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Clearance Rates and Particle Selectivity in the Hard Clam, Mercenaria mercenaria, from Warm Water Habitats


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CLEARANCE RATES AND PARTICLE SELECTIVITY IN THE HARD CLAM, Mercenaria mercenaria, FROM WARM WATER HABITATS By CARLA DANIELLE BEALS A THESIS PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE UNIVERSITY OF FLORIDA 2004

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Copyright 2004 by Carla Danielle Beals

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ACKNOWLEDGMENTS I would like to thank my advisor, Dr. Shirley Baker, for giving me the opportunity to conduct research under her guidance. My experience here has been invaluable. In addition, I would also like to thank Dr. Derk Bergquist for all his help, advice, and patience in answering my questions, especially the questions regarding statistics. I would also like to thank my committee members Dr. Edward Phlips, Dr. Thomas Frazier, and Dr. Debra Murie. In addition, I would also like to thank Neil Benson, UF Flow Cytometer Core Lab, for showing me how to use the FacScan Flow Cytometer and for help in analyzing the data. Also, I would like to thank Marinela Capanu, Graduate Assistant Consultant for the IFAS Department of Statistics, for her advice on the statistics used for this thesis. Thanks also go to Christina Jett-Richards and Erin Bledsoe for all their help and guidance. Likewise, special thanks also go to Stephanie Keller, Jamie Greenawalt, Daniel Goodfriend, Brooke Rimm-Hewitt, Edward DeCastro, and Karen Donnelly for help in the lab. Funding for this project was provided by the Sigma Xi Grants-in-Aid and the USDA Eutrophication Project. Last, but not least, I would like to thank Dr. Robert and Mrs. Doris Kline; whose unending kindness, patience, and support was invaluable to me. iii

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TABLE OF CONTENTS page ACKNOWLEDGMENTS .................................................................................................iii LIST OF TABLES .............................................................................................................vi LIST OF FIGURES ..........................................................................................................vii ABSTRACT .....................................................................................................................viii CHAPTER 1 INTRODUCTION........................................................................................................1 2 LITERATURE REVIEW.............................................................................................4 Description of the Study Area......................................................................................4 Sources of Nutrients to the Suwannee River Estuary...................................................4 Characteristics of Algal Blooms...................................................................................8 Bivalves and their Effect on the Water Column...........................................................8 Suspension Feeding in Bivalves...................................................................................9 The Effect of Temperature on Bivalve Clearance Rates............................................10 The Effect of Diet Composition and Concentration on Bivalve Clearance Rates......11 Selective Feeding in Bivalves.....................................................................................13 Controversies Concerning Bivalve Feeding Mechanisms..........................................14 3 METHODS AND MATERIALS...............................................................................17 Test Subjects...............................................................................................................17 Experimental Algae and Culture Protocols.........................................................17 Phytoplankton Combinations..............................................................................18 Experimental Organism.......................................................................................18 Experimental Protocol................................................................................................18 Additional Experiments.......................................................................................19 Calculation of Particle Selectivity.......................................................................20 Calculation of Clearance Rate.............................................................................21 4 RESULTS...................................................................................................................25 Electivity Indices........................................................................................................25 iv

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Clearance Rates..........................................................................................................26 5 DISCUSSION.............................................................................................................38 LIST OF REFERENCES...................................................................................................47 BIOGRAPHICAL SKETCH.............................................................................................56 v

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LIST OF TABLES Table page 1 Algal assemblages and the date(s) of replication(s) of each feeding trial at two different temperatures..............................................................................................23 2 Mean size and weight, and actual number of animals that opened in each feeding trial...........................................................................................................................24 vi

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LIST OF FIGURES Figure page 1 Electivity indices (means SE) for Mercenaria mercenaria at two different temperatures, 20 o C and 30 o C, when fed different combinations of algae at a total concentration of 10 5 cells/ml....................................................................................28 2 Electivity indices (means SE) of Mercenaria mercenaria for Isochrysis galbana when Synechococcus sp. (nonchainforming) is present, at two different temperatures, in clams from a single batch (IsoSyn-B)...........................................29 3 Electivity indices (means SE) of Mercenaria mercenaria for Isochrysis when Synechococcus sp. (non-chainforming) is present, at two temperatures, 20 o C and 30 o C, and two cell concentrations a) 10 5 and b) 10 6 cells/ml...................................30 4 Mean replication (or batch) electivity indices (mean SE) for Mercenaria mercenaria at two temperatures, 20 o C and 30 o C, when fed different combinations of algae.....................................................................................................................31 5 Mean electivity indices of Mercenaria mercenaria for Isochrysis galbana when Synechococcus sp. is present, at two temperatures, in clams acclimated for two weeks on either a) Synechococcus (IsoSyn-AS) or b) Isochrysis (IsoSyn-AI)...............................................................................................................33 6 Clearance rates by Mercenaria mercenaria (means SE ) at two temperatures (20 o C and 30 o C) when fed algal suspensions at 10 5 cells/ml...................................34 7 Clearance rates (means SE) of Mercenaria mercenaria fed Isochrysis galbana and the nonchainforming strains of Synechococcus (IsoSyn-B) at two temperatures (20 o C and 30 o C)..................................................................................35 8 Clearances rates (means SE) by Mercenaria mercenaria fed I. galbana and Synechococcus sp. at two temperatures (20 o C and 30 o C) and two concentrations...........................................................................................................36 9 Clearance rates (means SE) of Mercenaria mercenaria of the feeding trial Isochrysis galbana and the nonchain-forming species of Synechococcus (IsoSyn-AS and IsoSyn-AI) at two different temperatures (20 o C and 30 o C) when clams were acclimated to either a) Synechococcus or b) Isochrysis..................................37 vii

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Abstract of Thesis Presented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Master of Science CLEARANCE RATES AND PARTICLE SELECTIVITY IN THE HARD CLAM, Mercenaria mercenaria, FROM WARM WATER HABITATS By Carla Danielle Beals December 2004 Chair: Shirley M. Baker Major Department: Fisheries and Aquatic Sciences The objective of this study was to examine the effects of temperature and phytoplankton concentration on the feeding selectivity and clearance rates of the hard clam Mercenaria mercenaria. My hypothesis was that temperature has an effect on the ability of the hard clam to preferentially ingest certain particles over others. Adult clams obtained from a commercial supplier were subjected to laboratory manipulated phytoplankton assemblages of three different algae (Synechococcus sp., Isochrysis galbana, and Tetraselmis maculata) of different sizes (2-m, 5-m, and 10-m) at two temperatures (20 o C and 30 o C). One feeding treatment was conducted at two concentrations (10 5 cells/ml and 10 6 cells/ml). Clearance rates were determined from counting phytoplankton cells in water samples collected at the beginning and end of each feeding trial using a FacScan flow cytometer. Electivity indices were determined from initial water samples and clam pseudofeces using a FacScan flow cytometer. viii

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Temperature, algal combination, and concentration all had significant effects on feeding selectivity of clams. Clams had greater selectivity at 20 o C than at 30 o C. In addition, clams showed a trend to select for larger particles over smaller particles. Selectivity was greater at the lower concentration than the higher concentration. Within each temperature/algal combination, there was a high amount of variability in electivity indices between replicates. Temperature, algal combination, and concentration had no effect on clearance rates. There was, however, an interaction effect between temperature and algal combination. Feeding history, adaptation of clams to their environment, seasonal changes in digestive enzymes, and/or other parameters like changes in water viscosity due to temperature may account for high variability in electivity indices and clearance rates between replicates. Results obtained have implications for future selectivity and clearance rates studies. In addition, this study provides important information for the future productivity of cultured clams in semi-tropical environments by demonstrating that feeding preferences may be different for clams from cooler environments. ix

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CHAPTER 1 INTRODUCTION The Suwannee River begins in the Okeefonokee Swamp and meanders through southern Georgia and north central Florida before it discharges into the Gulf of Mexico, draining 28,500 km 2 (Wolfe & Wolfe, 1985). Its estuary and the surrounding regions (also known as the Big Bend) are home to a highly productive nursery of fish and marine invertebrates (SRWMD, 1979). Oysters (i.e. Crassostrea virginica) are regularly harvested and clam farming (i.e. Mercenaria mercenaria) has exploded as a new aquaculture industry. In 2001, hard clam aquaculture comprised over 18% of Floridas $99 million total aquaculture sales (USDA, 2002). As the human population increases in northern Florida, anthropogenic activities are more likely to have a serious impact on the shallow estuarine waters along the Big Bend. Surface water runoff and ground water are contributing high concentrations of nutrients which, in turn, may cause phytoplankton blooms in the estuary (Phlips & Bledsoe, 1997; Bledsoe & Phlips, 2000). Maintaining a balance between the nutrients needed for productivity and excessive eutrophication is important for the stability of the Big Bend ecosystem, and especially for the growth and stability of the newly emerging clam farm industries. Understanding this balance and the effects of eutrophication on bivalves is essential. Bivalves are adept suspension feeders and can modify seston in estuarine waters (Carlson et al., 1984; Asmus et al., 1990). On the other hand, seston quantity and composition can have effects on bivalve feeding behavior, particle selection, and 1

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2 clearance rates (Bayne et al., 1989, 1993; Navarro et al., 1996). Studies on bivalves have shown an ability to sort particles based on size (Stenton-Dozey & Brown, 1992; Defossez & Hawkins, 1997) and quality (Arifin & Bendell-Young, 1997; Ward et al., 1997). Furthermore, there appears to be variability among bivalve species in their ability to sort and preferentially ingest particles (Shumway et al., 1985; Prins et al., 1991; Ward et al., 1998). Studies by Bayne et al. (1989;1993) have corroborated the idea that changes in food concentration can have an effect on clearance rates and, ultimately, the growth and productivity of bivalves. Baker et al. (1998) demonstrated that the diversity of a plankton assemblage is important in determining clearance rates of the zebra mussel Dreissena polymorpha as well as selectivity for individual species of phytoplankton within the assemblage. While studies above have shown the composition of the seston to have an impact on clearance rates, temperature also has an effect. In a review of the physiological ecology of the hard clam, Grizzle et al. (2001) stated that temperature affected feeding rates. Feeding rates peaked at about 24-26 o C, but fell abruptly at temperatures above 27 o C. In this same review, temperatures between 20-24 o C were shown to be optimal for clam growth with decreasing growth rates outside this range. This is important because feeding rates are thought to be the physiological control on growth rates. While many studies have shown extremes in temperature to have a negative effect on clearance rates and growth of bivalves, there have been few, if any, studies to show what affect temperature may have on feeding selectivity. Selectivity studies are usually carried out at temperatures of 20 o C or less (e.g., Shumway et al., 1985; Bayne et al. 1989; Stenton-Dozey & Brown, 1992; Bayne et al. 1993; Arifin & Bendell-Young, 1997;

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3 Defossez & Hawkins, 1997; Ward et al., 1998). Surface waters in the Suwannee River estuary, however, commonly exceed 25 o C between March and October and temperatures around 30 o C are normal during summer months (T. Frazer, unpublished data; E. Phlips, unpublished data; Jett, 2004). This challenges what we know about factors important to clam aquaculture success and our understanding of bivalve feeding physiology. This study is part of a multi-faceted investigation determining the potential effects of coastal eutrophication on phytoplankton and bivalve communities of the Suwannee River estuary. The objective of this study was to examine the effects of temperature on particle selectivity and clearance rates of Mercenaria mercenaria feeding on bloom concentrations (10 5 and 10 6 cells/ml) of phytoplankton. Based on preliminary experiments, my hypothesis was that temperature would have an effect on the ability of hard clams to preferentially ingest smaller particles over larger particles. These effects may have a seasonal impact on the seston composition of the Suwannee River estuary.

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CHAPTER 2 LITERATURE REVIEW Description of the Study Area The Suwannee River estuary is among the ten largest estuaries along the Gulf Coast of Florida. It is located between latitudes 2913N and 2920N and longitudes 83 05 W and 83 12 W (Siegal et al., 1996). The estuary is home to a highly productive nursery of fish and marine invertebrates (SRWMD, 1979). The Suwannee River, which feeds the estuary, originates largely in the Okefenokee Swamp of southern Georgia and drains 28,500 km 2 of southern Georgia and north central Florida (Wolfe & Wolfe, 1985). It is fed, also, by the Alapaha, Withlacoochee, and Santa Fe Rivers. In addition, groundwater which emanates as spring discharges and diffuse seepage also feeds into the river. Groundwater contributions to riverflow are particularly important during low flow periods as a consequence of large-scale climate variation and reduced rainfall in the watershed. Due to the karst nature of the terrain, i.e. sinkholes, springs, and conduit systems in the underlying limestone, groundwater and surface water can have direct hydraulic and geochemical interactions (Katz et al., 1997; Crandall et al., 1999). These interactions are important because surface water and groundwater inputs contribute high concentrations of nutrients to the river which, in turn, discharges into the estuary (SRWMD, 2000b). Sources of Nutrients to the Suwannee River Estuary Nitrate concentrations in the river and loads to the Suwannee River Estuary have steadily increased over the years (Jones et al., 1996, 1997; Pittman et al., 1997). In fact, 4

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5 from 2001 to 2003, nitrate loads have more than doubled from 2676 tons to 4,591 tons (SRWMD, 2001b; SRWMD, 2003). Increased nitrates can cause a variety of problems. In drinking water it can cause health concerns like methelmoglobinemia or blue baby syndrome. Because nitrogen is a basic requirement for algae and other vegetation and can cause excessive growth, increased nitrogen loads are also an ecological concern. Studies have shown a positive correlation between nitrate concentration and growth of planktonic algae in this system (Phlips & Bledsoe, 1997; Bledsoe & Phlips, 2000). The years 1999 and 2000 were among the driest in the Suwannee River watershed since 1932. The annual mean stream flow was reduced to 28 to 52% of the long-term average (flow discharge of less than 142 m 3 *sec -1 ). As a consequence, elevated nutrient concentrations and extensive algal blooms occurred, with an appearance of some nuisance species like the red drift algae Gracilaria and filamentous brown algae Ectocarpus that are known to cause various water quality related problems (Bledsoe & Phlips, 2000; SRWMD, 2001b). Algal blooms can have an affect on the marine invertebrates and fish. For example, algal blooms can lead to extreme fluctuations in dissolved oxygen (DO) concentrations in the water column that can have damaging effects, sometimes fatal, on marine invertebrates and fish (Barica, 1980; Knights et al., 1995). In addition, hypoxic and microxic conditions can affect the feeding behavior of young post-settlement oysters which can limit or delay recruitment into the adult oyster population (Baker and Mann, 1994). Nitrogen enters the estuary in various ways: 1) the Floridian aquifer, 2) groundwater, and 3) the Suwannee River. The Floridian aquifer is impressive in that it is

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6 one of the largest underground freshwater aquifers in the United States. The surrounding environment of the aquifer is composed of carbonate rock, such as marine dolomite and limestone (Rosenau et al., 1977; SRWMD, 2001a). Sand or clay usually covers most of the carbonate rock. The aquifer extends from the southern portions of Alabama, Georgia, and South Carolina to the northeastern part of Florida to the Atlantic Ocean and the Gulf of Mexico. Weakly acidic rainwater dissolves the carbonate rock and creates cavities and caves in the aquifer. This type of terrain is known as a karst region and has many sinkholes and springs. In addition, it lacks a well-developed drainage system (SRWMD, 2001a). Groundwater can enter the surface water system, e.g., the Suwannee River, through spring discharges or breaches in the underlying aquifers. The river, in turn, discharges into the estuaries. In addition, groundwater can enter estuaries as seepage along the coastline (Cable et al., 1997). Spring water is usually a good indicator of the quality of the groundwater (SRWMD, 2001a). Human activities can have an effect on the quality and quantity of groundwater flow and these changes can have a significant impact on estuarine or coastal ecology because these areas receive large amounts of groundwater. Cable et al. (1996; 1997) have traced groundwater discharge by using 222 Rn and CH 4 and estimated that the seepage of groundwater into the northeastern coastal areas of the Gulf of Mexico (ca. 10 km 2 ) was equivalent to a first magnitude spring (i.e. a spring with > 2.8 m 3 sec -1 discharge). Studies over a 12-year period (1970-1991) have shown a statistically significant increase in nitrate concentration in the Suwannee River (Ham & Hatzell, 1996). In the upper part of the river, the major source of this increase was due to transport of nitrates

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7 by springs, while diffuse groundwater flow was the major source of transport in the lower portion (Pittman et al., 1997). Augmenting this increase is the karst nature of the aquifer, which allows leaching of nitrates into the aquifer making it more susceptible to contamination (Kreitler & Browning, 1983). Due to the karst topography of the terrain and increasing development along the Suwannee River, anthropogenic pollutants can also enter the estuary. High nitrate concentrations have been shown to come from numerous sources, highest among them being animal wastes, fertilizers, sewage effluent disposal, and residential and golf course landscapes (Jones et al., 1996; 1997; Katz et al., 1999). Katz et al. (1999) have shown that even though estimated nitrogen inputs from animal wastes have increased over the past 40 years in both Suwannee and Lafayette Counties, the nitrogen contributed by fertilizers is the highest input into the Suwannee River. Because the average residence time of groundwater discharge from springs is on the order of decades, there is little to be done to reduce the present nitrogen load to the estuary (Katz et al., 2001). While there are instances of denitrification by microorganisms, which can affect a decrease in the nitrogen load, especially in more eutrophic areas, studies have shown that the ability of ecosystems to regulate themselves can be exceeded by anthropogenic inputs into the ecosystems (Seitzinger & Nixon, 1985; Katz et al., 1997). In the water year 2000, 2676 tons of nitrate-nitrogen was delivered to the Gulf of Mexico by Florida rivers. The vast majority, 2620 tons, was supplied by the Suwannee River (SRWMD, 2001b). Since an immediate reduction of nitrate-nitrogen is unlikely, we need to examine the effects of the increased nitrogen loading in the estuary to determine what impacts are likely to occur.

