Particle removal by Northern bay scallops Argopecten irradians irradians in a semi-natural setting: Application of a flow-cytometric technique

Aquaculture 296 (2009) 237–245

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Aquaculture j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / a q u a - o n l i n e

Particle removal by Northern bay scallops Argopecten irradians irradians in a semi-natural setting: Application of a flow-cytometric technique Yaqin Li ⁎, David J. Veilleux, Gary H. Wikfors NOAA National Marine Fisheries Service, Northeast Fisheries Science Center, 212 Rogers Avenue, Milford, CT 06460, USA

a r t i c l e

i n f o

Article history: Received 9 April 2009 Received in revised form 11 August 2009 Accepted 14 August 2009 Keywords: Clearance rate Natural seston Scallop Flow-cytometry

a b s t r a c t The feeding activities of bivalve mollusks have direct impacts on suspended particles in nature. Feeding is also related to the carrying capacity of a particular system for maximal aquaculture production. Particle removal is known to be dependent upon particle characteristics, including size and type, because particle retention efficiency is size dependent, and bivalves are able to select particles actively to improve the quality of food ingested. Our goal was to quantify particle clearance by northern bay scallops of natural seston under semi-natural conditions, i.e., in raceway tanks receiving a constant flow of untreated, estuarine water. Particle clearance rate (CR) was determined based upon particle counts in tank inflow and outflow streams. A flow-cytometric technique was developed allowing rapid quantification of particles with different sizes and characterization by presence or absence of chlorophyll fluorescence simultaneously. CR varied greatly (ranging from 0.06 to 3.02 L h− 1 gDW− 1) among sizes and types of particles. CR of large particles was generally higher than that of the smaller particles. Under the specific conditions of this experiment, particle selectivity appeared to be ineffective because scallops did not show a higher CR on phytoplankton than on detritus. This study demonstrated the feasibility of this flow-cytometric approach to measure differential clearance of different particles in natural seston by suspension-feeding organisms. Published by Elsevier B.V.

1. Introduction Filter-feeding bivalves, such as scallops, oysters and clams, are able to remove considerable amounts of suspended particles from the water through filtration and feeding activities (Shumway et al., 1985; Cranford and Grant, 1990; MacDonald and Ward, 1994; Prins et al., 1994; Fanslow et al., 1995; Cranford et al., 1998; Shumway et al., 2003; Cranford et al., 2005; MacDonald et al., 2006). As a result, suspension-feeding bivalves affect phytoplankton dynamics directly by removal (e.g., Cloern and Alpine, 1991; Boegman et al., 2008), leading to suggestions that bivalve shellfish restoration and aquaculture can be considered as possible means of clearing water of microalgae and mitigating coastal eutrophication (e.g., Edebo et al., 2000; Lindahl et al., 2005; Newell et al., 2006; Xu et al., 2006). Bivalve feeding activities, however, can limit the system carrying capacity for aquaculture (Prins et al., 1997; Campbell and Newell, 1998; Grant et al., 2007), defined as the maximal shellfish stocking density either without food limitation or without compromising the health of the ecosystem (Campbell and Newell, 1998; Grant et al., 2005; Grant et al., 2007; Newell, 2007). Particle clearance rate (CR) by filter feeders, defined as the water volume cleared of particles per unit time per dry weight of animal soft tissues, is an important measurement of suspension-feeding activities.

