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CLEARANCE RATES AND PARTICLE SELECTIVITY IN THE HARD CLAM,
Mercenaria mercenaria, FROM WARM WATER HABITATS
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
Carla Danielle Beals
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
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
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
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
LIST OF TABLES
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
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
Carla Danielle Beals
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
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
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
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.
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
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,
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,
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
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
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.
METHODS AND MATERIALS
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.
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.
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.
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.
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
I. galbana + Synechococcus (no chains) at
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
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.
Trials n Height(mm) Length(mm) Width(mm) Weight(g) Weight(g)
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.
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
b) El for Tetraselmis in the feeding trial TetraSyn
c) El for Isochrysis in the feeding trial IsoSyn
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
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
Acceptance or rejection of Isochrysis when Synechococcus
two concentrations a) 105 and b) 106 cells/mi:
a) 10s cells/mi
is present at
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
1 b) Els for Tetraselmis in the feeding trial TetraSyn
n=5 n=8 n=8
1 c) Els for Isochrysis in the feeding trial IsoSyn
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
d) Els for Isochrysis in the feeding IsoSyn(Ch)
e) Els of Isochrysis in the feeding trial IsoSyn6
n=9 n=9 n=10
Figure 4. Continued.
Acceptance or rejection of Isochrysis when Synechococcus is present,
in clams acclimated to either a) Synechococcus or b) Isochrysis:
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).
n = 21
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.
two temperatures groups.
a) 10s cells/ml
b) 106 cells/ml
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).
n = 23
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.
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
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
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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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,