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8 Characteristics of Algal Blooms Phytoplankton are an important part of all estuarine and marine ecosystems and phytoplankton blooms are a naturally occurring phenomenon. Increased nutrient delivery to estuaries can result in an increase in the frequency and intensity of these blooms. Not all algal blooms are harmful, however. Blooms of some species may only cause water discoloration. Only when a phytoplankton species increases significantly in population size and has detrimental ecological and physiological effects on the surrounding area, it is considered a harmful algae bloom (HAB). Concentrations may vary depending on the species composition of the phytoplankton assemblages. For nontoxic species, biomass is the primary determinant of bloom conditions. For toxic species, the presence of a toxin in the water can determine the bloom status (Smayda, 1997). While the red tide dinoflagellate, Karenia brevis, has been documented at least once in the Suwannee River estuary, it is not a common occurrence (Bledsoe & Phlips, 2000). Instead, typical bloom species include cyanobacteria and diatoms in the genera Rhizoselenia, Thalassiosira, Cyclotella, and another unidentified small centric diatom ranging from 3 to 10 m in diameter (Bledsoe & Phlips, 2000; 2004). Bivalves and their Effect on the Water Column Bivalves are dominant suspension feeders in many estuarine ecosystems and are capable, in some cases, of maintaining phytoplankton at low levels. In a study on the freshwater clam, Corbicula fluminea, in the Potomac River, Cohen et al. (1984) found that the lowest concentrations of phytoplankton biomass were in the areas with the highest densities of clams. In the Chesapeake Bay, results from an estuarine model showed that resident bivalves could consume more than 50% of the primary production in shallow segments of the bay in spring and summer with 45% to 95% of the water

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9 column being filtered by the bivalves during spring, summer, and fall (Gerritsen et al., 1994). Werner and Hollibaugh (1993) also suggest that the clam, Potamocorbula amurensis, has a substantial impact on the phytoplankton biomass in northern San Francisco Bay. With a density of more than 2,000 clams m -2 and an average clearance rate of 267 ml/h per clam, they calculated that the bivalves at a depth of 10-m could filter the water column 1.28 times per day, while those in shallower waters (1-m) could filter the water column 12.8 times per day. Based on these findings and those mentioned above, it is evident that bivalves have the capacity to greatly influence the abundance of phytoplankton, especially in enclosed systems. Suspension Feeding in Bivalves According to LaBarbera (1981), suspension feeding is comprised of three separate processes: 1) movement of water past suspension-feeding structures, 2) removal of particles from the water, and 3) transport of food particles to the mouth. All three processes are accomplished by means of both mucociliary and hydrodynamic processes (Ward, 1996). Bernard (1974) related the pallial cavity (the latero-ventral space surrounding the visceral mass that includes the gills, labial palps, stomach, and rectum) of a bivalve to a simple pump housed in a chamber (inhalant chamber) provided with a restricted inlet (inhalant aperture) and a larger exit (exhalant aperture) in which the ctenidia (gills) functions as a large diaphragm which is also porous to water. Water is drawn through the inhalant siphon then encounters a partial obstruction, the ostial aperture, on its way through the ctenidium by water tubes and out the exhalant siphon (Bernard, 1974). The ostial aperture is the area that contains numerous pores (ostia) that connect the inhalant chamber with the water tubes, and comprises about 37% of the ctenidium (Jones et al.,

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10 1992). This obstruction of the ostial aperture decreases the water speed as it approaches the ctenidium. Bernard (1974) also suggests that this allows preingestive selection of particles by allowing larger particles, like minerals or inorganic particles, to settle directly on the inhalant chamber mantle surfaces, according to the influence of gravity, and to be ejected. Once particles are captured on the ctenidium, they are transported by the activity of the frontal cilia. Mucus may play a role in the transportation of particles (Bernard, 1974; Beninger et al., 1992; Ward, 1996; but see Jrgensen 1990). For most bivalves, the majority of particles > 4 m are completely retained on the ctenidium while smaller particles, 1 m to 4 m, are retained with various efficiencies (Mhlenberg & Riisgrd, 1978; Jrgensen, 1975). Some bivalves, like oysters, sort particles on the ctenidium. The rejected particles are excreted as pseudofeces (Ward et al., 1997). The particles selected for ingestion are moved dorsally to the labial palps for further selection and then to the mouth for ingestion. The remains of digested particles are excreted as feces after passing through the gut (Beninger et al., 1992; Ward et al., 1997). The concentration of the particles that the bivalve is exposed to appears to have an effect on the aforementioned feeding mechanisms. Beninger et al. (1992) found that as the concentration of particles increases, bivalves start to exhibit some ingestion volume control, among them being a reduction or a stoppage of movement of particles. In addition, bivalves start to exhibit lower selectivity where good particles are often rejected as pseudofeces (Beninger et al., 1992). The Effect of Temperature on Bivalve Clearance Rates Several investigations have shown temperature to have a hyperbolic effect on clearance rates of bivalves (e.g., Hibbert, 1977; Grizzle et al., 2001). Hence, clearance

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11 rates increase as temperatures increase until an optimum temperature is reached after which clearance rates start to decrease. Optimum temperatures may vary between bivalve species. For example, for the penshell, Atrina maura, Leyva-Valencia et al. (2001) found clearance rates to be highest at 29 o C when tested over a temperature range of 19-35 o C. The clearance rates for Crassostrea gigas reach a maximum at 19 o C (Bougrier et al., 1995). For the catarina scallop, Argopecten ventricosus, clearance rates are greatest at 19-22 o C (Sicard et al., 1999). Other investigations, however, have reported a different response. For instance, Sobral and Widdows (1997) showed that increasing temperature over the range 20 o C to 32 o C caused clearance rates to decrease in Ruditapes decussatus. Haure et al. (1998) showed clearance rates to increase for Ostrea edulis over a temperature range of 10-30 o C. In fact, rates were maximum at 30 o C. Doering and Oviatt (1986) also found clearance rates to have a linear relationship with temperature. On the other hand, some studies have shown clearance rates to be independent of temperature (Loosanoff, 1958; MacDonald et al., 1996). Loosanoff (1958) showed pumping rates to be independent of temperature for adult oysters (Crassostrea virginica) between 16 and 28 o C. Smaal et al. (1997) looked at clearance rates of mussels (Mytilus edulis) and cockles (Cerastoderma edule) and found that there was no relationship between temperature and clearance rates of mussels throughout the year. However, Smaal et al. (1997) found that temperature did have an effect on clearance rates for cockles. The Effect of Diet Composition and Concentration on Bivalve Clearance Rates There have been numerous experiments conducted to understand under what diet conditions bivalves perform best. These studies have determined that both seston quantity and quality may have an effect on bivalve feeding rates. In general, there is a

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12 positive correlation between seston concentration and clearance rate (Albentosa et al., 1996; Marsden, 1999; Hawkins et al., 2001; Ellis et al., 2002). Stenton-Dozey and Brown (1992) showed that Venerupis corrugatus displays an ability to alter its clearance rate in response to the quantity of the seston; as the seston concentration increased, the clearance rate of V. corrugatus increased. In another study, Mytilus edulis exhibited an increased rate of ingestion with increased particle concentration until the rate of ingestion reached an asymptotic value, which coincided with the threshold for pseudofeces production (Bayne et al., 1989; 1993). In some cases, however, clearance rates appear to be unaffected by increasing seston concentration (Cranford & Hargrave, 1994; Arifin & Bendell-Young, 1997). Rheault and Rice (1996), for example, found that clearance rates of C. virginica and A. irradians irradians did not vary significantly with fourfold tidal variations in food concentration. However, A. irradians did exhibit a reduction in clearance rates when the seston concentration was decreased by 88% compared to the original concentration. Cranford and Hargrave (1994) obtained similar results with ingestion rates (biodeposition rates) for Placopecten magellanicus using a new method for quantifying the feeding and absorption rates of suspension feeding bivalves. Arifin and Bendell-Young (1997) did not find a relationship between clearance rates and seston concentration, but did find that pseudofeces production was dependent on seston concentration which, as stated before, is another way for bivalves to regulate ingestion rates. In a study examining the effects of seston quality on bivalve physiology, raft mussels (Mytilus alloprovincialis) exhibited maximum clearance rates and absorption efficiencies on mixed diets in which phytoplankton and sediment were provided in

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13 similar proportions, with the phytoplankton being 30-40% of total particulate volume (Navarro et al., 1996). Gatenby et al. (1996) found that cultured freshwater mussels (Villosa iris and Pyganodon grandis) grew best on mixed diets of algae and sediment rather than on algae alone. Both of the above studies suggest that sediment could play a vital role in enhancing absorption of microalgae or could even help with digestion in the stomach. Generally, as seston quality increases, so does the clearance rate of a bivalve. In a study by Stenton-Dozey and Brown (1992), clearance rates were greatest during high tide when there was an abundance of particles greater than 9 m and the organic content of available food was higher than at low tide. Likewise, Gardner (2002) found that clearance rates for three species of bivalves (Aulacomya maoriana, Mytilus galloprovincialis, and Perna canaliculus) increased in a linear fashion, with the highest clearance rates at high levels of organic matter in mixed diets. Selective Feeding in Bivalves Although there is some question as to how bivalves select particles for ingestion, it is generally accepted that bivalves can selectively ingest particles (Shumway et al., 1985; Newell et al., 1989; Defossez & Hawkins, 1997). Selectivity can be divided into two categories: 1) separation of inorganic particles from organic ones and 2) selection between organic particles (Bernard, 1974). For the first category, experiments involving variations in quantity and organic content of bivalve diets show that when presented with a mixed diet, the pseudofeces often contain intact, nonorganic particles (Newell et al., 1989; Iglesias et al., 1992; Bayne et al., 1993; Arifin & Bendell-Young, 1997). This

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14 preingestive selection of organic versus inorganic particles could be based on size (Bernard, 1974; Tamburri & Zimmer-Faust, 1996; Defossez & Hawkins, 1997). Selectivity studies subjecting bivalves to organic particles of differing nutritional value have shown the pseudofeces to contain particles that are organic in nature, but not very nutritious, like detritus (Baker et al., 1998; Ward et al., 1998). In comparing selectivity between phytoplankton, the majority of the selection and clearance rate experiments have used few phytoplankton species, comparing preferential selection of nutritious versus less-nutritious phytoplankton. With regard to zooplankton, Wong et al. (2003) showed that rotifers in the microand mesozooplankton (140-210 m range) can play a significant role in the diet of zebra mussels (Dreissena polymorpha). There are some experiments, however, involving various organic particles that indicate that there is also selection by bivalves between nutritious particles. Baker et al. (1998) showed that zebra mussels preferentially selected the cyanobacterium Microcystis over other phytoplankton that are typically found in the Hudson River. Shumway et al. (1985) showed that there is preferential selection of the cryptomonad flagellate, Chroomonas salina, over other nutritious particles in the majority of bivalves they tested. Further information is needed to understand particle selectivity. Controversies Concerning Bivalve Feeding Mechanisms There is still a question among scientists concerning the mechanisms of particle capture and selection in bivalves. Ward et al. (1998) found that the ctenidia of Crassostrea virginica are responsible for particle sorting and the labial palps perform an accessory function in particle selection or they regulate the volume of the ingested material. Ward et al. claim that it is essential for food particles to approach the gill

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15 surface in a straight line at an angle of 30 degrees from the gill surface. They suggest that variations of this angle could have an effect on the interaction of particles with the gill filaments. A number of scientists disagree with the prerequisite of a low angle of approach (Beninger, 2000; Riisgrd & Larsen, 2000). For instance, Riisgrd and Larsen (2000) feel that because of the parallel arrangement of the lateral cilia, particles hit the ctendium in a curved path between 70-90 degrees and that none hit below 40 degrees. They feel that the 30 degrees that Ward et al. (1998) stated is not a prerequisite of particle capture, but rather a result of the existing flow fields caused by ciliary activity on the ctenidia in the bivalve. Another disagreement concerns the role of mucus in suspension feeding by bivalves. There are basically two hypotheses concerning the role of mucus. The first is that mucus only forms to entrap materials when the ingestive capacity of the bivalve is exceeded (Jrgensen, 1990). The second hypothesis is that mucus is present at all times and is a normal function of bivalve physiology (Beninger et al., 1993; Ward, 1996). According to Bernard (1974), there appears to be two types of mucus in bivalves that are involved in entrapment and rejection of particles. The mucus for entrapment of food particles headed for ingestion appears to be light-colored and almost transparent, while the rejection mucus appears to be darker and contains more particles. Beninger et al. (1992) also has observed two distinct mucus types with the denser mucus being associated with those particles that have been rejected. Because of their opaque shells, it is hard to determine what goes on during the normal functioning of living, intact bivalves. Different techniques for observation of bivalve physiological mechanisms have been employed, e.g., dissection with isolated gill

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16 filament preparations, the use of surgically altered animals, the use of confocal laser microscopy, and, most recently, the use of endoscopic techniques, have all been used to collect information. These methods have led to a different controversy involving the manner of data collection. Ward et al. (1998) have expressed some concerns about the data collected from techniques other than video endoscopy. These investigators feel that the manner of preparation of the organism for either methods of observation could change some critical interactions between the way particles interact with the gill filaments. However, some scientists disagree with this assessment (Beninger, 2000; Riisgrd & Larsen, 2000; Silverman et al., 2000). Beninger (2000) states that due to mechanical limitations endoscopy cannot in fact access the underlying mechanisms for the processes of particle capture, transport, and selection, although it can observe the net result of the processes and suggest where and what techniques to use to seek the sequence. These scientists suggest that results from previously mentioned techniques and endoscopy are similar and that all techniques should be used to balance the weaknesses and strengths of the individual methods.

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CHAPTER 3 METHODS AND MATERIALS Test Subjects Experimental Algae and Culture Protocols Four algal species were used in this study: Tetraselmis maculata (a large green flagellate), Isochrysis galbana, (a small brown flagellate) and two strains of Synechococcus (a cyanobacteria; one strain forms long chains and one consists primarily of single cells). Algal cultures were supplied by Jose Nunez (I. galbana and T. maculata), Whitney Laboratory at the University of Florida, Dr. Edward Phlips (I. galbana and both species of Synechococcus), Department of Fisheries and Aquatic Sciences at the University of Florida, and Dr. Gary Wikfors (T. maculata), Milford Laboratory. Algal species were chosen to represent different sizes, with T. maculata having cells that are ca. 10-m, I. galbana ca. 5-m, and Synechocccus spp. ca. 2-m. I. galbana is the primary algae used in shellfish hatcheries, but T. maculata is also used. Synechococcus spp., and other picoplankton-sized blue-green algae, are not typically used in hatcheries (Hadley et al., 1997) but are a major bloom forming taxa in coastal marine environments in Florida (Phlips et al., 1999; Bledsoe, 2003). Of the three, cyanobacteria are regularly found in the Suwannee River estuary (Bledsoe and Phlips, 2000). All algal species were cultured in 500-ml flasks containing 150 ml of sterilized L1-Si media at 28 ppt (Guillard and Hargraves, 1993) at a pH of 7.8-8.2. Cultures were kept at 25-28 C on a 14:10 light/dark photoperiod at 30-60 Einsteins/m 2 /s light flux except for the Synechococcus spp. which were kept at 20-30 Einsteins/m 2 /s light flux. Light 17

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18 intensity was measured using a quantum light meter with a Li-Cor data logger. Once cultures reached sufficient biomass they were transferred to either 1000-ml flasks or 2.8-L Fernbach flasks that contained 500 ml and 1.5 or 1.8 L of L1-Si, respectively. The 500-ml and 1000-ml flasks were swirled once a day. Fernbach flasks were aerated with 0.22-m filtered air containing 1/32 psi of added CO 2 Phytoplankton Combinations Experimental phytoplankton assemblages consisted of two of the algae species types combined at a 1:1 ratio based on numerical abundance (Table 1). Initial counts were determined by using either a flourescent microscope or a Coulter Multisizer II fitted with a 100-m orifice. Enough phytoplankton was added to the experimental water to bring the total experimental concentration to 10 5 cells/ml or 10 6 cells/ml. Experimental Organism Adults of the hard clam Mercenaria mercenaria were obtained from commercial suppliers operating out of Cedar Key, Florida, a coastal area influenced by the Suwannee River. Clams were allowed to purge in 0.-m filtered seawater (28 ppt) for 72 hours at the experimental temperature with no prior temperature acclimation. All water in the holding tanks was changed twice during this period. Shell length, height, and width were measured according to Fritz (2001). Data on size, weight, and number of animals used in each feeding trial are provided in Table 2. Experimental Protocol Between December 2003 and March 2004, feeding selectivity and clearance rates were examined through a series of laboratory feeding trials in static systems subjected to two different temperatures (20C and 30C). Particle selection and clearance rates were

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19 measured following Baker et al. (1998). Ten individual clams were individually placed in 2-L clear glass Pyrex beakers containing the experimental phytoplankton assemblage in 28 ppt 0.2-m filtered seawater. The experimental beakers were run concurrently with two control beakers, containing no clams. To lessen the occurrence of phytoplankton settling to the bottom, beakers were gently aerated throughout the experiment. Clearance rates were determined by analyzing 1.0-ml water samples at the start of each feeding trial and again after 45 minutes. Feeding selectivity was determined by examining the composition of the pseudofeces produced during the feeding trials (see below). At the end of each feeding trial, clams were measured, weighed wet, and then tissues were dried to a constant weight (60C). Each feeding trial was replicated three times on different days. Hurlbert (1984) wrote about the dangers of pseudoreplication in ecological field experiments; replicates that are spatially or temporally separated are not independent and the data should not be used as such. In the experimental design, time was not taken into account. For example, one replicate of IsoSyn at 30C was performed in January, and another in February. The clams used for each replicate were from different batches and had been exposed to different environmental parameters. Therefore, according to Hurlbert (1984), the replicates were not true replicates of each temperature/algal combination but rather pseudoreplicates. In addition, the replicates were not performed randomly; feeding trials at 30C were completed before the 20C feeding trials. Additional Experiments Two additional types of feeding trials were conducted. To determine whether clams would prefer algae to which they had been acclimated, one batch of clams was split

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20 into two groups. Batch refers to clams obtained at the same date and time and from the same area. Prior to purging and conducting feeding trials, the two groups of clams were kept in separate holding tanks for two weeks. One group of clams was fed I. galbana and the other group of clams was fed Synechococcus at a rate of 2% of their dry body weight every other day (http:// www.reedmariculture.com ). Feeding trials were conducted following the experimental protocol outlined above. To eliminate time as a factor, a second type of feeding trial was performed. One batch of clams was obtained and split into two groups. The clams were allowed to purge for three days; one group at 20 o C and one group at 30 o C. Feeding trials for each group were subsequently performed following the experimental protocol described above. Ideally, this is how the experiment protocol should have been designed. Calculation of Particle Selectivity Phytoplankton species in the pseudofeces and water samples were identified and counted using a FACSCAN flow cytometer (BD BioSciences, San Jose, CA) at the University of Florida Flow Cytometry Core Lab. Samples were vortexed to disrupt aggregations immediately prior to analysis. Samples were shaken and filtered through a 40-m mesh to remove large particles to prevent clogging of the flow cytometer. In the cytometer, cells flow singly through a 488 nm laser beam while their forward light scatter and red fluorescence emission above 650 nm are measured. Forward light scatter was used to eliminate small particles. Data was collected for one minute at the high flow rate (approximately 60 L/min). Regions identifying phytoplankton were set based on forward light scatter and red fluorescence using CellQuest 3.3 software running on a

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21 Macintosh computer (Apple Computer, Cupertino, CA). Counts of cells in each region were collected in a text file and imported into a Microsoft Excel spreadsheet. To determine feeding selectivity, a modified electivity index (EI) was calculated. The equation for EI is based on Ivlevs index of electivity for freshwater fish and was modified as followed: EI = -[(P S) / ((P + S) (2*P*S))] where P is the ratio of a particular algal species in the pseudofeces and S is the ratio in the suspension (Ivlev, 1961; Bayne et al., 1977). The electivity index can range from .0 to1.0. If the EI is positive for a given algal species, then there is a preferential ingestion of that species. If the EI is negative, then it indicates a rejection of that algal species. Since electivity indices are not continuous, the indices were arcsine transformed. The mean of the means for each replication within each temperature feeding trial (20 o C and 30 o C) was compared to zero using a one-sample t-test. For the feeding trials of IsoSyn, TetraIso, TetraSyn, and IsoSyn(Ch), the indices were compared using a 2-way ANOVA (temperature and feeding trial). To compare IsoSyn and IsoSyn6, a 2-way ANOVA (temperature and concentration) was used. Since there was only one replication for IsoSyn-AI, IsoSyn-AS, and IsoSyn-B, no statistical procedures were conducted. Instead the mean clearance rates were graphed. Feeding trials, statistical tests used, and p-values are summarized in Table 3 in the Results section. Calculation of Clearance Rate Clearance rates were also determined using a flow cytometer. The reduction in the concentration of particles over 45 minutes was used to calculate net clearance rates according to Coughlan (1969) as follows: CR= V [(ln(C o )-ln(C 1 ))/t) A); where CR is clearance rate, V is the volume of the experimental suspension, C o is the initial concentration, C 1 is the concentration after time

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22 t, and A is the average of the controls (A = ([(ln(C o )-ln(C 1 )])/n, where n is the number of controls). Clearance rates were corrected for particle abundance changes in the controls and for the amount of time the bivalves were open. Clearance rates were standardized to 1-g of dry tissue mass using an allometric exponent for bivalves of 0.75 (Grizzle et al., 2001) as follows: CR (1g) = CR/b 0.75 ; where CR is the uncorrected clearance rate of the experimental clam expressed in L*h -1 and b is the dry weight of the experimental clam expressed in grams. The means for each replicate feeding trial at 20 o C and 30 o C involving mixed algal assemblages, i.e. IsoSyn, TetraIso, TetraSyn, and IsoSyn(Ch), were analyzed with a 2-way ANOVA. Clearance rates for IsoSyn and IsoSyn6 were compared also with a 2-way ANOVA. Since there was only one replication for IsoSyn-AI, IsoSyn-AS, and IsoSyn-B, no statistical procedures were conducted. Instead the mean clearance rates were graphed. Feeding trials, statistical tests used, and p-values are summarized in Table 4 in the Results section. Clearance rate data was not used if the particle concentration in the beakers declined below 40% of the initial concentration (Baker and Levinton, 2003).

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23 Table 1. Algal assemblages and the date(s) of replication(s) of each feeding trial at two different temperatures. Unless stated otherwise, concentrations were 10 5 cells/ml. Phytoplankton Trial Temperature Abbreviation 20C 30C I. galbana + Synechococcus (no chains) IsoSyn I. 2/26/04 II. 3/04/04 III. 3/25/04 I. 1/29/04 II. 2/05/04 III. 2/19/04 T. maculata + Synechococcus (no chains) TetraSyn I. 3/04/04 II. 3/11/04 III. 3/18/04 I. 1/29/04 II. 2/12/04 III. 2/12/04 T. maculata + I. galbana TetraIso I. 3/04/04 II. 3/11/04 III. 3/18/04 I. 3/04/04 II. 3/11/04 III. 3/18/04 I. galbana + Synechococcus(Chain-forming) IsoSyn(Ch) I. 2/26/04 II. 2/26/04 III. 3/11/04 I. 1/15/04 II. 1/15/04 III. 1/22/04 I. galbana + Synechococcus (no chains) at 10 6 cells/ml IsoSyn6 I. 3/04/04 II. 3/11/04 III. 3/18/04 I. 1/22/04 II. 2/05/04 III. 2/12/04 I. galbana + Synechococcus (no chains) (acclimated to Synechococcus-no chains) IsoSyn-AS I. 3/25/04 I. 2/19/04 I. galbana + Synechococcus (no chains) (acclimated to I. galbana) IsoSyn-AI I. 3/25/04 I. 2/19/04 I. galbana + Synechococcus (no chains) (Same batch, 2 temperatures) at 10 6 cells/ml IsoSyn-B I. 4/08/04 I. 4/08/04

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Table 2. Mean size and weight, and actual number of animals that opened in each feeding trial. Numbers in parantheses are SD. 24 Trials n Height(mm) Length(mm) Width(mm) Wet Weight(g) Dry Weight(g) Experimental Clams 124 38.11 (0.19) 44.13 (0.21) 24.04 (0.19) 27.5 (0.40) 0.87 (0.01) IsoSyn-AI 10 39.41 (1.48) 46.11 (1.75) 26.37 (4.18) 31.2 (3.0) 1.01 (0.17) IsoSyn-AS 9 37.46 (1.75) 42.61 (2.00) 23.94 (0.89) 26.6 (2.8) 0.77 (0.08) 20 o C (0.47) IsoSyn-B 14 37.68 (2.18) 43.22 (1.81) 24.52 (0.76) 26.5 (3.0) 0.74 (0.12) Experimental Clams 150 37.43 (0.32) 43.50 (0.45) 23.31 (0.27) 26.18 (0.54) 0.76 (0.02) IsoSyn-AI 10 37.52 (3.59) 44.95 (1.75) 24.09 (1.06) 27.5 (2.7) 0.75 (0.10) IsoSyn-AS 9 38.70 (2.72) 45.46 (3.32) 24.03 (1.39) 28.1 (5.8) 0.82 (0.18) 30 o C (0.75) IsoSyn-B 19 38.42 (2.39) 44.66 (3.04) 25.06 (1.19) 29.4 (4.9) 0.80 (0.13)

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CHAPTER 4 RESULTS Electivity Indices Based on results, clams sorted particles for ingestion or rejection. Out of eight algal combinations, three had EIs that were significantly different than zero (Fig 1). Specifically, there was preferential ingestion of Isochrysis in all three trials. Algal combination had a significant effect on EIs (p-value = 0.000). Clams showed greater selectivity between Synechococcus (either chainforming or not) and Isochrysis than between Tetraselmis and Synechococcus or Tetraselmis and Isochrysis (Fig 1). In particular, IsoSyn(Ch) had much higher EIs than TetraIso (p-value = 0.0029) and TetraSyn (p-value = 0.0175). In addition, IsoSyn had significantly higher EIs than TetraIso (p-value = 0.0001). Although there was no interaction between algal combination and temperature, within the 20 o C particle combinations, TetraIso had a significantly lower EI than IsoSyn (p-value = 0.0325). At 30 o C, TetraIso had a significantly lower EI than either IsoSyn (p-value = 0.0021) or IsoSyn(Ch) (p-value = 0.0190) (Fig 1). Based on these results, there appears to be a pattern in which clams select the larger algal species (either Tetraselmis or Isochrysis) over the smaller Synechococcus. Temperature had a significant effect on particle selectivity (p-value = 0.035); clams were more selective at 20 o C than they were at 30 o C (Fig 1). However, when a single batch of clams was used for both temperature experiments, there appeared to be no difference in EIs (Fig 2). Cell concentration had a significant effect on electivity. Electivity indices were significantly greater at the lower concentration (p-value = 0.012) (Fig 3). 25

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26 Within each temperature/algal combination, there was a high amount of variability in EIs between replicate trials (batches of clams) (Fig 4). For example, when three different batches of clams were fed Tetraselmis and Synechococcus at 30 o C, one batch strongly selected for Tetraselmis, one batch slightly selected for Tetraselmis, and one batch selected for Synechococcus (Fig 4b). When the three batches were combined, this resulted in a small positive EI, but no active selection for either alga (Fig 1b). Prior feeding history did not appear to account for differences between batches (Fig 5). When a single batch of clams was split and two groups acclimated to different algae, there was no difference in their preference for those algae. Clearance Rates Temperature had no effect on clearance rate when clams were fed any of the four particle combinations (Fig 6). However, when a single batch of clams was used for both temperature experiments, mean clearance rates were greater at 30 o C than at 20 o C (Fig 7). Interestingly, there was a significant interaction (p-value = 0.0461) between temperature and algal combination due mainly to TetraSyn at 20 o C having a lower clearance rate than TetraIso at 30 o C. Cell concentration did not have a significant effect on clearance rates (Fig 8). However, temperature did have a significant effect when comparing IsoSyn and IsoSyn6, with clearance rates higher at 20 o C than at 30 o C (p = 0.003). Prior feeding history may have had an impact on clearance rates. Clams acclimated to Synechococcus exhibited a higher mean clearance rates for a combination of Isochrysis and Synechococcus than did those clams acclimated to Isochrysis (Fig 9).

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Figure 1. Electivity indices (means SE) for Mercenaria mercenaria at two different temperatures, 20 o C and 30 o C, when fed different combinations of algae at a total concentration of 10 5 cells/ml. A positive EI indicates selection of an algal species. A negative EI indicates rejection of an algal species. Symbol (*) indicates which EIs were significantly different than zero (p<0.05). a) Acceptance (+) or rejection (-) of Tetraselmis when Isochrysis is present. b) Acceptance or rejection of Tetraselmis when Synechococcus sp. is present. c) Acceptance or rejection of Isochrysis when Synechococcus is present. d) Acceptance or rejection of Isochrysis when the chainforming strain of Synechococcus is present.

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28 -1-0.6-0.20.20.61n=3n=3a) EI for Tetraselmis in the feeding trial TetraIso 20oC30oC -1-0.6-0.20.20.61n=3n=3b) EI for Tetraselmis in the feeding trial TetraSyn -1-0.6-0.20.20.61n=3n=3c) EI for Isochrysis in the feeding trial IsoSyn* -1-0.6-0.20.20.61n=3n=3d) EI for Isochrysis in the feeding trial IsoSyn(Ch)*

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29 -1-0.6-0.20.20.61Acceptance or rejection of Isochrysis when Synechococcus is present in clams from a single batch (106 cells/ml):n=14n=15 20oC30oC Figure 2. Electivity indices (means SE) of Mercenaria mercenaria for Isochrysis galbana when Synechococcus sp. (nonchainforming) is present, at two different temperatures, in clams from a single batch (IsoSyn-B). Total cell concentration was 10 6 cells/ml. A negative EI indicates rejection of Isochrysis.

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30 -1-0.6-0.20.20.61Acceptance or rejection of Isochrysis when Synechococcus is present at two concentrations a) 105 and b) 106 cells/ml:n=3n=3 20oC30oCn=3n=3a) 105 cells/mlb) 106 cells/ml Figure 3. Electivity indices (means SE) of Mercenaria mercenaria for Isochrysis when Synechococcus sp. (non-chainforming) is present, at two temperatures, 20 o C and 30 o C, and two cell concentrations a) 10 5 and b) 10 6 cells/ml. Symbol (*) indicates which replication(s) were significantly different than zero (p<0.05).

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31 -1-0.6-0.20.20.61n=6n=4n=5n=5n=8n=8a) EIs for Tetraselmis in the feeding trial TetraIso 20oC30oC -1-0.6-0.20.20.61n=4n= 5 n=3n=8n=7n=6b) EIs for Tetraselmis in the feeding trial TetraSyn -1-0.6-0.20.20.61n=8n=8n= 7n=8n=5n=10c) EIs for Isochrysis in the feeding trial IsoSyn Figure 4. Mean replication (or batch) electivity indices (mean SE) for Mercenaria mercenaria at two temperatures, 20 o C and 30 o C, when fed different combinations of algae. There were three replicates for each temperature. A positive EI indicates acceptance of an algal species. A negative EI indicates rejection of an algal species. a) Acceptance or rejection of Tetraselmis when Isochrysis is present at a total concentration of 10 5 cells/ml. b) Acceptance or rejection of Tetraselmis when Synechococcus sp. is present at a total concentration of 10 5 cells/ml. c) Acceptance or rejection of Isochrysis when Synechococcus is present at a total concentration of 10 5 cells/ml. d) Acceptance or rejection of Isochrysis when the chainforming species of Synechococcus is present at a total concentration of 10 5 cells/ml. e) Acceptance or rejection of Isochrysis when Synechococcus is present at a total concentration of 10 6 cells/ml.

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32 -1-0.6-0.20.20.61n=6n=8n= 5n=6n=6n=9d) EIs for Isochrysis in the feeding IsoSyn(Ch) -1-0.6-0.20.20.61n=9n=9n=10n=10n=8n=7e) EIs of Isochrysis in the feeding trial IsoSyn6 Figure 4. Continued.

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33 -1-0.6-0.20.20.61Acceptance or rejection of Isochrysis when Synechococcus is present, in clams acclimated to either a) Synechococcusor b) Isochrysis:n=7n=8a) Synechococcusb) Isochrysisn=3n=9 20oC30oC Figure 5. Mean electivity indices of Mercenaria mercenaria for Isochrysis galbana when Synechococcus sp. is present, at two temperatures, in clams acclimated for two weeks on either a) Synechococcus (IsoSyn-AS) or b) Isochrysis (IsoSyn-AI). Total cell concentration was 10 5 cells/ml. A positive EI indicates selection of Isochrysis. (Means SE).

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34 00.511.522.53 00.511.522.53 00.511.522.53 00.511.522.53 Clearance Rates ( L h -1 g -1 ) 20 o C 30 o C 20 o C 30 o C b) TetraSyn a) TetraIso n = 16 n = 9 n = 13 n = 18 d) IsoSyn(Ch) c) IsoSyn n = 18 n = 21 n = 18 n = 21 Figure 6. Clearance rates by Mercenaria mercenaria (means SE ) at two temperatures (20 o C and 30 o C) when fed algal suspensions at 10 5 cells/ml. All clearance rates were standardized to 1 gram of dry weight.

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35 00.511.522.5330oC20oCClearance Rates ( L h -1 g -1 )n=13n=15 Figure 7. Clearance rates (means SE) of Mercenaria mercenaria fed Isochrysis galbana and the nonchainforming strains of Synechococcus (IsoSyn-B) at two temperatures (20 o C and 30 o C). A single batch of clams was split between the two temperatures groups.

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36 00.511.522.53 00.511.522.53 Clearance Rates (L h -1 g -1 ) 20 o C 30 o C 20 o C 30 o C a) 10 5 cells/ml b) 10 6 cells/ml n = 23 n = 27 n = 13 n = 18 Figure 8. Clearances rates (means SE) by Mercenaria mercenaria fed I. galbana and Synechococcus sp. at two temperatures (20 o C and 30 o C) and two concentrations: a) 10 5 cells/ml (IsoSyn) and b) 10 6 cells/ml (IsoSyn6).

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37 00.511.522.5330oC20oCClearance Rates (L h -1 g -1 )n=7n=8n=8n=320oC30oC a) Synechococcus b) Isochrysis Figure 9. Clearance rates (means SE) of Mercenaria mercenaria of the feeding trial Isochrysis galbana and the nonchain-forming species of Synechococcus (IsoSyn-AS and IsoSyn-AI) at two different temperatures (20 o C and 30 o C) when clams were acclimated to either a) Synechococcus or b) Isochrysis.

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CHAPTER 5 DISCUSSION This is the first study to conduct feeding selectivity experiments with bivalves at temperatures above 20 o C Most feeding selectivity studies have been conducted at temperatures between 12 o C and 20 o C (Shumway et al., 1985; Bayne et al. 1989; Stenton-Dozey & Brown, 1992; Bayne et al. 1993; Arifin & Bendell-Young, 1997; Defossez & Hawkins, 1997; Ward et al., 1998); appropriate given the cooler temperatures of collection sites. For example, summer water temperatures at northeastern locations such as Wells, Maine; Hudson River, New York; and Narragansett Bay, Rhode Island, reach no more than 16.5 o C, 22.6 o C, and 23.3 o C, respectively (NERRS, 2004). In the mid-Atlantic, such as Chesapeake Bay, Bay Bridge, Maryland, temperatures reach about 27.5 o C in August (NERRS, 2004). In the shallow waters along Floridas Gulf coast, however, temperatures exceed 30 o C in the summer and are routinely > 25 o C for half the year (Jett, 2004; NERRS, 2004; Frazer, unpublished data; Phlips, unpublished data). In the Suwannee River Estuary, near Cedar Key, Florida (a prominent site for clam aquaculture), water temperatures can also get up to 30 o C (Jett, 2004). The warmest National Estuarine Research Reserve (NERRS, 2004) sites in Florida can be found in Rookery Bay, Naples (about 70 miles south of Charlotte Harbor which is another site for clam aquaculture), where waters reach 35.6 o C (NERRS, 2004). Therefore, this study is important because it is the first to examine how high temperatures (30 o C) may influence feeding selectivity of bivalves, and Mercenaria mercenaria in particular. 38

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39 The results of this study indicate that temperature affects feeding selectivity of Florida hard clams. Clams exhibited greater selection at 20 o C than they did at 30 o C. This finding may be the result of the acclimatizing protocol. When acclimatizing the clams to the experimental temperature, they were immediately placed in either 20 o C or 30 o C water, depending on the feeding trial, and held for three days. Since the lower temperature trials were done in late February and March, the experimental water was within a typical range of water temperatures for that time of year in Cedar Key. In the feeding trials at 30 o C, there was a 10-20 o C difference between the collection water temperature and the experimental water temperature ( www.floridaaquaculture.com ). For fish, acclimation to the experimental water temperature should be gradual to reduce shock which could change physiological states like hormone levels or blood chemistry concentrations (Stickney and Kohler, 1990; Wedemeyer et al., 1990). Fry (1971) suggests a gradual acclimation schedule of 1 o C/day until the experimental temperature is reached. While intertidal organisms can withstand daily temperature variations of 20-30 o C, subjecting the clams to a temperature change of more than 10 o C have shocked the clams (Hochachka & Somero, 2002). Therefore, the lack of acclimation to 30 o C may have rendered the clams less efficient in their selective abilities. Another reason for the difference in selectivity between temperatures may have been a batch effect. Again, a batch refers to a quantity of clams collected at one time from the same area and exposed to the same environmental history. Since they were collected at different times of the year, the clams used in the low temperature trials may have been subjected to different environmental parameters than those used in higher temperature trials. In the batch experiment (IsoSyn-B), in which one batch of clams was

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40 split between two temperatures, temperature did not appear to have an effect on selectivity (Fig 2). This suggests that the difference in selectivity between 20 o C and 30 o C in the main experiment may largely be due to the batch used rather than to the lack of acclimation. More study is needed, however. In addition to temperature, algal combination had a significant effect on electivity indices of hard clams. At algal concentrations of 10 5 cells/ml, there was greater selection for larger particles (Tetraselmis at 10 m and Isochrysis at 5 m) over smaller particles (Synechococcus sp. at 2 m and with chains) (Fig 1). Tetraselmis and Isochrysis are both well within the size range to be completely retained by a bivalve while Synechococcus is on the lower end of the size range (Jrgensen, 1975; Mhlenberg & Riisgrd, 1978). Bass et al. (1990) examined the growth of M. mercenaria on picoplankton which included Nannochloris (ca. 3 m) and two species of Synechococcus sp. (both ca. 1 m) in length. They found that, while the picoplankton were filtered out of suspension by the clam, it was assimilated with low efficiency (17.6-31.1%) compared to the 4 m Pseudoisochrysis paradoxa (86.5%). The results from this study, however, showed preferential ingestion of the smaller Synechoccococus (2 m) over the larger Isochrysis (5 m) in four of the eight feeding trials (Fig 2; Fig 4) done at 10 6 cells/ml. Bass et al. (1990) worked with smaller picoplankton and lower concentrations (5 x 10 4 to 10 5 cells/ml) which could be why a difference was seen in this study. Although the mean values of replicates indicated no preference for Synechococcus, there was a high amount of variability in electivity indices between batches of clams (Fig 2). In addition, although statistics were not done in Fig 4, the electivity indices were high (0.7). Because of these conflicting results, further studies would be interesting.

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41 Algal concentration had a significant effect on selectivity, with clams exhibiting active selection at the lower concentration and no sorting at the higher concentration. A study by Levinton et al. (2001) suggests that rate-limiting steps within a bivalves digestive process may affect how a bivalve processes particles. As the gut becomes full, bivalves may start to increase rejection of nutritious particles as pseudofeces that they would otherwise ingest. As a result, bivalves may show no preferential ingestion between algal species when presented at high concentrations. There was no significant effect of temperature on clearance rates when clams were fed any of the four algal combinations at 10 5 cells/ml (Fig 6). This is in agreement with findings reported by Smaal et al. (1997), who showed clearance rates for mussels (M. edulis) to be independent of temperature. In addition, MacDonald et al. (1996) showed clearance rates for Placopecten magellanicus to be independent of temperatures over the range of 0 to 15 o C. In a field study, Paterson et al. (2003) examined growth in the rock oyster Saccostrea glomerata in Australia for a year and found that, although there was variation in temperature between study sites, growth (a good indicator of feeding rates) was independent of temperature. It is interesting to note that while statistics were unable to be done on the clearance rates for the batch experiment, the mean clearance rate for 20 o C was higher than that at 30 o C. Again this could be because of a batch effect. However, when comparing the two different concentrations (10 5 and 10 6 cells/ml) of IsoSyn, temperature was found to be significant with higher clearance rates at 20 o C than at 30 o C (Fig 8). This is in agreement with studies of northeastern clams. Hamwi (1969) showed that temperature had an inverted parabolic effect on pumping rates, i.e. the rate at which water flows through the mantle cavity, with pumping rates at 30 o C being

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42 much less than the pumping rates at 20 o C. Other bivalves are reported to react much the same way. Over the temperature range of 16-28 o C scallops (Arogpecten ventricosus circularis) had maximum clearance rates at 19 o C and 22 o C (Sicard et al., 1999). In the clam Rupitapes decussates (L.), increasing temperature has a negative effect on clearance rates, leading to a reduction in scope for growth (Sobral & Widdows, 1997). In contrast, cockles appear to increase clearance rates with increasing temperature (Smaal et al., 1997). Levya-Valencia et al. (2001) showed that temperatures of 29 o C or higher were optimum for clearance, ingestion, and growth rates for the penshell Atrina maura. In contrast to other studies, concentration did not have a significant effect on clearance rates temperature was held constant. Bayne et al. (1989) showed that ingestion rates (equivalent to clearance rates when no pseudofeces are produced) of Mytilus edulis increased with increasing seston concentration. Tenore and Dunstan (1973) showed that although feeding rates of M. mercenaria increased with increasing food concentration, they were still lower than the feeding rates observed in M. edulis and C. virginica. This could be the result of a combination of reduction in pumping rate or filtering efficiency and an increase in pseudofeces production as concentrations increase. In addition, each species response to increasing food concentration may reflect adaptation of bivalves to the areas where they were collected. Additionally, Tenore and Dunstan showed that feeding rates in the three bivalves exhibit a slight inverted parabolic effect in association with seston concentration. In contrast, Bricelj and Malouf (1984) found a negative relationship between seston concentration and clearance rates in M. mercenaria, with clearance rates decreasing as seston concentration increases. In this study, however, effect of concentration had no effect on clearance rates. Both concentrations used in the

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43 present study were relatively high. Tenore and Dunstan (1973) suggest that hard clams may not be well suited for feeding at high particle concentrations, compared to other bivalves. While there were no differences between clearance rate replicates, there was a high amount of variability among replicates or batches in the selectivity experiments. Differences between replicates may be due to a variety of parameters including feeding history, adaptation of clams to their environment, seasonal changes in digestive enzymes, and/or other factors, e.g., changes in water viscosity due to temperature. Feeding history, for example, may have an impact on feeding selectivity and digestion. Bayne (1993) noted that bivalves can shift feeding preferences as an adaptative strategy to seasonal changes in food availability. In addition, Ibarrola et al. (1998) showed that there is an apparent seasonal pattern of digestive enzyme activity that may be affected by past feeding history and could potentially affect food selection. However, in the feeding trials IsoSyn-AI and IsoSyn-AS where clams were acclimated for two weeks on either Isochrysis or Synechococcus sp., there appeared to be no difference in the feeding selectivity between clams previously fed I. galbana and those previously fed Synechococcus (Fig 5). While the clams were fed 2% of their dry weight per day during the acclimation period, a ration generally recommended for bivalves ( www.reedmariculture.com ), this resulted in concentrations of only 317 or 6349 cells/ml Isochrysis and Synechococcus, respectively. Therefore, total particle concentrations may have been too low to have a significant acclimatory effect on the clams. Additional studies with higher cell concentrations are warranted.

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44 Although the two-week study was inconclusive, bivalves may become adapted to exploit the specific suite of food available to them in the field. For example, bivalves from areas that are dominated by high bacterial counts had higher rates of clearance of bacteria compared to bivalves from other areas not dominated by bacteria (Wright et al., 1982; Berry & Schleyer, 1983). The high variability in selectivity between batches of hard clams in this study, with four out of eight feeding trials at 10 6 cells/ml able to select small particles (2 m), suggests that these batches had adapted to high counts of small particles in the environment. There have been no studies to date that compare the clearance rates of clams from Cedar Key with clams from other areas along the Atlantic coast that may typically feed on other particle types and sizes. The absorption efficiency of particular phytoplankton species is determined by digestive enzymes, and enzymatic activity may be influenced by season or food availability (Bayne et al., 1993; Ibarrola et al., 1998). For example, Seiderer and Newell (1979) reported that Choromytilus meridionalis changed the activity rate of a-amylase in response to changes in temperature and, coincidentally, with phytoplankton composition. Ibarrola et al. (1998) also found seasonal variation of digestive enzyme activities in the cockle C. edule, in northern Spain, where their spring/summer diet is predominantly living phytoplankton while in fall it consists mainly of kelp detritus. There is speculation that assimilation efficiency corresponds to selectivity; phytoplankton that are easily assimilated are selected for ingestion (Baker et al., 1998). It follows that if digestive enzymatic activity changes seasonally and in response to available food, then selectivity should change also. This offers a further explanation for differences in the ability of batches to select for the smaller algae, Synechococcus sp.

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45 Temperature may affect the physiology of bivalves and, as a consequence, the ability to process certain food items. Temperature may also have an effect on the mechanical aspects of suspension feeding. For example, because temperature is inversely related to viscosity, suspension feeding echinoderm larvae (Dendraster excentricus) are more apt to ingest large particles when the water has a high viscosity (colder) (Podolsky, 1994). Podolsky (1994) suggested that changes in viscosity might also affect retention efficiencies of bivalves. Since waters in the Suwannee River Estuary change seasonally from cool (11 o C) to hot (30 o C) (Jett, 2003; Frazer, unpublished data; Phlips, unpublished data), water viscosity could play a role in what hard clams are able to filter and ingest. In conclusion, temperature was found to have an effect on food selectivity, with Cedar Key hard clams exhibiting greater selection at 20 o C than at 30 o C. In addition, I found that temperature had almost no effect on clearance rates. However, due to the wide variability in results, more studies are needed to further test the effects of temperature and batch effect on particle selectivity and clearance rates for Cedar Key hard clams and bivalves in general. For example, it would be interesting to determine whether a batch effect is unique to Cedar Key hard clams, common to all hard clams, or common to all bivalves. In addition, it would be interesting to see how much the batch effect changed over a year and whether it corresponded to phytoplankton abundance or composition in the Suwannee River Estuary. It is documented that cyanobacteria like Synechococcus are common in Florida waters, especially in the summer (Phlips et al., 1999; Bledsoe, 2003) when the waters are warm, and bigger sized phytoplankton are common in the winter. It would make sense, then, that the clams tested in July 2003 would prefer the smaller sized

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46 cyanobacteria and why they rejected it when the experiment was performed again in January through March 2004 (Figure 3). Temperature may have additional indirect effects by either affecting phytoplankton composition in the estuary or by being a conditioning factor for bivalves living in the area. This study is important in that it is one of the first to examine feeding selectivity of bivalves in association with changes in temperature. The results suggest important additional avenues of research which will be essential to improving aquaculture practices in warmer climates, especially for the growth and stability of the clam farms located in the vicinity Suwannee River Estuary.

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55 USDA United States Department of Agriculture. 2002. Florida aquaculture sales exceed $99 million in 2001. Florida Agriculture, Florida Agriculture Statistic Service: June. Ward JE. 1996. Biodynamics of suspension-feeding in adult bivalve molluscs: Particle capture, processing, and fate. Invert Biol 115(3): 218-31. Ward JE, Danford LP, Newell RIE, and BA MacDonald. 1988. A new explanation of particle capture in suspension-feeding mollusks. Limnol Oceanogr 43: 741-52. Ward JE, Levinton JS, Shumway SE, and T Cucci. 1997. Direct identification of the locus of particle selection in a bivalve mollusc. Nature 390: 131-2. Ward JE, Levinton JS, Shumway SE, and T Cucci. 1998. Particle sorting in bivalves: In vivo determination of the pallial organs of selection. Mar Biol 131: 283-92. Wedemeyer GA, Barton BA, and DJ McLeay. 1990. Stress and acclimation. In CB Schreck and PB Moyle (eds), Methods for fish biology, American Fisheries Society, Bethesda, MD, pp 451-490. Werner I and JT Hollibaugh. 1993. Potamocorbula amurensis: Comparison of clearance rates and assimilation efficiencies for phytoplankton and bacterioplankton. Limnol Oceanogr 38(5): 949-64. Wolfe, LE and SH Wolfe. 1985. The ecology of the Suwannee River Estuary: An analysis of ecological data from the Suwannee River Water Management District Study of the Suwannee River estuary, 1982-1983. Florida Department of Environmental Regulation. 188pp. Wong WH, Levinton JS, Twining BS, and NS Fisher. 2003. Assimilation of microand mesozooplankton by zebra mussels: A demonstration of the food web link between zooplankton and benthic suspension feeders. Limnol Oceanogr 48(1): 308-12. Wright RT, Coffin RB, Ersing CP and D Pearson. 1982. Field and laboratory measurements of bivalve filtration of natural marine bacterioplankton. Limnol Oceanogr 27(1): 91-8.

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BIOGRAPHICAL SKETCH Carla Danielle Beals was born in Charleston, South Carolina, on February 14, 1976. In high school, she attended the South Carolina Governors School for Science and Mathematics for her 11 th and 12 th grade years. She received her BS in marine science and graduated cum laude from the University of South Carolina, Columbia, SC, in December 1998. In December of 2004, she will receive her MS from the University of Florida, Gainesville, Florida. 56


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Permanent Link: http://ufdc.ufl.edu/UFE0008841/00001

Material Information

Title: Clearance Rates and Particle Selectivity in the Hard Clam, Mercenaria mercenaria, from Warm Water Habitats
Physical Description: Mixed Material
Copyright Date: 2008

Record Information

Source Institution: University of Florida
Holding Location: University of Florida
Rights Management: All rights reserved by the source institution and holding location.
System ID: UFE0008841:00001

Permanent Link: http://ufdc.ufl.edu/UFE0008841/00001

Material Information

Title: Clearance Rates and Particle Selectivity in the Hard Clam, Mercenaria mercenaria, from Warm Water Habitats
Physical Description: Mixed Material
Copyright Date: 2008

Record Information

Source Institution: University of Florida
Holding Location: University of Florida
Rights Management: All rights reserved by the source institution and holding location.
System ID: UFE0008841:00001


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CLEARANCE RATES AND PARTICLE SELECTIVITY IN THE HARD CLAM,
Mercenaria mercenaria, FROM WARM WATER HABITATS

















By

CARLA DANIELLE BEALS


A THESIS PRESENTED TO THE GRADUATE SCHOOL
OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT
OF THE REQUIREMENTS FOR THE DEGREE OF
MASTER OF SCIENCE

UNIVERSITY OF FLORIDA


2004

































Copyright 2004

by

Carla Danielle Beals















ACKNOWLEDGMENTS

I would like to thank my advisor, Dr. Shirley Baker, for giving me the opportunity

to conduct research under her guidance. My experience here has been invaluable. In

addition, I would also like to thank Dr. Derk Bergquist for all his help, advice, and

patience in answering my questions, especially the questions regarding statistics.

I would also like to thank my committee members Dr. Edward Phlips, Dr. Thomas

Frazier, and Dr. Debra Murie.

In addition, I would also like to thank Neil Benson, UF Flow Cytometer Core Lab,

for showing me how to use the FacScan Flow Cytometer and for help in analyzing the

data. Also, I would like to thank Marinela Capanu, Graduate Assistant Consultant for the

IFAS Department of Statistics, for her advice on the statistics used for this thesis.

Thanks also go to Christina Jett-Richards and Erin Bledsoe for all their help and

guidance. Likewise, special thanks also go to Stephanie Keller, Jamie Greenawalt,

Daniel Goodfriend, Brooke Rimm-Hewitt, Edward DeCastro, and Karen Donnelly for

help in the lab.

Funding for this project was provided by the Sigma Xi Grants-in-Aid and the

USDA Eutrophication Project.

Last, but not least, I would like to thank Dr. Robert and Mrs. Doris Kline; whose

unending kindness, patience, and support was invaluable to me.
















TABLE OF CONTENTS

page

A C K N O W L E D G M E N T S ................................................................................................. iii

LIST OF TABLES ...... ......... ........ .......................... vi

LIST OF FIGURES ............. ........................................ vii

ABSTRAC T ............. ............ ............................... viii

CHAPTER

1 INTRODUCTION ................... ............................ ......... .. .......... 1

2 LITERATURE REVIEW .................................................. ...............4

D description of the Study A rea ................................................................................. 4
Sources of Nutrients to the Suwannee River Estuary.................................................4
Characteristics of A lgal Bloom s....................... ............................. ...... ... ..........8
Bivalves and their Effect on the Water Column ................ ........................8
Suspension Feeding in Bivalves .................. ........ .....................9
The Effect of Temperature on Bivalve Clearance Rates ..........................................10
The Effect of Diet Composition and Concentration on Bivalve Clearance Rates...... 11
Selective Feeding in Bivalves..............................................13
Controversies Concerning Bivalve Feeding Mechanisms...............................14

3 M ETHODS AND M ATERIALS ........................................................ 17

Test Subjects............................................................17
Experimental Algae and Culture Protocols ........................................... 17
Phytoplankton Com binations ................................... ...... ............... 18
Experim ental O rganism ................................................... 18
E xperim mental Protocol .............................................................18
Additional Experiments....................... ..............................19
Calculation of Particle Selectivity ........................... ......... 20
Calculation of Clearance Rate ................. .......... ........... 21

4 RESULTS .................................................25

Electivity Indices ................................................25










Clearance Rates ............................................... ........ 26

5 DISCUSSION ............ .............. .... ...............3 8

LIST OF REFERENCES ....................... ......... ........47

BIOGRAPHICAL SKETCH .................................................. ............... 56




















































v
















LIST OF TABLES

Table page

1 Algal assemblages and the date(s) of replication(s) of each feeding trial at two
different temperatures ...................... .......... .......... .. ........ 23

2 Mean size and weight, and actual number of animals that opened in each feeding
trial .............................................................. 24
















LIST OF FIGURES


Figure page

1 Electivity indices (means SE) for Mercenaria mercenaria at two different
temperatures, 20'C and 30'C, when fed different combinations of algae at a total
concentration of 105 cells/m l............................................................ 28

2 Electivity indices (means SE) of Mercenaria mercenaria for Isochrysis galbana
when Synechococcus sp. (nonchainforming) is present, at two different
temperatures, in clams from a single batch (IsoSyn-B). .......................................29

3 Electivity indices (means SE) of Mercenaria mercenaria for Isochrysis when
Synechococcus sp. (non-chainforming) is present, at two temperatures, 20'C and
30oC, and two cell concentrations a) 105 and b) 106 cells/ml.............................30

4 Mean replication (or batch) electivity indices (mean SE) for Mercenaria
mercenaria at two temperatures, 20'C and 30C, when fed different combinations
of algae. ................................................... 3 1

5 Mean electivity indices of Mercenaria mercenaria for Isochrysis galbana when
Synechococcus sp. is present, at two temperatures, in clams acclimated for two
weeks on either a) Synechococcus (IsoSyn-AS) or b) Isochrysis
(IsoSyn-A I)...................................... ............................... ......... 33

6 Clearance rates by Mercenaria mercenaria (means SE ) at two temperatures
(20'C and 30C) when fed algal suspensions at 105 cells/ml...............................34

7 Clearance rates means SE) of Mercenaria mercenaria fed Isochrysis galbana
and the nonchainforming strains of Synechococcus (IsoSyn-B) at two
tem peratures (20'C and 30 C).......................................................... 35

8 Clearances rates means SE) by Mercenaria mercenaria fed I. galbana and
Synechococcus sp. at two temperatures (200C and 30C) and two
concentrations...................................... ................................ ......... 36

9 Clearance rates means SE) of Mercenaria mercenaria of the feeding trial
Isochrysis galbana and the nonchain-forming species of Synechococcus (IsoSyn-
AS and IsoSyn-AI) at two different temperatures (20'C and 30C) when clams
were acclimated to either a) Synechococcus or b) Isochrysis. ..............................37















Abstract of Thesis Presented to the Graduate School
of the University of Florida in Partial Fulfillment of the
Requirements for the Degree of Master of Science

CLEARANCE RATES AND PARTICLE SELECTIVITY IN THE HARD CLAM,
Mercenaria mercenaria, FROM WARM WATER HABITATS


By

Carla Danielle Beals

December 2004

Chair: Shirley M. Baker
Major Department: Fisheries and Aquatic Sciences

The objective of this study was to examine the effects of temperature and

phytoplankton concentration on the feeding selectivity and clearance rates of the hard

clam Mercenaria mercenaria. My hypothesis was that temperature has an effect on the

ability of the hard clam to preferentially ingest certain particles over others. Adult clams

obtained from a commercial supplier were subjected to laboratory manipulated

phytoplankton assemblages of three different algae (Synechococcus sp., Isochrysis

galbana, and Tetraselmis maculata) of different sizes (2-[im, 5-[im, and 10-[im) at two

temperatures (20'C and 30'C). One feeding treatment was conducted at two

concentrations (105 cells/ml and 106 cells/ml). Clearance rates were determined from

counting phytoplankton cells in water samples collected at the beginning and end of each

feeding trial using a FacScan flow cytometer. Electivity indices were determined from

initial water samples and clam pseudofeces using a FacScan flow cytometer.









Temperature, algal combination, and concentration all had significant effects on feeding

selectivity of clams. Clams had greater selectivity at 200C than at 300C. In addition,

clams showed a trend to select for larger particles over smaller particles. Selectivity was

greater at the lower concentration than the higher concentration. Within each

temperature/algal combination, there was a high amount of variability in electivity

indices between replicates. Temperature, algal combination, and concentration had no

effect on clearance rates. There was, however, an interaction effect between temperature

and algal combination. Feeding history, adaptation of clams to their environment,

seasonal changes in digestive enzymes, and/or other parameters like changes in water

viscosity due to temperature may account for high variability in electivity indices and

clearance rates between replicates. Results obtained have implications for future

selectivity and clearance rates studies. In addition, this study provides important

information for the future productivity of cultured clams in semi-tropical environments

by demonstrating that feeding preferences may be different for clams from cooler

environments.














CHAPTER 1
INTRODUCTION

The Suwannee River begins in the Okeefonokee Swamp and meanders through

southern Georgia and north central Florida before it discharges into the Gulf of Mexico,

draining 28,500 km2 (Wolfe & Wolfe, 1985). Its estuary and the surrounding regions

(also known as the Big Bend) are home to a highly productive nursery of fish and marine

invertebrates (SRWMD, 1979). Oysters (i.e. Crassostrea virginica) are regularly

harvested and clam farming (i.e. Mercenaria mercenaria) has exploded as a new

aquaculture industry. In 2001, hard clam aquaculture comprised over 18% of Florida's

$99 million total aquaculture sales (USDA, 2002).

As the human population increases in northern Florida, anthropogenic activities are

more likely to have a serious impact on the shallow estuarine waters along the Big Bend.

Surface water runoff and ground water are contributing high concentrations of nutrients

which, in turn, may cause phytoplankton blooms in the estuary (Phlips & Bledsoe, 1997;

Bledsoe & Phlips, 2000). Maintaining a balance between the nutrients needed for

productivity and excessive eutrophication is important for the stability of the Big Bend

ecosystem, and especially for the growth and stability of the newly emerging clam farm

industries. Understanding this balance and the effects of eutrophication on bivalves is

essential.

Bivalves are adept suspension feeders and can modify seston in estuarine waters

(Carlson et al., 1984; Asmus et al., 1990). On the other hand, seston quantity and

composition can have effects on bivalve feeding behavior, particle selection, and









clearance rates (Bayne et al., 1989, 1993; Navarro et al., 1996). Studies on bivalves have

shown an ability to sort particles based on size (Stenton-Dozey & Brown, 1992; Defossez

& Hawkins, 1997) and quality (Arifin & Bendell-Young, 1997; Ward et al., 1997).

Furthermore, there appears to be variability among bivalve species in their ability to sort

and preferentially ingest particles (Shumway et al., 1985; Prins et al., 1991; Ward et al.,

1998). Studies by Bayne etal. (1989;1993) have corroborated the idea that changes in

food concentration can have an effect on clearance rates and, ultimately, the growth and

productivity of bivalves. Baker et al. (1998) demonstrated that the diversity of a plankton

assemblage is important in determining clearance rates of the zebra mussel Dreissena

polymorpha as well as selectivity for individual species of phytoplankton within the

assemblage.

While studies above have shown the composition of the seston to have an impact

on clearance rates, temperature also has an effect. In a review of the physiological

ecology of the hard clam, Grizzle et al. (2001) stated that temperature affected feeding

rates. Feeding rates peaked at about 24-26oC, but fell abruptly at temperatures above

27C. In this same review, temperatures between 20-24oC were shown to be optimal for

clam growth with decreasing growth rates outside this range. This is important because

feeding rates are thought to be the physiological control on growth rates.

While many studies have shown extremes in temperature to have a negative effect

on clearance rates and growth of bivalves, there have been few, if any, studies to show

what affect temperature may have on feeding selectivity. Selectivity studies are usually

carried out at temperatures of 20'C or less (e.g., Shumway et al., 1985; Bayne et al. 1989;

Stenton-Dozey & Brown, 1992; Bayne et al. 1993; Arifin & Bendell-Young, 1997;









Defossez & Hawkins, 1997; Ward et al., 1998). Surface waters in the Suwannee River

estuary, however, commonly exceed 25oC between March and October and temperatures

around 30'C are normal during summer months (T. Frazer, unpublished data; E. Phlips,

unpublished data; Jett, 2004). This challenges what we know about factors important to

clam aquaculture success and our understanding of bivalve feeding physiology.

This study is part of a multi-faceted investigation determining the potential effects

of coastal eutrophication on phytoplankton and bivalve communities of the Suwannee

River estuary. The objective of this study was to examine the effects of temperature on

particle selectivity and clearance rates of Mercenaria mercenaria feeding on bloom

concentrations (105 and 106 cells/ml) of phytoplankton. Based on preliminary

experiments, my hypothesis was that temperature would have an effect on the ability of

hard clams to preferentially ingest smaller particles over larger particles. These effects

may have a seasonal impact on the seston composition of the Suwannee River estuary.














CHAPTER 2
LITERATURE REVIEW

Description of the Study Area

The Suwannee River estuary is among the ten largest estuaries along the Gulf Coast

of Florida. It is located between latitudes 29013'N and 29020'N and longitudes 830 05'

W and 830 12' W (Siegal et al., 1996). The estuary is home to a highly productive

nursery of fish and marine invertebrates (SRWMD, 1979). The Suwannee River, which

feeds the estuary, originates largely in the Okefenokee Swamp of southern Georgia and

drains 28,500 km2 of southern Georgia and north central Florida (Wolfe & Wolfe, 1985).

It is fed, also, by the Alapaha, Withlacoochee, and Santa Fe Rivers. In addition,

groundwater which emanates as spring discharges and diffuse seepage also feeds into the

river. Groundwater contributions to riverflow are particularly important during low flow

periods as a consequence of large-scale climate variation and reduced rainfall in the

watershed. Due to the karst nature of the terrain, i.e. sinkholes, springs, and conduit

systems in the underlying limestone, groundwater and surface water can have direct

hydraulic and geochemical interactions (Katz et al., 1997; Crandall et al., 1999). These

interactions are important because surface water and groundwater inputs contribute high

concentrations of nutrients to the river which, in turn, discharges into the estuary

(SRWMD, 2000b).

Sources of Nutrients to the Suwannee River Estuary

Nitrate concentrations in the river and loads to the Suwannee River Estuary have

steadily increased over the years (Jones et al., 1996, 1997; Pittman et al., 1997). In fact,









from 2001 to 2003, nitrate loads have more than doubled from 2676 tons to 4,591 tons

(SRWMD, 2001b; SRWMD, 2003). Increased nitrates can cause a variety of problems.

In drinking water it can cause health concerns like methelmoglobinemia or "blue baby"

syndrome. Because nitrogen is a basic requirement for algae and other vegetation and

can cause excessive growth, increased nitrogen loads are also an ecological concern.

Studies have shown a positive correlation between nitrate concentration and growth of

planktonic algae in this system (Phlips & Bledsoe, 1997; Bledsoe & Phlips, 2000).

The years 1999 and 2000 were among the driest in the Suwannee River watershed

since 1932. The annual mean stream flow was reduced to 28 to 52% of the long-term

average (flow discharge of less than 142 m3 sec-1). As a consequence, elevated nutrient

concentrations and extensive algal blooms occurred, with an appearance of some

nuisance species like the red drift algae Gracilaria and filamentous brown algae

Ectocarpus that are known to cause various water quality related problems (Bledsoe &

Phlips, 2000; SRWMD, 2001b).

Algal blooms can have an affect on the marine invertebrates and fish. For example,

algal blooms can lead to extreme fluctuations in dissolved oxygen (DO) concentrations in

the water column that can have damaging effects, sometimes fatal, on marine

invertebrates and fish (Barica, 1980; Knights et al., 1995). In addition, hypoxic and

microxic conditions can affect the feeding behavior of young post-settlement oysters

which can limit or delay recruitment into the adult oyster population (Baker and Mann,

1994).

Nitrogen enters the estuary in various ways: 1) the Floridian aquifer, 2)

groundwater, and 3) the Suwannee River. The Floridian aquifer is impressive in that it is









one of the largest underground freshwater aquifers in the United States. The surrounding

environment of the aquifer is composed of carbonate rock, such as marine dolomite and

limestone (Rosenau et al., 1977; SRWMD, 200 l1a). Sand or clay usually covers most of

the carbonate rock. The aquifer extends from the southern portions of Alabama, Georgia,

and South Carolina to the northeastern part of Florida to the Atlantic Ocean and the Gulf

of Mexico. Weakly acidic rainwater dissolves the carbonate rock and creates cavities and

caves in the aquifer. This type of terrain is known as a karst region and has many

sinkholes and springs. In addition, it lacks a well-developed drainage system (SRWMD,

2001 a).

Groundwater can enter the surface water system, e.g., the Suwannee River, through

spring discharges or breaches in the underlying aquifers. The river, in turn, discharges

into the estuaries. In addition, groundwater can enter estuaries as seepage along the

coastline (Cable et al., 1997). Spring water is usually a good indicator of the quality of

the groundwater (SRWMD, 2001a). Human activities can have an effect on the quality

and quantity of groundwater flow and these changes can have a significant impact on

estuarine or coastal ecology because these areas receive large amounts of groundwater.

Cable et al. (1996; 1997) have traced groundwater discharge by using 222Rn and CH4 and

estimated that the seepage of groundwater into the northeastern coastal areas of the Gulf

of Mexico (ca. 10 km2) was equivalent to a first magnitude spring (i.e. a spring with > 2.8

m3 sec-1 discharge).

Studies over a 12-year period (1970-1991) have shown a statistically significant

increase in nitrate concentration in the Suwannee River (Ham & Hatzell, 1996). In the

upper part of the river, the major source of this increase was due to transport of nitrates









by springs, while diffuse groundwater flow was the major source of transport in the lower

portion (Pittman et al., 1997). Augmenting this increase is the karst nature of the aquifer,

which allows leaching of nitrates into the aquifer making it more susceptible to

contamination (Kreitler & Browning, 1983).

Due to the karst topography of the terrain and increasing development along the

Suwannee River, anthropogenic pollutants can also enter the estuary. High nitrate

concentrations have been shown to come from numerous sources, highest among them

being animal wastes, fertilizers, sewage effluent disposal, and residential and golf course

landscapes (Jones et al., 1996; 1997; Katz et al., 1999). Katz et al. (1999) have shown

that even though estimated nitrogen inputs from animal wastes have increased over the

past 40 years in both Suwannee and Lafayette Counties, the nitrogen contributed by

fertilizers is the highest input into the Suwannee River.

Because the average residence time of groundwater discharge from springs is on

the order of decades, there is little to be done to reduce the present nitrogen load to the

estuary (Katz et al., 2001). While there are instances of denitrification by

microorganisms, which can affect a decrease in the nitrogen load, especially in more

eutrophic areas, studies have shown that the ability of ecosystems to regulate themselves

can be exceeded by anthropogenic inputs into the ecosystems (Seitzinger & Nixon, 1985;

Katz et al., 1997). In the water year 2000, 2676 tons of nitrate-nitrogen was delivered to

the Gulf of Mexico by Florida rivers. The vast majority, 2620 tons, was supplied by the

Suwannee River (SRWMD, 200 Ib). Since an immediate reduction of nitrate-nitrogen is

unlikely, we need to examine the effects of the increased nitrogen loading in the estuary

to determine what impacts are likely to occur.









Characteristics of Algal Blooms

Phytoplankton are an important part of all estuarine and marine ecosystems and

phytoplankton blooms are a naturally occurring phenomenon. Increased nutrient delivery

to estuaries can result in an increase in the frequency and intensity of these blooms. Not

all algal blooms are harmful, however. Blooms of some species may only cause water

discoloration. Only when a phytoplankton species increases significantly in population

size and has detrimental ecological and physiological effects on the surrounding area, it is

considered a harmful algae bloom (HAB). Concentrations may vary depending on the

species composition of the phytoplankton assemblages. For nontoxic species, biomass is

the primary determinant of bloom conditions. For toxic species, the presence of a toxin in

the water can determine the bloom status (Smayda, 1997).

While the "red tide" dinoflagellate, Karenia brevis, has been documented at least

once in the Suwannee River estuary, it is not a common occurrence (Bledsoe & Phlips,

2000). Instead, typical bloom species include cyanobacteria and diatoms in the genera

Rhizoselenia, Thalassiosira, Cyclotella, and another unidentified small centric diatom

ranging from 3 to 10 tm in diameter (Bledsoe & Phlips, 2000; 2004).

Bivalves and their Effect on the Water Column

Bivalves are dominant suspension feeders in many estuarine ecosystems and are

capable, in some cases, of maintaining phytoplankton at low levels. In a study on the

freshwater clam, Corbiculafluminea, in the Potomac River, Cohen et al. (1984) found

that the lowest concentrations of phytoplankton biomass were in the areas with the

highest densities of clams. In the Chesapeake Bay, results from an estuarine model

showed that resident bivalves could consume more than 50% of the primary production

in shallow segments of the bay in spring and summer with 45% to 95% of the water









column being filtered by the bivalves during spring, summer, and fall (Gerritsen et al.,

1994). Werner and Hollibaugh (1993) also suggest that the clam, Potamocorbula

amurensis, has a substantial impact on the phytoplankton biomass in northern San

Francisco Bay. With a density of more than 2,000 clams m-2 and an average clearance

rate of 267 ml/h per clam, they calculated that the bivalves at a depth of 10-m could filter

the water column 1.28 times per day, while those in shallower waters (1-m) could filter

the water column 12.8 times per day. Based on these findings and those mentioned

above, it is evident that bivalves have the capacity to greatly influence the abundance of

phytoplankton, especially in enclosed systems.

Suspension Feeding in Bivalves

According to LaBarbera (1981), suspension feeding is comprised of three separate

processes: 1) movement of water past suspension-feeding structures, 2) removal of

particles from the water, and 3) transport of food particles to the mouth. All three

processes are accomplished by means of both mucociliary and hydrodynamic processes

(Ward, 1996).

Bernard (1974) related the pallial cavity (the latero-ventral space surrounding the

visceral mass that includes the gills, labial palps, stomach, and rectum) of a bivalve to a

"simple pump housed in a chamber (inhalant chamber) provided with a restricted inlet

(inhalant aperture) and a larger exit exhalantt aperture)" in which the ctenidia (gills)

"functions as a large diaphragm which is also porous to water." Water is drawn through

the inhalant siphon then encounters a partial obstruction, the ostial aperture, on its way

through the ctenidium by water tubes and out the exhalant siphon (Bernard, 1974). The

ostial aperture is the area that contains numerous pores (ostia) that connect the inhalant

chamber with the water tubes, and comprises about 37% of the ctenidium (Jones et al.,









1992). This obstruction of the ostial aperture decreases the water speed as it approaches

the ctenidium. Bernard (1974) also suggests that this allows preingestive selection of

particles by allowing larger particles, like minerals or inorganic particles, to settle directly

on the inhalant chamber mantle surfaces, according to the influence of gravity, and to be

ejected. Once particles are captured on the ctenidium, they are transported by the activity

of the frontal cilia. Mucus may play a role in the transportation of particles (Bernard,

1974; Beninger et al., 1992; Ward, 1996; but see Jiprgensen 1990). For most bivalves,

the majority of particles > 4 |tm are completely retained on the ctenidium while smaller

particles, 1 |tm to 4 |tm, are retained with various efficiencies (Mohlenberg & Riisgdrd,

1978; Jiprgensen, 1975). Some bivalves, like oysters, sort particles on the ctenidium.

The rejected particles are excreted as pseudofeces (Ward et al., 1997). The particles

selected for ingestion are moved dorsally to the labial palps for further selection and then

to the mouth for ingestion. The remains of digested particles are excreted as feces after

passing through the gut (Beninger et al., 1992; Ward et al., 1997).

The concentration of the particles that the bivalve is exposed to appears to have an

effect on the aforementioned feeding mechanisms. Beninger et al. (1992) found that as

the concentration of particles increases, bivalves start to exhibit some ingestion volume

control, among them being a reduction or a stoppage of movement of particles. In

addition, bivalves start to exhibit lower selectivity where "good" particles are often

rejected as pseudofeces (Beninger et al., 1992).

The Effect of Temperature on Bivalve Clearance Rates

Several investigations have shown temperature to have a hyperbolic effect on

clearance rates of bivalves (e.g., Hibbert, 1977; Grizzle et al., 2001). Hence, clearance









rates increase as temperatures increase until an optimum temperature is reached after

which clearance rates start to decrease. Optimum temperatures may vary between

bivalve species. For example, for the penshell, Atrina maura, Leyva-Valencia et al.

(2001) found clearance rates to be highest at 290C when tested over a temperature range

of 19-3 5C. The clearance rates for Crassostrea gigas reach a maximum at 190C

(Bougrier et al., 1995). For the catarina scallop, Argopecten ventricosus, clearance rates

are greatest at 19-220C (Sicard et al., 1999).

Other investigations, however, have reported a different response. For instance,

Sobral and Widdows (1997) showed that increasing temperature over the range 20TC to

32TC caused clearance rates to decrease in Ruditapes decussatus. Haure et al. (1998)

showed clearance rates to increase for Ostrea edulis over a temperature range of 10-30oC.

In fact, rates were maximum at 300C. Doering and Oviatt (1986) also found clearance

rates to have a linear relationship with temperature. On the other hand, some studies

have shown clearance rates to be independent of temperature (Loosanoff, 1958;

MacDonald et al., 1996). Loosanoff (1958) showed pumping rates to be independent of

temperature for adult oysters (Crassostrea virginica) between 16 and 28TC. Smaal et al.

(1997) looked at clearance rates of mussels (Mytilus edulis) and cockles (Cerastoderma

edule) and found that there was no relationship between temperature and clearance rates

of mussels throughout the year. However, Smaal et al. (1997) found that temperature did

have an effect on clearance rates for cockles.

The Effect of Diet Composition and Concentration on Bivalve Clearance Rates

There have been numerous experiments conducted to understand under what diet

conditions bivalves perform best. These studies have determined that both seston

quantity and quality may have an effect on bivalve feeding rates. In general, there is a









positive correlation between seston concentration and clearance rate (Albentosa et al.,

1996; Marsden, 1999; Hawkins et al., 2001; Ellis et al., 2002). Stenton-Dozey and

Brown (1992) showed that Venerupis corrugatus displays an ability to alter its clearance

rate in response to the quantity of the seston; as the seston concentration increased, the

clearance rate of V corrugatus increased. In another study, Mytilus edulis exhibited an

increased rate of ingestion with increased particle concentration until the rate of ingestion

reached an asymptotic value, which coincided with the threshold for pseudofeces

production (Bayne et al., 1989; 1993).

In some cases, however, clearance rates appear to be unaffected by increasing

seston concentration (Cranford & Hargrave, 1994; Arifin & Bendell-Young, 1997).

Rheault and Rice (1996), for example, found that clearance rates of C. virginica and A.

irradians irradians did not vary significantly with "fourfold tidal variations in food

concentration." However, A. irradians did exhibit a reduction in clearance rates when the

seston concentration was decreased by 88% compared to the original concentration.

Cranford and Hargrave (1994) obtained similar results with ingestion rates (biodeposition

rates) for Placopecten magellanicus using a new method for quantifying the feeding and

absorption rates of suspension feeding bivalves. Arifin and Bendell-Young (1997) did

not find a relationship between clearance rates and seston concentration, but did find that

pseudofeces production was dependent on seston concentration which, as stated before, is

another way for bivalves to regulate ingestion rates.

In a study examining the effects of seston quality on bivalve physiology, raft

mussels (Mytilus alloprovincialis) exhibited maximum clearance rates and absorption

efficiencies on mixed diets in which phytoplankton and sediment were provided in









similar proportions, with the phytoplankton being 30-40% of total particulate volume

(Navarro et al., 1996). Gatenby et al. (1996) found that cultured freshwater mussels

(Villosa iris and Pyganodon grandis) grew best on mixed diets of algae and sediment

rather than on algae alone. Both of the above studies suggest that sediment could play a

vital role in enhancing absorption of microalgae or could even help with digestion in the

stomach.

Generally, as seston quality increases, so does the clearance rate of a bivalve. In a

study by Stenton-Dozey and Brown (1992), clearance rates were greatest during high tide

when there was an abundance of particles greater than 9 |tm and the organic content of

available food was higher than at low tide. Likewise, Gardner (2002) found that

clearance rates for three species of bivalves (Aulacomya maoriana, Mytilus

galloprovincialis, and Perna canaliculus) increased in a linear fashion, with the highest

clearance rates at high levels of organic matter in mixed diets.

Selective Feeding in Bivalves

Although there is some question as to how bivalves select particles for ingestion, it

is generally accepted that bivalves can selectively ingest particles (Shumway et al., 1985;

Newell et al., 1989; Defossez & Hawkins, 1997). Selectivity can be divided into two

categories: 1) separation of inorganic particles from organic ones and 2) selection

between organic particles (Bernard, 1974). For the first category, experiments involving

variations in quantity and organic content of bivalve diets show that when presented with

a mixed diet, the pseudofeces often contain intact, nonorganic particles (Newell et al.,

1989; Iglesias et al., 1992; Bayne et al., 1993; Arifin & Bendell-Young, 1997). This









preingestive selection of organic versus inorganic particles could be based on size

(Bernard, 1974; Tamburri & Zimmer-Faust, 1996; Defossez & Hawkins, 1997).

Selectivity studies subjecting bivalves to organic particles of differing nutritional

value have shown the pseudofeces to contain particles that are organic in nature, but not

very nutritious, like detritus (Baker et al., 1998; Ward et al., 1998). In comparing

selectivity between phytoplankton, the majority of the selection and clearance rate

experiments have used few phytoplankton species, comparing preferential selection of

nutritious versus less-nutritious phytoplankton. With regard to zooplankton, Wong et al.

(2003) showed that rotifers in the micro- and mesozooplankton (140-210 |tm range) can

play a significant role in the diet of zebra mussels (Dreissenapolymorpha). There are

some experiments, however, involving various organic particles that indicate that there is

also selection by bivalves between nutritious particles. Baker et al. (1998) showed that

zebra mussels preferentially selected the cyanobacterium Microcystis over other

phytoplankton that are typically found in the Hudson River. Shumway et al. (1985)

showed that there is preferential selection of the cryptomonad flagellate, Chroomonas

salina, over other nutritious particles in the majority of bivalves they tested. Further

information is needed to understand particle selectivity.

Controversies Concerning Bivalve Feeding Mechanisms

There is still a question among scientists concerning the mechanisms of particle

capture and selection in bivalves. Ward et al. (1998) found that the ctenidia of

Crassostrea virginica are responsible for particle sorting and the labial palps perform an

accessory function in particle selection or they regulate the volume of the ingested

material. Ward et al. claim that it is essential for food particles to approach the gill









surface in a straight line at an angle of 30 degrees from the gill surface. They suggest

that variations of this angle could have an effect on the interaction of particles with the

gill filaments. A number of scientists disagree with the prerequisite of a low angle of

approach (Beninger, 2000; Riisgard & Larsen, 2000). For instance, Riisgard and Larsen

(2000) feel that because of the parallel arrangement of the lateral cilia, particles hit the

ctendium in a curved path between 70-90 degrees and that none hit below 40 degrees.

They feel that the 30 degrees that Ward et al. (1998) stated is not a prerequisite of particle

capture, but rather a result of the existing flow fields caused by ciliary activity on the

ctenidia in the bivalve.

Another disagreement concerns the role of mucus in suspension feeding by

bivalves. There are basically two hypotheses concerning the role of mucus. The first is

that mucus only forms to entrap materials when the ingestive capacity of the bivalve is

exceeded (Jiprgensen, 1990). The second hypothesis is that mucus is present at all times

and is a normal function of bivalve physiology (Beninger et al., 1993; Ward, 1996).

According to Bernard (1974), there appears to be two types of mucus in bivalves that are

involved in entrapment and rejection of particles. The mucus for entrapment of food

particles headed for ingestion appears to be light-colored and almost transparent, while

the rejection mucus appears to be darker and contains more particles. Beninger et al.

(1992) also has observed two distinct mucus types with the denser mucus being

associated with those particles that have been rejected.

Because of their opaque shells, it is hard to determine what goes on during the

normal functioning of living, intact bivalves. Different techniques for observation of

bivalve physiological mechanisms have been employed, e.g., dissection with isolated gill-









filament preparations, the use of surgically altered animals, the use of confocal laser

microscopy, and, most recently, the use of endoscopic techniques, have all been used to

collect information. These methods have led to a different controversy involving the

manner of data collection. Ward et al. (1998) have expressed some concerns about the

data collected from techniques other than video endoscopy. These investigators feel that

the manner of preparation of the organism for either methods of observation could

change some critical interactions between the way particles interact with the gill

filaments. However, some scientists disagree with this assessment (Beninger, 2000;

Riisgdrd & Larsen, 2000; Silverman et al., 2000). Beninger (2000) states that due to

mechanical limitations "endoscopy cannot in fact access the underlying mechanisms for

the processes of particle capture, transport, and selection, although it can... observe the

net result of the processes and suggest where and what techniques to use to seek the

sequence." These scientists suggest that results from previously mentioned techniques

and endoscopy are similar and that all techniques should be used to balance the

weaknesses and strengths of the individual methods.














CHAPTER 3
METHODS AND MATERIALS

Test Subjects

Experimental Algae and Culture Protocols

Four algal species were used in this study: Tetraselmis maculata (a large green

flagellate), Isochrysis galbana, (a small brown flagellate) and two strains of

Synechococcus (a cyanobacteria; one strain forms long chains and one consists primarily

of single cells). Algal cultures were supplied by Jose Nunez (I. galbana and T maculata),

Whitney Laboratory at the University of Florida, Dr. Edward Phlips (I. galbana and both

species of Synechococcus), Department of Fisheries and Aquatic Sciences at the

University of Florida, and Dr. Gary Wikfors (T maculata), Milford Laboratory. Algal

species were chosen to represent different sizes, with T maculata having cells that are ca.

10-[Im, I. galbana ca. 5-[im, and Synechocccus spp. ca. 2-[im. I. galbana is the primary

algae used in shellfish hatcheries, but T maculata is also used. Synechococcus spp., and

other picoplankton-sized blue-green algae, are not typically used in hatcheries (Hadley et

al., 1997) but are a major bloom forming taxa in coastal marine environments in Florida

(Phlips et al., 1999; Bledsoe, 2003). Of the three, cyanobacteria are regularly found in

the Suwannee River estuary (Bledsoe and Phlips, 2000).

All algal species were cultured in 500-ml flasks containing 150 ml of sterilized Ll-

Si media at 28 ppt (Guillard and Hargraves, 1993) at a pH of 7.8-8.2. Cultures were kept

at 25-28 'C on a 14:10 light/dark photoperiod at 30-60 [tEinsteins/m2/s light flux except

for the Synechococcus spp. which were kept at 20-30 [tEinsteins/m2/s light flux. Light









intensity was measured using a quantum light meter with a Li-Cor data logger. Once

cultures reached sufficient biomass they were transferred to either 1000-ml flasks or 2.8-

L Fernbach flasks that contained 500 ml and 1.5 or 1.8 L of Li-Si, respectively. The

500-ml and 1000-ml flasks were swirled once a day. Fernbach flasks were aerated with

0.22-|jm filtered air containing 1/32 psi of added CO2.

Phytoplankton Combinations

Experimental phytoplankton assemblages consisted of two of the algae species

types combined at a 1:1 ratio based on numerical abundance (Table 1). Initial counts

were determined by using either a flourescent microscope or a Coulter Multisizer II fitted

with a 100-|jm orifice. Enough phytoplankton was added to the experimental water to

bring the total experimental concentration to 105 cells/ml or 106 cells/ml.

Experimental Organism

Adults of the hard clam Mercenaria mercenaria were obtained from commercial

suppliers operating out of Cedar Key, Florida, a coastal area influenced by the Suwannee

River. Clams were allowed to purge in 0.2-|jm filtered seawater (28 ppt) for 72 hours at

the experimental temperature with no prior temperature acclimation. All water in the

holding tanks was changed twice during this period. Shell length, height, and width were

measured according to Fritz (2001). Data on size, weight, and number of animals used in

each feeding trial are provided in Table 2.

Experimental Protocol

Between December 2003 and March 2004, feeding selectivity and clearance rates

were examined through a series of laboratory feeding trials in static systems subjected to

two different temperatures (200C and 300C). Particle selection and clearance rates were









measured following Baker et al. (1998). Ten individual clams were individually placed

in 2-L clear glass Pyrex 9 beakers containing the experimental phytoplankton

assemblage in 28 ppt 0.2-|jm filtered seawater. The experimental beakers were run

concurrently with two control beakers, containing no clams. To lessen the occurrence of

phytoplankton settling to the bottom, beakers were gently aerated throughout the

experiment. Clearance rates were determined by analyzing 1.0-ml water samples at the

start of each feeding trial and again after 45 minutes. Feeding selectivity was determined

by examining the composition of the pseudofeces produced during the feeding trials (see

below). At the end of each feeding trial, clams were measured, weighed wet, and then

tissues were dried to a constant weight (600C). Each feeding trial was replicated three

times on different days.

Hurlbert (1984) wrote about the dangers of pseudoreplication in ecological field

experiments; replicates that are spatially or temporally separated are not independent and

the data should not be used as such. In the experimental design, time was not taken into

account. For example, one replicate of IsoSyn at 300C was performed in January, and

another in February. The clams used for each replicate were from different batches and

had been exposed to different environmental parameters. Therefore, according to

Hurlbert (1984), the replicates were not "true" replicates of each temperature/algal

combination but rather pseudoreplicates. In addition, the replicates were not performed

randomly; feeding trials at 300C were completed before the 200C feeding trials.

Additional Experiments

Two additional types of feeding trials were conducted. To determine whether

clams would prefer algae to which they had been acclimated, one batch of clams was split









into two groups. "Batch" refers to clams obtained at the same date and time and from the

same area. Prior to purging and conducting feeding trials, the two groups of clams were

kept in separate holding tanks for two weeks. One group of clams was fed I. galbana and

the other group of clams was fed Synechococcus at a rate of 2% of their dry body weight

every other day (http://www.reedmariculture.com). Feeding trials were conducted

following the experimental protocol outlined above.

To eliminate time as a factor, a second type of feeding trial was performed. One

batch of clams was obtained and split into two groups. The clams were allowed to purge

for three days; one group at 200C and one group at 300C. Feeding trials for each group

were subsequently performed following the experimental protocol described above.

Ideally, this is how the experiment protocol should have been designed.

Calculation of Particle Selectivity

Phytoplankton species in the pseudofeces and water samples were identified and

counted using a FACSCAN flow cytometer (BD BioSciences, San Jose, CA) at the

University of Florida Flow Cytometry Core Lab. Samples were vortexed to disrupt

aggregations immediately prior to analysis. Samples were shaken and filtered through a

40-|jm mesh to remove large particles to prevent clogging of the flow cytometer. In the

cytometer, cells flow singly through a 488 nm laser beam while their forward light scatter

and red fluorescence emission above 650 nm are measured. Forward light scatter was

used to eliminate small particles. Data was collected for one minute at the "high" flow

rate (approximately 60 [LL/min). Regions identifying phytoplankton were set based on

forward light scatter and red fluorescence using CellQuest 3.3 software running on a









Macintosh computer (Apple Computer, Cupertino, CA). Counts of cells in each region

were collected in a text file and imported into a Microsoft Excel spreadsheet.

To determine feeding selectivity, a modified electivity index (EI) was calculated.

The equation for El is based on Ivlev's index of electivity for freshwater fish and was

modified as followed: El = -[(P S) / ((P + S) (2*P*S))] where P is the ratio of a

particular algal species in the pseudofeces and S is the ratio in the suspension (Ivlev,

1961; Bayne et al., 1977). The electivity index can range from -1.0 tol.O. If the El is

positive for a given algal species, then there is a preferential ingestion of that species. If

the El is negative, then it indicates a rejection of that algal species.

Since electivity indices are not continuous, the indices were arcsine transformed.

The mean of the means for each replication within each temperature feeding trial (200C

and 30C) was compared to zero using a one-sample t-test. For the feeding trials of

IsoSyn, Tetralso, TetraSyn, and IsoSyn(Ch), the indices were compared using a 2-way

ANOVA (temperature and feeding trial). To compare IsoSyn and IsoSyn6, a 2-way

ANOVA (temperature and concentration) was used. Since there was only one replication

for IsoSyn-AI, IsoSyn-AS, and IsoSyn-B, no statistical procedures were conducted.

Instead the mean clearance rates were graphed. Feeding trials, statistical tests used, and

p-values are summarized in Table 3 in the Results section.

Calculation of Clearance Rate

Clearance rates were also determined using a flow cytometer. The reduction in the

concentration of particles over 45 minutes was used to calculate net clearance rates

according to Coughlan (1969) as follows:

CR= V [(ln(Co)-ln(C1))/t) -A); where CR is clearance rate, V is the volume of the

experimental suspension, Co is the initial concentration, C1 is the concentration after time









t, and A is the average of the controls (A = (Z[(ln(Co)-ln(Ci)])/n, where n is the number

of controls). Clearance rates were corrected for particle abundance changes in the

controls and for the amount of time the bivalves were open. Clearance rates were

standardized to 1-g of dry tissue mass using an allometric exponent for bivalves of 0.75

(Grizzle et al., 2001) as follows: CR(ig) = CR/b 0.75; where CR is the uncorrected

clearance rate of the experimental clam expressed in L*h-1 and b is the dry weight of the

experimental clam expressed in grams.

The means for each replicate feeding trial at 200C and 30'C involving mixed algal

assemblages, i.e. IsoSyn, Tetralso, TetraSyn, and IsoSyn(Ch), were analyzed with a 2-

way ANOVA. Clearance rates for IsoSyn and IsoSyn6 were compared also with a 2-way

ANOVA. Since there was only one replication for IsoSyn-AI, IsoSyn-AS, and IsoSyn-B,

no statistical procedures were conducted. Instead the mean clearance rates were graphed.

Feeding trials, statistical tests used, and p-values are summarized in Table 4 in the

Results section. Clearance rate data was not used if the particle concentration in the

beakers declined below 40% of the initial concentration (Baker and Levinton, 2003).









Table 1. Algal assemblages and the date(s) of replication(s) of each feeding trial at two
different temperatures. Unless stated otherwise, concentrations were 105
cells/ml.


I. galbana + Synechococcus (no chains) at
106Cells/ml


I. galbana + Synechococcus (no chains)
(acclimated to Synechococcus-no chains)
I. galbana + Synechococcus (no chains)
(acclimated to I. galbana)
I. galbana + Synechococcus (no chains)
(Same batch, 2 temperatures) at 106Cells/ml


IsoSyn




TetraSyn




Tetralso


IsoSyn6




IsoSyn-AS


IsoSyn-AI


IsoSyn-B


Phytoplankton Trial
Abbreviation


I. 3/25/04


I. 4/08/04


I. 2/19/04


I. 4/08/04


Temperature

200C 300C

I. 2/26/04 I. 1/29/04
II. 3/04/04 II. 2/05/04
III. 3/25/04 III. 2/19/04
I. 3/04/04 I. 1/29/04
II. 3/11/04 II. 2/12/04
III. 3/18/04 III. 2/12/04
I. 3/04/04 I. 3/04/04
II. 3/11/04 II. 3/11/04
III. 3/18/04 III. 3/18/04
I. 2/26/04 I. 1/15/04
II. 2/26/04 II. 1/15/04
III. 3/11/04 III. 1/22/04
I. 3/04/04 I. 1/22/04
II. 3/11/04 II. 2/05/04
III. 3/18/04 III. 2/12/04
I. 3/25/04 I. 2/19/04


I. galbana + Synechococcus_(no chains)




T. maculata + Synechococcus (no chains)




T. maculata + I. galbana


I. galbana + Synechococcus(Chain-forming) IsoSyn(Ch)














Table 2. Mean size and weight, and actual number of animals that opened in each feeding trial. Numbers in parentheses are SD.
Wet Dry
Trials n Height(mm) Length(mm) Width(mm) Weight(g) Weight(g)


200C Experimental
(0.47) Clams
IsoSyn-Al
IsoSyn-AS
IsoSyn-B
300C Experimental
(0.75) Clams
IsoSyn-Al
IsoSyn-AS
IsoSyn-B


38.11 (0.19)
39.41 (1.48)
37.46 (1.75)
37.68 (2.18)

37.43 (0.32)
37.52 (3.59)
38.70 (2.72)
38.42 (2.39)


44.13 (0.21)
46.11 (1.75)
42.61 (2.00)
43.22 (1.81)

43.50 (0.45)
44.95 (1.75)
45.46 (3.32)
44.66 (3.04)


24.04 (0.19)
26.37 (4.18)
23.94 (0.89)
24.52 (0.76)

23.31 (0.27)
24.09 (1.06)
24.03 (1.39)
25.06 (1.19)


27.5 (0.40)
31.2 (3.0)
26.6 (2.8)
26.5 (3.0)

26.18 (0.54)
27.5 (2.7)
28.1 (5.8)
29.4 (4.9)


0.87 (0.01)
1.01 (0.17)
0.77 (0.08)
0.74 (0.12)

0.76 (0.02)
0.75 (0.10)
0.82 (0.18)
0.80 (0.13)















CHAPTER 4
RESULTS

Electivity Indices

Based on results, clams sorted particles for ingestion or rejection. Out of eight algal

combinations, three had Els that were significantly different than zero (Fig 1). Specifically,

there was preferential ingestion oflsochrysis in all three trials.

Algal combination had a significant effect on Els (p-value = 0.000). Clams showed greater

selectivity between Synechococcus (either chainforming or not) and Isochrysis than between

Tetraselmis and Synechococcus or Tetraselmis and Isochrysis (Fig 1). In particular, IsoSyn(Ch)

had much higher Els than Tetralso (p-value = 0.0029) and TetraSyn (p-value = 0.0175). In

addition, IsoSyn had significantly higher Els than Tetralso (p-value = 0.0001). Although there

was no interaction between algal combination and temperature, within the 20'C particle

combinations, Tetralso had a significantly lower El than IsoSyn (p-value = 0.0325). At 30'C,

Tetralso had a significantly lower El than either IsoSyn (p-value = 0.0021) or IsoSyn(Ch) (p-

value = 0.0190) (Fig 1). Based on these results, there appears to be a pattern in which clams

select the larger algal species (either Tetraselmis or Isochrysis) over the smaller Synechococcus.

Temperature had a significant effect on particle selectivity (p-value = 0.035); clams were

more selective at 200C than they were at 300C (Fig 1). However, when a single batch of clams

was used for both temperature experiments, there appeared to be no difference in Els (Fig 2).

Cell concentration had a significant effect on electivity. Electivity indices were

significantly greater at the lower concentration (p-value = 0.012) (Fig 3).









Within each temperature/algal combination, there was a high amount of variability in Els

between replicate trials (batches of clams) (Fig 4). For example, when three different batches of

clams were fed Tetraselmis and Synechococcus at 300C, one batch strongly selected for

Tetraselmis, one batch slightly selected for Tetraselmis, and one batch selected for

Synechococcus (Fig 4b). When the three batches were combined, this resulted in a small positive

EI, but no active selection for either alga (Fig Ib). Prior feeding history did not appear to

account for differences between batches (Fig 5). When a single batch of clams was split and two

groups acclimated to different algae, there was no difference in their preference for those algae.

Clearance Rates

Temperature had no effect on clearance rate when clams were fed any of the four particle

combinations (Fig 6). However, when a single batch of clams was used for both temperature

experiments, mean clearance rates were greater at 300C than at 200C (Fig 7). Interestingly, there

was a significant interaction (p-value = 0.0461) between temperature and algal combination due

mainly to TetraSyn at 200C having a lower clearance rate than Tetralso at 300C. Cell

concentration did not have a significant effect on clearance rates (Fig 8). However, temperature

did have a significant effect when comparing IsoSyn and IsoSyn6, with clearance rates higher at

20'C than at 300C (p = 0.003).

Prior feeding history may have had an impact on clearance rates. Clams acclimated to

Synechococcus exhibited a higher mean clearance rates for a combination oflsochrysis and

Synechococcus than did those clams acclimated to Isochrysis (Fig 9).









Figure 1. Electivity indices (means SE) for Mercenaria mercenaria at two different
temperatures, 20'C and 30TC, when fed different combinations of algae at a
total concentration of 105 cells/ml. A positive El indicates selection of an
algal species. A negative El indicates rejection of an algal species. Symbol
(*) indicates which Els were significantly different than zero (p<0.05). a)
Acceptance (+) or rejection (-) of Tetraselmis when Isochrysis is present. b)
Acceptance or rejection of Tetraselmis when Synechococcus sp. is present. c)
Acceptance or rejection of Isochrysis when Synechococcus is present. d)
Acceptance or rejection of Isochrysis when the chainforming strain of
Synechococcus is present.











a) El for Tetraselmis in the feeding trial Tetralso


................ T,


E 200C

M 300C


b) El for Tetraselmis in the feeding trial TetraSyn


I________


c) El for Isochrysis in the feeding trial IsoSyn


n=3 n=3


d) El for Isochrysis in the feeding trial IsoSyn(Ch)










Acceptance or rejection of Isochrysis when Synechococcus is present
in clams from a single batch (106 cells/ml): 200


300C


n=14 n=15


Figure 2. Electivity indices (means SE) of Mercenaria mercenaria for Isochrysis
galbana when Synechococcus sp. (nonchainforming) is present, at two
different temperatures, in clams from a single batch (IsoSyn-B). Total cell
concentration was 106 cells/ml. A negative El indicates rejection of
Isochrysis.


0.6


0.2


-0.2


-0.6


-1










Acceptance or rejection of Isochrysis when Synechococcus
two concentrations a) 105 and b) 106 cells/mi:


-0.6


-1


a) 10s cells/mi


is present at

Z 200C

300C




T


b)106 cells/mi


n=3


n=3


Figure 3. Electivity indices (means SE) of Mercenaria mercenaria for Isochrysis when
Synechococcus sp. (non-chainforming) is present, at two temperatures, 20'C
and 30TC, and two cell concentrations a) 105 and b) 106 cells/ml. Symbol (*)
indicates which replication(s) were significantly different than zero (p<0.05).











a) Els for Tetraselmis in the feeding trial Tetralso


n=4 n=5


1 b) Els for Tetraselmis in the feeding trial TetraSyn
.6
.2 -


n=3


n=5 n=8 n=8


1 c) Els for Isochrysis in the feeding trial IsoSyn

0.6
0.2

-0.2
-0.6
n=8 n=8 n= 7


n=8 n=5 n=10


Figure 4. Mean replication (or batch) electivity indices (mean SE) for Mercenaria
mercenaria at two temperatures, 20'C and 30C, when fed different
combinations of algae. There were three replicates for each temperature. A
positive El indicates acceptance of an algal species. A negative El indicates
rejection of an algal species. a) Acceptance or rejection of Tetraselmis when
Isochrysis is present at a total concentration of 105 cells/ml. b) Acceptance or
rejection of Tetraselmis when Synechococcus sp. is present at a total
concentration of 105 cells/ml. c) Acceptance or rejection of Isochrysis when
Synechococcus is present at a total concentration of 105 cells/ml. d)
Acceptance or rejection of Isochrysis when the chainforming species of
Synechococcus is present at a total concentration of 105 cells/ml. e)
Acceptance or rejection of Isochrysis when Synechococcus is present at a total
concentration of 106 cells/ml.


-1 J n=6


200C

300C


n=4


n=8


EM

MM


I












d) Els for Isochrysis in the feeding IsoSyn(Ch)


n=6


n= 5


e) Els of Isochrysis in the feeding trial IsoSyn6


n=9 n=9 n=10


n=10 n=8


Figure 4. Continued.


n=6


n=9


n=7











Acceptance or rejection of Isochrysis when Synechococcus is present,
in clams acclimated to either a) Synechococcus or b) Isochrysis:


-0.2


-0.6


a) Synechococcus


b) Isochrysis


-1 n=7


Figure 5. Mean electivity indices of Mercenaria mercenaria for Isochrysis galbana
when Synechococcus sp. is present, at two temperatures, in clams acclimated
for two weeks on either a) Synechococcus (IsoSyn-AS) or b) Isochrysis
(IsoSyn-AI). Total cell concentration was 105 cells/ml. A positive El
indicates selection of Isochrysis. (Means SE).


2000C

300C














b) TetraSyn


n=13


n= 18


c) IsoSyn


n= 21


200C


n= 18


300C


0.5


n=9


3


2.5


2


1.5


d) IsoSyn(Ch)


n = 21


200C


Figure 6. Clearance rates by Mercenaria mercenaria (means SE ) at two temperatures
(20'C and 30'C) when fed algal suspensions at 105 cells/ml. All clearance
rates were standardized to 1 gram of dry weight.


1


0.5


0


n= 16


3


2.5
2-
2


1.5
1 -
1


0.5


0


.\\\\\\\\\\\\\\\\\\\\\\\\
.\\\\\\\\\\\\\\\\\\\\\\\\
.\\\\\\\\\\\\\\\\\\\\\\\\
.\\\\\\\\\\\\\\\\\\\\\\\\
.\\\\\\\\\\\\\\\\\\\\\\\\


n= 18


300C


a) Tetralso










3

2.5

2

1.5




two temperatures groups.














a) 10s cells/ml


n=13


200C


n=18


300C


3


2.5


2


1.5


b) 106 cells/ml


n =27


200C


Figure 8. Clearances rates means SE) by Mercenaria mercenaria fed I. galbana and
Synechococcus sp. at two temperatures (200C and 30oC) and two
concentrations: a) 105 cells/ml (IsoSyn) and b) 106 cells/ml (IsoSyn6).


3


2.5


2


1.5


n = 23


300C


,,,,,,,,,,,,,,,,,,,,,
,,,,,,,,,,,,,,,,,,,,,
,,,,,,,,,,,,,,,,,,,,,
,,,,,,,,,,,,,,,,,,,,,
,,,,,,,,,,,,,,,,,,,,,
,,,,,,,,,,,,,,,,,,,,,
,,,,,,,,,,,,,,,,,,,,,


_I_














a) Synechococcus


b) Isochrysis


3

c02.5

c 2
-J
,1.5

a 1

a 0.5

(0


n=3
200C


n=8
300C


Figure 9. Clearance rates means SE) of Mercenaria mercenaria of the feeding trial
Isochrysis galbana and the nonchain-forming species of Synechococcus
(IsoSyn-AS and IsoSyn-AI) at two different temperatures (20'C and 30'C)
when clams were acclimated to either a) Synechococcus or b) Isochrysis.


n=7 n=8
200C 300C














CHAPTER 5
DISCUSSION

This is the first study to conduct feeding selectivity experiments with bivalves at

temperatures above 20'C Most feeding selectivity studies have been conducted at

temperatures between 12 'C and 20'C (Shumway et al., 1985; Bayne et al. 1989;

Stenton-Dozey & Brown, 1992; Bayne et al. 1993; Arifin & Bendell-Young, 1997;

Defossez & Hawkins, 1997; Ward et al., 1998); appropriate given the cooler

temperatures of collection sites. For example, summer water temperatures at

northeastern locations such as Wells, Maine; Hudson River, New York; and Narragansett

Bay, Rhode Island, reach no more than 16.5C, 22.60C, and 23.3oC, respectively

(NERRS, 2004). In the mid-Atlantic, such as Chesapeake Bay, Bay Bridge, Maryland,

temperatures reach about 27.5C in August (NERRS, 2004).

In the shallow waters along Florida's Gulf coast, however, temperatures exceed

30'C in the summer and are routinely > 25C for half the year (Jett, 2004; NERRS, 2004;

Frazer, unpublished data; Phlips, unpublished data). In the Suwannee River Estuary, near

Cedar Key, Florida (a prominent site for clam aquaculture), water temperatures can also

get up to 300C (Jett, 2004). The warmest National Estuarine Research Reserve (NERRS,

2004) sites in Florida can be found in Rookery Bay, Naples (about 70 miles south of

Charlotte Harbor which is another site for clam aquaculture), where waters reach 35.6 TC

(NERRS, 2004). Therefore, this study is important because it is the first to examine how

high temperatures (30oC) may influence feeding selectivity of bivalves, and Mercenaria

mercenaria in particular.









The results of this study indicate that temperature affects feeding selectivity of

Florida hard clams. Clams exhibited greater selection at 200C than they did at 300C.

This finding may be the result of the acclimatizing protocol. When acclimatizing the

clams to the experimental temperature, they were immediately placed in either 20'C or

30'C water, depending on the feeding trial, and held for three days. Since the lower

temperature trials were done in late February and March, the experimental water was

within a typical range of water temperatures for that time of year in Cedar Key. In the

feeding trials at 300C, there was a 10-20'C difference between the collection water

temperature and the experimental water temperature (www.floridaaquaculture.com). For

fish, acclimation to the experimental water temperature should be gradual to reduce

shock which could change physiological states like hormone levels or blood chemistry

concentrations (Stickney and Kohler, 1990; Wedemeyer et al., 1990). Fry (1971)

suggests a gradual acclimation schedule of 1oC/day until the experimental temperature is

reached. While intertidal organisms can withstand daily temperature variations of 20-

30oC, subjecting the clams to a temperature change of more than 10oC have shocked the

clams (Hochachka & Somero, 2002). Therefore, the lack of acclimation to 300C may

have rendered the clams less efficient in their selective abilities.

Another reason for the difference in selectivity between temperatures may have

been a "batch effect." Again, a "batch" refers to a quantity of clams collected at one time

from the same area and exposed to the same environmental history. Since they were

collected at different times of the year, the clams used in the low temperature trials may

have been subjected to different environmental parameters than those used in higher

temperature trials. In the batch experiment (IsoSyn-B), in which one batch of clams was









split between two temperatures, temperature did not appear to have an effect on

selectivity (Fig 2). This suggests that the difference in selectivity between 20'C and 30'C

in the main experiment may largely be due to the batch used rather than to the lack of

acclimation. More study is needed, however.

In addition to temperature, algal combination had a significant effect on electivity

indices of hard clams. At algal concentrations of 105 cells/ml, there was greater selection

for larger particles (Tetraselmis at 10 [im and Isochrysis at 5 [im) over smaller particles

(Synechococcus sp. at 2 ism and with chains) (Fig 1). Tetraselmis and Isochrysis are both

well within the size range to be completely retained by a bivalve while Synechococcus is

on the lower end of the size range (JQprgensen, 1975; Mohlenberg & Riisgdrd, 1978).

Bass et al. (1990) examined the growth of M mercenaria on picoplankton which

included Nannochloris (ca. 3 [im) and two species of Synechococcus sp. (both ca. 1 [im)

in length. They found that, while the picoplankton were filtered out of suspension by the

clam, it was assimilated with low efficiency (17.6-31.1%) compared to the 4 .im

Pseudoisochrysisparadoxa (86.5%). The results from this study, however, showed

preferential ingestion of the smaller Synechoccococus (2 [im) over the larger Isochrysis

(5 pm) in four of the eight feeding trials (Fig 2; Fig 4) done at 106 cells/ml. Bass et al.

(1990) worked with smaller picoplankton and lower concentrations (5 x 104 to 105

cells/ml) which could be why a difference was seen in this study. Although the mean

values of replicates indicated no preference for Synechococcus, there was a high amount

of variability in electivity indices between batches of clams (Fig 2). In addition, although

statistics were not done in Fig 4, the electivity indices were high (0.7). Because of these

conflicting results, further studies would be interesting.









Algal concentration had a significant effect on selectivity, with clams exhibiting

active selection at the lower concentration and no sorting at the higher concentration. A

study by Levinton et al. (2001) suggests that rate-limiting steps within a bivalve's

digestive process may affect how a bivalve processes particles. As the gut becomes full,

bivalves may start to increase rejection of nutritious particles as pseudofeces that they

would otherwise ingest. As a result, bivalves may show no preferential ingestion

between algal species when presented at high concentrations.

There was no significant effect of temperature on clearance rates when clams were

fed any of the four algal combinations at 105 cells/ml (Fig 6). This is in agreement with

findings reported by Smaal et al. (1997), who showed clearance rates for mussels (M

edulis) to be independent of temperature. In addition, MacDonald et al. (1996) showed

clearance rates for Placopecten magellanicus to be independent of temperatures over the

range of 0 to 150C. In a field study, Paterson et al. (2003) examined growth in the rock

oyster Saccostrea glomerata in Australia for a year and found that, although there was

variation in temperature between study sites, growth (a good indicator of feeding rates)

was independent of temperature. It is interesting to note that while statistics were unable

to be done on the clearance rates for the batch experiment, the mean clearance rate for

20TC was higher than that at 300C. Again this could be because of a batch effect.

However, when comparing the two different concentrations (105 and 106 cells/ml)

of IsoSyn, temperature was found to be significant with higher clearance rates at 200C

than at 300C (Fig 8). This is in agreement with studies of northeastern clams. Hamwi

(1969) showed that temperature had an inverted parabolic effect on pumping rates, i.e.

the rate at which water flows through the mantle cavity, with pumping rates at 300C being









much less than the pumping rates at 200C. Other bivalves are reported to react much the

same way. Over the temperature range of 16-280C scallops (Arogpecten ventricosus

circularis) had maximum clearance rates at 190C and 220C (Sicard et al., 1999). In the

clam Rupitapes decussates (L.), increasing temperature has a negative effect on clearance

rates, leading to a reduction in scope for growth (Sobral & Widdows, 1997). In contrast,

cockles appear to increase clearance rates with increasing temperature (Smaal et al.,

1997). Levya-Valencia et al. (2001) showed that temperatures of 290C or higher were

optimum for clearance, ingestion, and growth rates for the penshell Atrina maura.

In contrast to other studies, concentration did not have a significant effect on

clearance rates temperature was held constant. Bayne et al. (1989) showed that ingestion

rates (equivalent to clearance rates when no pseudofeces are produced) ofMytilus edulis

increased with increasing seston concentration. Tenore and Dunstan (1973) showed that

although feeding rates ofM. mercenaria increased with increasing food concentration,

they were still lower than the feeding rates observed in M. edulis and C. virginica. This

could be the result of a combination of reduction in pumping rate or filtering efficiency

and an increase in pseudofeces production as concentrations increase. In addition, each

species' response to increasing food concentration may reflect adaptation of bivalves to

the areas where they were collected. Additionally, Tenore and Dunstan showed that

feeding rates in the three bivalves exhibit a slight inverted parabolic effect in association

with seston concentration. In contrast, Bricelj and Malouf (1984) found a negative

relationship between seston concentration and clearance rates in M. mercenaria, with

clearance rates decreasing as seston concentration increases. In this study, however,

effect of concentration had no effect on clearance rates. Both concentrations used in the









present study were relatively high. Tenore and Dunstan (1973) suggest that hard clams

may not be well suited for feeding at high particle concentrations, compared to other

bivalves.

While there were no differences between clearance rate replicates, there was a high

amount of variability among replicates or batches in the selectivity experiments.

Differences between replicates may be due to a variety of parameters including feeding

history, adaptation of clams to their environment, seasonal changes in digestive enzymes,

and/or other factors, e.g., changes in water viscosity due to temperature.

Feeding history, for example, may have an impact on feeding selectivity and

digestion. Bayne (1993) noted that bivalves can shift feeding preferences as an

adaptative strategy to seasonal changes in food availability. In addition, Ibarrola et al.

(1998) showed that there is an apparent seasonal pattern of digestive enzyme activity that

may be affected by past feeding history and could potentially affect food selection.

However, in the feeding trials IsoSyn-AI and IsoSyn-AS where clams were acclimated

for two weeks on either Isochrysis or Synechococcus sp., there appeared to be no

difference in the feeding selectivity between clams previously fed I. galbana and those

previously fed Synechococcus (Fig 5). While the clams were fed 2% of their dry weight

per day during the acclimation period, a ration generally recommended for bivalves

(www.reedmariculture.com), this resulted in concentrations of only 317 or 6349 cells/ml

Isochrysis and Synechococcus, respectively. Therefore, total particle concentrations may

have been too low to have a significant acclimatory effect on the clams. Additional

studies with higher cell concentrations are warranted.









Although the two-week study was inconclusive, bivalves may become adapted to

exploit the specific suite of food available to them in the field. For example, bivalves

from areas that are dominated by high bacterial counts had higher rates of clearance of

bacteria compared to bivalves from other areas not dominated by bacteria (Wright et al.,

1982; Berry & Schleyer, 1983). The high variability in selectivity between batches of

hard clams in this study, with four out of eight feeding trials at 106 cells/ml able to select

small particles (2 .im), suggests that these batches had adapted to high counts of small

particles in the environment. There have been no studies to date that compare the

clearance rates of clams from Cedar Key with clams from other areas along the Atlantic

coast that may typically feed on other particle types and sizes.

The absorption efficiency of particular phytoplankton species is determined by

digestive enzymes, and enzymatic activity may be influenced by season or food

availability (Bayne et al., 1993; Ibarrola et al., 1998). For example, Seiderer and Newell

(1979) reported that Choromytilus meridionalis changed the activity rate of a-amylase in

response to changes in temperature and, coincidentally, with phytoplankton composition.

Ibarrola et al. (1998) also found seasonal variation of digestive enzyme activities in the

cockle C. edule, in northern Spain, where their spring/summer diet is predominantly

living phytoplankton while in fall it consists mainly of kelp detritus. There is speculation

that assimilation efficiency corresponds to selectivity; phytoplankton that are easily

assimilated are selected for ingestion (Baker et al., 1998). It follows that if digestive

enzymatic activity changes seasonally and in response to available food, then selectivity

should change also. This offers a further explanation for differences in the ability of

batches to select for the smaller algae, Synechococcus sp.









Temperature may affect the physiology of bivalves and, as a consequence, the

ability to process certain food items. Temperature may also have an effect on the

mechanical aspects of suspension feeding. For example, because temperature is inversely

related to viscosity, suspension feeding echinoderm larvae (Dendraster excentricus) are

more apt to ingest large particles when the water has a high viscosity (colder) (Podolsky,

1994). Podolsky (1994) suggested that changes in viscosity might also affect retention

efficiencies of bivalves. Since waters in the Suwannee River Estuary change seasonally

from cool (< 11C) to hot (>30'C) (Jett, 2003; Frazer, unpublished data; Phlips,

unpublished data), water viscosity could play a role in what hard clams are able to filter

and ingest.

In conclusion, temperature was found to have an effect on food selectivity, with

Cedar Key hard clams exhibiting greater selection at 200C than at 300C. In addition, I

found that temperature had almost no effect on clearance rates. However, due to the wide

variability in results, more studies are needed to further test the effects of temperature and

batch effect on particle selectivity and clearance rates for Cedar Key hard clams and

bivalves in general. For example, it would be interesting to determine whether a batch

effect is unique to Cedar Key hard clams, common to all hard clams, or common to all

bivalves. In addition, it would be interesting to see how much the batch effect changed

over a year and whether it corresponded to phytoplankton abundance or composition in

the Suwannee River Estuary. It is documented that cyanobacteria like Synechococcus are

common in Florida waters, especially in the summer (Phlips et al., 1999; Bledsoe, 2003)

when the waters are warm, and bigger sized phytoplankton are common in the winter. It

would make sense, then, that the clams tested in July 2003 would prefer the smaller sized









cyanobacteria and why they rejected it when the experiment was performed again in

January through March 2004 (Figure 3). Temperature may have additional indirect

effects by either affecting phytoplankton composition in the estuary or by being a

conditioning factor for bivalves living in the area. This study is important in that it is one

of the first to examine feeding selectivity of bivalves in association with changes in

temperature. The results suggest important additional avenues of research which will be

essential to improving aquaculture practices in warmer climates, especially for the growth

and stability of the clam farms located in the vicinity Suwannee River Estuary.
















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BIOGRAPHICAL SKETCH

Carla Danielle Beals was born in Charleston, South Carolina, on February 14,

1976. In high school, she attended the South Carolina Governor's School for Science and

Mathematics for her 11th and 12th grade years. She received her BS in marine science and

graduated cum laude from the University of South Carolina, Columbia, SC, in December

1998. In December of 2004, she will receive her MS from the University of Florida,

Gainesville, Florida.