⁎ Corresponding author. Tel.: +1 203 882 6561; fax: +1 203 882 6570. E-mail address: [email protected] (Y. Li). 0044-8486/$ – see front matter. Published by Elsevier B.V. doi:10.1016/j.aquaculture.2009.08.026

CR is related to the rate of material and energy transfer between planktonic and benthic compartments in coastal ecosystems; the term “pelagic–benthic coupling” is used to describe this ecological process. Most of the existing CR measurements have been made by putting an individual bivalve in a closed container and measuring the decline in suspended particles in the container water over time (Petersen et al., 2004; Järnegren and Altin, 2006). Alternatively, CR has been measured with a flow-through chamber wherein CR was determined by the flow rate and counts of particles in the inflow and outflow of the chamber (Bacon et al., 1998; Petersen et al., 2004). Another method that has been used more commonly in nature is the biodeposition method, in which CR was estimated on the basis of egested inorganic material as true faeces and pseudofaeces (Hawkins et al., 1996; Petersen et al., 2004). Reliable in situ measurements of particle clearance are needed because it is uncertain if laboratory-measured clearance rates reflect those occurring in situ (Urrutia et al., 1996; Cranford, 2001; Riisgård, 2001), where constantly-changing environmental conditions may influence the physiological status and other responses of the bivalve. It appears that acclimation time and the composition of particles in the water were the two main factors causing discrepancies between measurements in the laboratory and in situ (Velasco and Navarro, 2005). Indeed, natural seston often contains a more diverse array of particles than standardized, sometimes artificial, particles used in laboratory studies, and natural seston composition may vary over short periods of time (Velasco and Navarro, 2005). There are different opinions as to whether or not the ambient detritus present in nature

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affects bivalve clearance rates. Some believe that the CR should not be affected because bivalves are able to reject detritus, through action of the labial palps, as pseudofaeces (Kiørboe and Møhlenberg, 1981). Others reported that CR decreased exponentially with high ambient detritus (Shumway et al., 2003; Velasco and Navarro, 2005). Few studies have been conducted measuring in situ clearance rates of bivalve filter feeders in nature (Hawkins et al., 1996; Cranford et al., 1998; Dionisio Pires et al., 2004; Cranford et al., 2005; Grizzle et al., 2006). The commonly-used biodeposit method is thought to underestimate actual CR (Petersen et al., 2004). Another factor that affects the in situ measurements of CR is the manipulation of animals and disturbance of their environment during the experiment (Petersen et al., 2004). It is also difficult to determine if bivalves function at full clearing capacity in nature (Cranford and Hill, 1999); thus, most in situ measurements in nature may only represent snap shots of bivalve clearing activities under disturbed conditions. Recently, Grizzle et al. (2006) developed an in situ, fluorometric method to estimate seston uptake based upon upstream and downstream concentrations of chlorophyll a. No information on the removal of detritus, however, was available from this approach. Accordingly, quantitative information on clearance rates of bivalve species under natural conditions, exposed to the full range of particle types present in the environment, is needed. Quantitative information on bivalve clearance rates of different particles, such as detritus vs. phytoplankton, is necessary to describe fully the fundamental ecosystem function of bivalve shellfish in pelagic–benthic coupling. Different techniques have been used in quantification and differentiation of particles in bivalve CR measurements. Light microscope observations and counting can quantify and determine the taxonomic groups of phytoplankton, but is very time consuming. The Coulter Counter particle-counting method can quantify particles very rapidly in a fluid environment, but cannot distinguish between phytoplankton and non-phytoplankton particles within the same size range. A bio-medical technology, flow-cytometry, also has been used to quantify phytoplankton and particle uptake by bivalves (Cucci et al., 1985; Shumway et al., 1985; Dubelaar et al., 1989; Hofstraat et al., 1994; Li, 1997; Dubelaar and Jonker, 2000; Dionisio Pires et al., 2004). Flow-cytometry has three advantages over other techniques. First, it is able to process vast numbers of particles in a very short period of time (about 105 per second); thus, analyzing large numbers of samples is possible. Second, flow-cytometry is able to measure fluorescence at one or more wavelengths; therefore, it is able to differentiate between phytoplankton and detritus and may be able to differentiate phytoplankton groups to some extent (Dubelaar and Jonker, 2000). Third, flow-cytometry is able to quantify the relative size of particles by the measurement of forward light scatter (Shapiro, 1988). