoidea ) did not affect the survival rate or development of oyster larvae at the same concentrations. Interestingly, mortality did not necessarily relate to the toxicity of the dinoflagellate. Alexandrium taylori had an extreme lethal effect on C. gigas larvae, but HPLC analysis of A. taylori cultures revealed no PSP toxins. The PSP producer Gymnodinium catenatum however, caused no harmful effects on oyster larvae even at abnormally high (above bloom) concentrations. Exposure of C. gigas embryos to unfiltered seawater containing Gyrodinium aureolum for two days

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99 resulted in poor (ten percent) survival to the veliger stage (Helm et al ., 1974), suggesting evidence of toxicity. The process by which K. brevis affects larval survival is not clear, but several possible mechanisms have been suggested and may be involved. The earliest studies on interactions between bivalve larvae and harmful algae suggested that a direct cell-to-cell contact with microalgae was responsible for larval mortality, either through exposure to toxins present on cell surfaces or through mechanical damage to sensitive organs, particularly gills. Gallager et al (1989) suggested that Argopecten irradians larvae must ingest or be in contact with whole cells of Aureococcus anophagefferens before elevated larval mortality is observed. This same microalgae, however, had no effect on survival for larvae of the northern quahog, M. mercenaria even at bloom conditions (Padilla et al ., 2006). Mortality of C. virginica larvae in the presence of the dinoflagellate Cochlodinium heterolobatum was thought to be a result of increased direct contact between larvae and algal cells (Ho and Zubkoff, 1979). Cell-free filtrates of the two Alexandrium species ( A tamarense and A taylori ) had less effect on mortality of C. gigas larvae than their whole-cell counterparts (Matsuyama et al ., 2001), implying the cause of toxicity was localized on the cell surface. Yan et al (2001) suggested that direct contact with toxic algal cells may also release an unknown inhibitory factor which could negatively affect survival. Ultrasonic disruption (=lysing), which produces cellular fragments as well as releasing intracellular toxins, should make the toxins more available for encounters with bivalve larvae. Collectively, these studies strongly suggest that physical cell-to-cell contact between bivalve larvae and toxic dinoflagellates is, at least partially, responsible for observed increases in larval mortality.

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100 In this study, lysed cultures produced higher mortality in M. mercenaria and C. virginica than whole cultures at the same cell concentrations. Cell fragments in the lysed treatments were not removed from the experi mental medium (either by centrifugation or filtration) after sonication. Thus, these fragments (and any released intracellular toxins) were available to interact with the exposed larvae, thereby increasing the frequency of physical contact between algae and larvae. This scenario might explain, at least partially, the observed increase in larval mortality associated with lysed treatments of K brevis Consumption (or ingestion) of harmful algal cells by bivalve larvae is dependent upon a variety of factors, including algal species, cell size and concentration, and larval species and age. Consumption of K. brevis cells could also explain the observed inhibitory effects on larval survival in this study. Larvae of the mussel, Mytilus galloprovincialis readily ingested cells of several sp ecies of red-tide dinoflagellates with mean equivalent spherical diameters of 12-38 m (Jeong et al ., 2004). However, mussel larvae did not feed on any dinoflagellate until at least nine days after fertilization. Eastern oyster ( C. virginica ) larvae ingested P. minimum cells, although filtration was depressed in the presence of this toxic algae (Wikfors and Smolowitz, 1995), and ingestion of these cells resulted in cytological changes in digestive tissues, including the deleterious development of cuboidal and squamous epithelial cells in the stomach and intestine, reductions in the size of absorptive cells, and the presence of dense inclusions in the cytoplasm. All of these symptoms indicate possible phagolytic reactions to dinoflagellate debris (Wikfors and Smolowitz, 1995). Early D-shape larvae of two scallop species ( Argopecten irradians concentricus and Chlamys farreri ) were unable to

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101 feed on Alexandrium tamarense cells due to the relatively large algal cell size (Yan et al ., 2001; Yan et al ., 2003). During this study, larvae were fed an optimal ration (Lu and Blake, 1996) of the chrysophyte, I. galbana in addition to K. brevis to ensure that the larvae were well-fed throughout the experiment and that any observed mortality was not due to starvation. Although larval feeding rates were not measured nor K. brevis consumption investigated, ingestion of K. brevis cells was most likely negligible due to the large cell size (ESD = 14-26 m) and low density compared to I. galbana ; however, the presence of K. brevis especially at higher concentrations, could have altered activity patterns (Yan et al ., 2003) and/or feeding rates (Jeong et al ., 2004), resulting in increased mortality and retarded metamorphosis (Matsuyama et al ., 2001). The numerous cellular fragments in the lysed treatments could have been of an appropriate size for filtration and ingestion, further complicating feeding patterns and possibly initiating phagolytic reactions similar to those reported by Wikfors and Smolowitz (1995). In addition to affecting larval survival, K. brevis also negatively impacted larval development and metamorphosis. For example, even though overall survival was identical in C. virginica larvae exposed to 100 and 1,000 cells ml-1, a higher proportion from 100 cells ml-1 had reached the pediveliger stage and completed larval development (i.e., settled as spat) than larvae from 1,000 cells ml-1. Almost ninety percent of larvae subjected to 5,000 cells ml-1 did not live beyond the umboveliger stage. Development of M. mercenaria larvae was also affected by the presence of K. brevis cells. In this case, progress to the pediveliger stage was inversely related to K. brevis concentration. Similarly, larvae of the Pacific oyster, C. gigas which did not show significant mortality when exposed to Cochlodinium polykrikoides did suffer retarded metamorphosis to the

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102 D-shaped larvae (Matsuyama et al ., 2001). Development of C. virginica larvae was also delayed when exposed to a laboratory clone of the dinoflagellate, P. minimum (Wikfors and Smolowitz, 1995). Heavy metals have also been shown to delay metamorphosis in bivalve larvae. Settlement of oyster ( Crassostrea gigas ) and bay scallop ( Argopecten irradians ) larvae was delayed in the presence of zinc (Boyden et al., 1975; Watling, 1983) and development in northern quahog larvae ( Mercenaria mercenaria ) was delayed by exposure to nickel (Calabrese and Nelson, 1974). While the mechanism for increased mortality of bivalve larvae remains unanswered, it is easy to see how the added stress associated with K. brevis and/or its toxins could be reflected in suboptimum development. Since delayed metamorphosis has been observed in bivalve larvae exposed to heavy metals as well, these results may reflect a more general toxic response rather than one that is attributable to brevetoxins. Matsuyama et al (2001) organized the effects of harmful algae on (oyster) larvae into three categories: Type 1 = lethal to larvae at visible bloom density (red tide) Type 2 = non-lethal effects but induce a delay in metamorphosis Type 3 = no effect. Based on these categories, K. brevis exhibits a combination of Type 1 and Type 2 responses in the three species of bivalve larvae in these studies. Sixty percent of brevetoxins in laboratory cultures of K. brevis are extracellular in nature (Pierce et al ., 2001). Ultrasonic disruption, which releases the remaining intracellular toxins, resulted in a 20-24% increase in total brevetoxin in the current study. Three brevetoxin compounds were present in each culture: PbTx-2, PbTx-3, and

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103 brevenal, a recently identified brevetoxin antagonist (Bourdelais et al ., 2004). The proportion of each brevetoxin remained unchanged after the cultures were lysed. Except for the absence of PbTx-1, the relative brevetoxin composition of laboratory cultures closely resembled that from water samples collected in 2003 during a red tide outbreak along the Gulf coast of Sarasota, FL (Pierce et al ., 2005). Larvae of all three bivalve species in this study responded similarly, but with different sensitivities, to cells of K. brevis and its suite of toxins. Mortality was not necessarily dependent on ingestion of algal cells; rather it appears that the toxins were at least partially responsible for increased mortality and delayed larval development. The presence of K. brevis cells at high densities may interfere with larval feeding processes, resulting in suboptimal clearance, inhibited growth and development, and mortality. Our results clearly indicate that when K. brevis and its toxins persist, shellfish larvae are at greater risk of mortality and may continue to be adversely affected even after the disappearance of K. brevis cells. While K. brevis blooms may not directly cause mortality in adult shellfish, they do have the ability to disrupt a critical phase in the life cycle and consequently have important ramifications for recruitment and population stability. The collapse of bay scallop populations in North Carolina, USA, in 1989 was attributed to a bloom of Ptychodiscus brevis (= K. brevis ), which was blamed for higher than natural mortality in adults the previous year (Summerson and Peterson, 1990). Depletion of the adult spawner stock led to poor recruitment and failure of local populations ability to recover quickly to previous levels of abundance (Peterson and Summerson, 1992). These observations on population dynamics following a red tide did not even consider the additional, negative effects of K. brevis on larval growth and

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104 survival that were ascertained in the present study. Our demonstrated effects of K. brevis on the larvae of northern quahogs (= hard clams) and eastern oysters point to the potential for this toxic dinoflagellate to negatively impact recruitment in these species as well. Thus, there is a clear need for continued research on the relationship between K. brevis and bivalve larvae, ranging from the mechanisms of toxicity to the effects on recruitment and population stability. Juvenile Bivalves There are surprisingly few studies which have focused on the interaction between K. brevis and bivalve molluscs. Our current knowledge is limited to general field observations on the toxicity and/or mortality of shellfish during red tides (Gunter et al ., 1947; Cummins et al ., 1971; Simon & Dauer, 1972; Hemmert, 1975) and a few laboratory studies on behavioral responses to K. brevis cultures (Sievers, 1969; Roberts et al 1979). More recently, a series of studies have focused on brevetoxin uptake and metabolism in the eastern oyster (Dickey et al ., 1999; Poli et al ., 2000; Plakas et al ., 2002; Pierce et al ., 2004; Wang et al ., 2004). Several general relationships between individual bivalve species and K. brevis have emerged from these collective studies. During a bloom of K. brevis (= red tide), the eastern oyster becomes toxic through the accumulation and metabolizing of brevetoxins, northern quahogs also become toxic and bay scallops succumb to mortality. The current research lends further support to the species-specific response of bivalve molluscs in the presence of toxic or noxious algae (Shumway and Cucci, 1987;

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105 see Table 1 in Shumway, 1990). Each of the four species responded differently when exposed to K. brevis at different concentrations and culture preparations. Furthermore, we found that each species responded similarly under two very different exposure regimes: short-term (1 hr) exposure to a non-replenished supply of K. brevis and longterm (2 day) exposure to a continuous supply of K. brevis The bay scallop ( A. irradians ) was the most sensitive species to the presence of K. brevis in terms of clearance rate. This was the only species that showed a significant reduction in clearance rate when fed K. brevis at a concentration of 100 cells ml-1 independent of culture preparation. The response was immediate when exposed to intact cells, but took 24 hr to be manifested with lysed cells. The delayed feeding response to lysed K. brevis in our study likely indicates an unknow n cytotoxic or neurotoxic effect. Although no data are available for Florida populations of A. irradians Summerson and Peterson (1990) implicated a bloom of K. brevis in massive mortalities of bay scallops in North Carolina. This mass mortality led to recruitment failure of bay scallops in subsequent years (Peterson and Summerson, 1992). No other information is available on how bay scallops are effected by K. brevis Our results showed that K. brevis had an appreciable effect on survival or development of A. irradians larvae at high (bloom) concentrations. In addition, clearance rates in juvenile bay scallops exposed to K. brevis were the most sensitive of the bivalve species we tested. These laboratory findings support reports from North Carolina on bay scallop recruitment failure after a red tide outbreak in that larval mortality is high and feeding in juvenile scallops is compromised. Longer-term exposure of adult A. irradians to K. brevis revealed deleterious histological changes in the digestive diverticula; most notably an accumulation of

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106 hemocytes, but also cellular changes in the epithelial layer surrounding the lumen. These observations strongly suggest a combination of poor nutrition and toxic effects from exposure to K. brevis Additional studies are necessary to elucidate these differences. The ability of green mussels to feed upon and metabolize K brevis cultures was confirmed by Ishida et al (2004) using an EPA strain of K. brevis and New Zealand populations of the greenshell mussel, Perna canaliculus Several brevetoxin metabolites have been identified and biosynthetic pathways proposed by Morohashi et al (1995), Murata et al (1998) and Ishida et al (2004). However, no other studies involving K. brevis or brevetoxin analyses have been conducted on the greenshell mussel congener, P. viridis which has been a resident of Tampa Bay, FL since 1999. Our data, along with personal observations of local populations, indicate that Florida green mussel populations may be susceptible to blooms of K. brevis In the laboratory, the clearance rate in juvenile P. viridis was significantly reduced at moderate (100 cells ml-1) K. brevis cell concentrations, but not by lysed K. brevis culture. In the field, a high degree of mortality was observed in green mussel populations throughout lower Tampa Bay during a prolonged red tide outbreak in 2005 (personal observation). Additional studies are clearly needed to better understand the interactions between K. brevis and P. viridis During a 1973-74 red tide in Sarasota, FL, shellfish suspected of being contaminated from a bloom of K. brevis were processed and analyzed for the presence of toxins (Hemmert, 1975). Local surfclams ( Spisula solidissima raveneli ) and southern quahogs ( Mercenaria campechaenis ) were found to be toxic. Poli et al (2000) found toxic Mercenaria sp. during a rare red tide in the northern Gulf of Mexico. Clams were less toxic than oysters taken at the same time from the same location during an unusual

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107 red tide event in North Carolina (Tester and Fowler, 1990). Pierce et al (2004) found two brevetoxin metabolites in M. mercenaria in Sarasota Bay during a 2001 red tide, indicating that quahogs, like oysters, have the ability to consume and metabolize K. brevis and its toxins. These are the only published data regarding the ability of clams to filter, ingest and accumulate brevetoxins from exposure to K. brevis Our results are in agreement with this limited information on M. mercenaria While clearance rate was depressed at high K. brevis concentrations, quahogs did continue to feed. As a result, no mortality was observed in juvenile quahogs in our studies. Early laboratory studies showed that eastern oysters exhibited normal feeding behavior during exposure to K. brevis (Ray and Aldrich, 1967; Sievers, 1969), while oysters exposed to K. brevis during a bloom become toxic (Cummins et al ., 1971). More recently, Plakas et al (2002) and Wang et al (2004) documented brevetoxin uptake and metabolism in C. virginica in the laboratory. Oysters also eliminated brevetoxins once the oysters were removed from the algal source. In the present study, oysters removed the highest percentage of K. brevis cells from the surrounding media of all bivalve species examined. We showed that clearance rates in eastern oysters were reduced the least by exposure to K. brevis which supports the general conclusion that C. virginica are not critically impacted by K. brevis (Shumway et al ., 1990). Overall, whole cultures of K. brevis (intact cells) had a greater effect than lysed cultures (disrupted cells) on clearance rate in all species except C. virginica even though the amount of total brevetoxin was similar between the two preparations, suggesting that encounters with the dinoflagellate interfered with filtering capability. The New Zealand cockle ( Austrovenus stutchbury ) and the greenshell mussel ( P. viridis ) were shown to

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108 assimilate brevetoxins from K. brevis culture as well as from the supernatant from disrupted culture (Ishida et al ., 2004), but the effects of these preparations on feeding was not investigated. Additional studies using recently isolated strains of K. brevis including a non-toxic Wilson clone and two new isolates from Sarasota Bay (Florida, USA), could further elucidate these differences in bivalve feeding behavior. There was close within-species agreement in clearance rates between static and flow-through systems; however, the effects of K. brevis on A. irradians was shown to be significantly affected by exposure time, whereas clearance rates at both medium (100 cells ml-1) and high (1,000 cells ml-1) densities declined only after 24 hr exposure. For this reason, continuous flow-through systems are generally preferred over static systems when measuring physiological performance. With static systems, conditions are not held constant and therefore clearance rates may be affected if algal concentrations fall below a critical level (Widdows and Salkeld, 1993). Conditions in flow-through systems can be held constant (i.e., algal concentration), thus enabling continuous monitoring of clearance rate over extended time periods which more closely reflect environmental conditions during algal blooms. Additionally, flow-through systems allow for the monitoring of possible behavioral or physiological changes a ssociated with long term exposure to toxic algae (Lassus et al ., 1999). Bardouil et al (1996) suggested that longer exposure times are necessary to assess the effects of toxic algae on algal ingestion and toxin absorption in bivalve shellfish. Conclusion and Significance

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109 Impacts from Different Culture Preparations This research sought to determine whether there were differences in larval mortality and juvenile feeding for individual bivalve species, as well as bay scallop cytohistology, when exposed to two different culture preparations of K. brevis (Wilson clone). The culture preparations included 1) whole intact cell cultures and 2) cultures where the cells had been disrupted, or lysed by ultrasound. Each culture was analyzed for brevetoxin composition and concentration. In essentially every case, lysed cultures had higher reported brevetoxin amounts than the corresponding whole culture. Since the two preparations were obtained from the same batch culture, these differences in brevetoxin amounts are best explained by the extraction and recovery procedures. One explanation for these differences is the possibility that there is better extraction efficiency when cultures are lysed prior to extraction. An important observation from these data, however, is verification that a particular culture of K. brevis was not only toxic, but contained ratios of the major brevetoxins (PbTx-2 and PbTx-3) corresponding to previously analyzed cultures (Landsberg, 2002; Pierce et al ., 2005). These cultures also contained brevenal, a recently discovered polyether compound which, in fish, has been shown to competitively displace brevetoxin from its binding site thereby inhibiting the toxic effects of brevetoxins (Bourdelais et al ., 2004). The absolute amount of brevenal reported in each sample is calculated in PbTx-3 equivalents; whereby PbTx-3 is the standard used to develop the response factor upon which all fractions were quantified (Pierce, personal communication). Thus, the amount

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110 of brevenal indicated in a given sample is a relative, rather than absolute concentration. An important observation about the relative amount of brevenal in these cultures is that their concentration was approximately 10x higher than brevenal concentrations from water samples collected off Siesta Key, FL during a 2005 red tide (Pierce, unpublished). These reported differences in brevenal composition suggest that this laboratory culture of K. brevis may not be as toxic as natural blooms. Results from this study, therefore, may be considered as conservative estimates of the effects of this toxic dinoflagellate on bivalve molluscs. Lysed cultures of K. brevis did not undergo any additional processing, such as centrifugation or filtering, after sonification. As a result, the solution contained byproducts of the lysing process, including cellular debris and fragments in addition to the liberated toxins. It is reasonable to assume that these toxins, since they are lipophyllic, would adsorb onto these particles as they came in contact. Juvenile bivalves exposed to these conditions during feeding experiments would conceivably have to process this toxin-laden particulate matter in addition to the nontoxic algae. While it could be argued that removing this particulate matter prior to experimentation would have eliminated this variable, in reality, bivalves in nature must deal with intact toxic cells, particulate matter, extracellular toxins and whatever else is present during a bloom; consequently, what is learned from exposing bivalves to lysed treatments as they were prepared in this study are representative of natural exposure conditions. Juvenile bivalve feeding studies showed a significant effect of culture preparation on clearance rate in the bay scallop, green mussel and northern quahog. This difference was most noticeable in the green mussel and northern quahog at higher cell

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111 concentrations. In both cases, intact K. brevis produced significantly lower clearance rates while lysed K brevis had only a slight effect on clearance rate Two possible explanations for these results are: 1) lysed cultures made the toxins less bioavailable, or 2) the observed effects were due to the presence of the dinoflagellate and not the associated toxins. Since bivalves in the field are exposed to a combination of these conditions during a bloom of K. brevis it could be argued that the observed differences in clearance rate between culture preparations in the laboratory would not be as great in the wild. Possible Mechanisms of Toxic Activity Brevetoxins are polyether ladder neurotoxins that bind to voltage-sensitive sodium channels in cell membranes. Binding results in persistent activation of neuronal cells, skeletal muscle cells and cardiac cells (Baden, 1988). The manners in which brevetoxins affect mollusc tissues, or specific ways in which molluscs may respond to brevetoxin exposure, have not been thoroughly investigated. Observations from this research may be a direct result of brevetoxin interactions with specific tissues, particularly nerve cells, or they may be due to a secondary effect which may or may not include behavioral a nd physiological responses. What follows is an attempt to explain what these interactions might include. Juvenile bay scallops ( A. irradians ) were the most sensitive species to the presence of K. brevis in terms of clearance rate. Bay scallops are non-siphonate filter feeders and remain partially open when at rest. Scallops also live in seagrass meadows of

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112 the open coast and lower estuary where daily fluctuations in water quality are reduced. Northern quahogs and eastern oysters, on the other hand, can close their valves and survive anaerobically for extended periods of time. Oysters also inhabit the intertidal zone and regularly experience long periods out of the water while quahogs bury in sediments throughout the coastal zone. Oysters and quahogs are subjected to relatively greater daily fluctuations in water quality, particularly oxygen and temperature, than bay scallops. Differences in morphology and ecology among these bivalves may partially explain how each species responds to Karenia brevis in the field. Summerson and Peterson (1990) reported on recruitment failure of the bay scallop during an outbreak of K. brevis in North Carolina in 1987. The authors did not, however, mention the mechanism behind these observations. Since there are no other published reports on the effects of K. brevis on bay scallops in the field, insight into the physiological response might be obtained by looking at the response of scallops during exposure to brown tides, caused by the picoplantonic alga Aureococcus anophagefferens in eastern Long Island, NY over the past several decades. These responses also included recruitment failure, growth inhibition and decimation of local populations. The negative impacts of this alga were attributed to an unknown, dopamine-mimetic, bioactive/toxic metabolite which suppresses the activity of gill lateral cilia (Gainey and Shumway, 1991) and thus negatively impacted clearance rates. These effects were observed even in the presence of a mixed phytoplankton assemblage containing non-toxic algae similar to results from this research (Bricelj et al., 2004). Unlike the results from this study, however, toxic effects from brown tides required direct contact with the algal cell and did not appear to be

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113 associated with dissolved toxic exudates (Ward and Targett, 1989). Gill cilliary inhibition by brown tide was not demonstrated during in vitro trials (Gainey and Shumway, 1991) even though natural populations of this species are known to be adversely affected by brown tides. The most reasonable conclusion to draw from this body of information is that K. brevis also suppresses gill cilliary activity in the scallop. This could occur by direct action on the gill neuronal cells similar to the mechanism described above or by a secondary, indirect action. Perhaps more importantly, the effect of reducing gill cilliary activity also would affect oxygen uptake, thereby compounding the effects of K. brevis and/or its toxins on the bay scallop. Scallop mortality in the field has been observed within the first two days after the onset of a red tide (personal observation), indicating that the cause of death is more likely due to a lack of oxygen rather than starvation. Scallops, unlike oysters and quahogs, do not have the ability to close their shells for extended periods and undergo facultative anaerobiosis. Therefore, scallops are more vulnerable to blooms of K. brevis than other bivalves, in part, because of their inability to reduce or eliminate their exposure to this toxic dinoflagellate and its toxins.

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114 Implications for Fisheries Management Recurring blooms (= red tides) of K. brevis are common along the Florida west coast (Tester and Steidinger, 1997; Kirkpatrick et al ., 2004). Results of these studies clearly demonstrate short-term, negative impacts of K. brevis on resident bivalve species, suggesting that the ecological and fisheries impacts from these algal blooms could be quite significant, depending upon bloom intensity and duration, and which bivalve species are exposed. Results from these studies on bivalve larvae and K. brevis are the first reported and while we plainly demonstrated a negative impact of K. brevis on larval survival and development, there is a clear need for continued research into the mechanisms underlying these interactions. We also showed that feeding rates in juvenile bivalves were also negatively impacted by exposure to K. brevis and that the response was species-specific. The most sensitive species in the present study was the bay scallop, A. irradians A rare bloom of K. brevis in North Carolina during 1987-88 was implicated in the massive mortality and subsequent recruitment failure of local bay scallop populations (Summerson and Peterson, 1990). Recently, bay scallops have been the focus of restoration activities in several southwest Florida estuaries (Geiger and Arnold, 2003; Wilbur et al ., 2005; Leverone et al ., 2005). In 2001, a restoration project was irrevocably compromised when a dense (105 107 cells L-1) bloom of K. brevis infiltrated Sarasota Bay, FL, resulting in complete mortality of captive scallops (Leverone, unpublished). While more precise studies are necessary to resolve the relationship between red tide intensity and duration on bay scallop mortality, prediction and monitoring of algal blooms would be beneficial in identifying potential restorations sites that are less prone

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115 to chronic K. brevis blooms. Floridas hard clam ( M. mercenaria ) aquaculture industry would also benefit from improved red tide prediction and monitoring. Relocating lease sites to areas less susceptible to red tides would benefit the industry twofold: reduce the deleterious effects of high K. brevis concentrations on feeding rates which, in turn, would affect growth rates, and 2) reduce the probability that cultured clams will be prevented from reaching the market due to harvest closures (Shumway, 1990). Locating aquaculture sites in lower salinity waters might reduce the frequency and duration of exposure to red tides, which typically initiate in more saline offshore waters. If a red tide does penetrate the estuary, the lower salinity further into the bay could serve as a potentially effective salinity barrier to a bloom of K. brevis Similarly, reduced feeding rates in the green mussel ( P. viridis ) at high K. brevis concentrations should theoretically make it more difficult for mussel populations to remain established in estuaries where red tides are more frequent and/or severe. Emperical observations, however, suggest a different outcome. An intense red tide during 2005-06 resulted in high mortality of green mussels attached to pilings and other structures in lower Tampa Bay (personal observation). Intense recolonization by juvenile green mussels, however, was observed in late 2006, several months after the bloom had dissipated. The prolific and dynamic recruitment rates of green mussels and their ability to rapidly recolonize a previously inhabited space after a red tide has disappeared suggests populations of this exotic species have no difficulty overcoming the temporary effects of exposure to K. brevis Finally, the relative insensitivity of C. virginica feeding rates to K. brevis suggests that the structure and function of Eastern oyster habitats in southwest Florida should not suffer serious negative impacts from K. brevis blooms.

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116 Table 14. Reported impacts of Karenia brevis on molluscs Species Common name Field/ Laboratory Effect/ Observation1 Location K. brevis (cells L-1) Reference Bivalves Argopecten irradians bay scallop Field Mortality (?); Recruitment failure North Carolina 8.2 x 105 Summerson & Peterson (1990) Austrovenus stutchburyi New Zealand cockle Laboratory Toxic; Brevetoxin metabolism New Zealand 6-12 x 106 Ishida et al (2004) Brachidontes recurvus hooked mussel Laboratory Unaffected 9.9 x 106 Sievers (1969) Chione cancellata Field Toxic Sarasota, FL N.R. Poli et al (2000) Crassostrea virginica eastern oyster Laboratory Laboratory Field Field Field Laboratory Field Laboratory Unaffected (Normal feeding) Unaffected (Normal behavior) Toxic Toxic Toxic Toxic; Brevetoxin metabolism Toxic; Brevetoxin metabolism Toxic; Brevetoxin metabolism Galveston, TX Venice, FL Beaufort Inlet, NC Mississippi Sound, Gulf of Mexico Sarasota, FL N.R. 9.9 x 106 8.2 x 105 ~ 105 5.6 x 105 1.3 x 106 6.6 x 105 1.5 x 107 Ray & Aldrich (1967) Sievers (1969) Cummins et al (1971) Tester & Fowler, 1990) Dickey et al (1999) Plakas et al (2002) Pierce et al (2004) Wang et al (2004) Crassostrea gigas Pacific oyster Laboratory Toxic New Zealand 1.0 2.5 x 107 Fletcher et al (1998) Mulinia lateralis Cross-barred venus Field Mortality Tampa Bay < 1.8 x 107 Simon & Dauer (1972)

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117 Table 14. (Continued). Donax variabilis Variable coquina Field Toxic Venice, FL 8.2 x 105 Cummins et al (1971) Mercenaria campechiensis Southern quahog Field Field Field Laboratory Toxic Toxic Toxic Toxic Venice, FL Sarasota Bay, FL Englewood, FL Tampa Bay, FL 8.2 x 105 N.R. N.R. N.R. Cummins et al (1971) Hemmert (1975) Hemmert (1975) Roberts et al (1979) Mercenaria mercenaria Northern quahog Field Field Toxic Toxic; Brevetoxin metabolism Beaufort Inlet, NC Sarasota, FL ~ 105 6.6 x 105 Tester & Fowler, 1990) Pierce et al (2004) Mercenaria sp. Quahog Field Toxic Sarasota, FL N.R. Poli et al (2000) Macrocallista nimbosa Sunray venus Field Toxic Venice, FL 8.2 x 105 Cummins et al (1971) Perna canaliculus Greenshell mussel Laboratory Toxic; Brevetoxin metabolism New Zealand 6-12 x 106 Ishida et al (2004) Spisula solidissma raveneli Atlantic surfclam Field Toxic Sies ta Key, FL N.R. Hemmert (1975) Oysters Field Mortality Naples to Boca Grande, FL N.R. Gunter et al (1947) Clams Field Mortality Naples to Boca Grande, FL N.R. Gunter et al (1947) Gastropods Busycon contrarium Whelk Field Toxic Sarasota, FL N.R. Poli et al (2000) Busycon sp. Whelk Field Toxic Sarasota, FL 6.6 x 105 Pierce et al (2004) Fasciolaria lilium hunteria Banded tulip Laboratory Loss of muscle control Tampa Bay, FL N.R. Roberts et a l. (1979) Melongena corona Crown conch Laboratory Loss of muscle control Tampa Bay, FL N.R. Roberts et al (1979) Oliva sayana Lettered olive Laboratory Loss of muscle control Tampa Bay, FL N.R. Roberts et al (1979) 1Toxic = containing toxins using the mouse bioassay or analyti cal methods. Brevetoxin metabolism = the ability to metabolize pa rent toxins found in Karenia brevis N.R. = not reported.

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131 About the Author Jay Leverone received a B.A. in Biology in 1976 and a M.S. in Zoology in 1990 from the University of South Florida. He became a Staff Biologist at Mote Marine Laboratory in Sarasota in 1980 and has ma intained his staff position during both his Masters and Doctoral Degrees. He entered the Ph. D. program at the University of South Florida in 1996. While in the Ph.D. program, Mr. Leverone has been a student member of the National Shellfisheries Association and has presented portions of his doctoral research at the annual meetings. He also presented results from several shellfish restoration projects which he supervised while conducting his doctoral research. Mr. Leverone has made presentations at the International Conference on Shellfish Restoration and the International Pectinid Workshop. Two manuscripts have been published on his doctoral research. Leverone, Jay R., Norman J. Blake, Richard H. Pierce and Sandra E. Shumway. 2006. Effects of the dinoflagellate Karenia brevis on larval development in three species of bivalve mollusc from Florida. Toxicon 48: 75-84. Leverone, Jay R., Norman J. Blake and Sandra E. Shumway. Comparative effect of the toxic dinoflagellate Karenia brevis on clearance rates in juveniles of four bivalve molluscs from Florida, USA. Toxicon (In press). Mr. Leverone has been married to his wife, Barbara, for 23 years and has two wonderful children; a daughter, Donna and a son, Jason.


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ABSTRACT: The effects of the toxic dinoflagellate, karenia brevis (Wilson clone), on larval survival and development of the northern quahog (=hard clam, Mercenaria mercenaria), eastern oyster (Crassostrea virginica) and bay scallop (Argopecten irradians) were studied in the laboratory. The effects of K. brevis on feeding activities of juveniles from these species plus the green mussel (Perna viridis) were also examined. Finally, adult bay scallops were exposed to K. brevis for two weeks to investigate possible cytotoxic effects. Survival of 3-day-old larvae was generally > 85% for all shellfish species at Karenia brevis densities of 100 cells ml-1 or less, and not significantly different between whole and lysed culture. At 1,000 cells ml-1, survival was significantly less in lysed culture than whole culture for both M. mercenaria and C. virginica. Survival of 7-day-old larvae in all species was not significantly affected at densities up to 1,000 cells ml-1.^ ^At 5,000 cells ml-1, however, survival was reduced to 37, 26 and 19% for A. irradians, M. mercenaria and C. virginica, respectively. Development of C. virginica and M. mercenaria larvae was protracted at K. brevis densities of 1,000 cells ml-1. Clearance rates of juveniles were determined under static and flow-through conditions using whole and lysed cultures of K. brevis. The bay scallop was most sensitive, exhibiting a 79% reduction in clearance rate at 1,000 cells ml-1 of whole culture. The eastern oyster was least responsive, showing a 38% reduction in clearance rate between the same treatments. The green mussel and the northern quahog displayed intermediate responses. Similar results were observed during longer (2 day) exposures to a continuous supply of K. brevis. Bay scallops showed a significant decline in clearance rate at 100 cells ml-1 after 24 hr exposure; clearance rate of oysters was not affected by K. brevis at this concentration.^ ^No mortality was observed for any species during these brief exposures. Adult bay scallops exposed to K. brevis for two weeks showed degenerative and inflammatory changes in the digestive gland, including reduced thickness of the epithelium, increased size of digestive tubule lumens and hemocytic infiltration. The prospect for recovery of bay scallop populations in Florida may be hampered by recurring blooms of K. brevis.
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Histopathology.
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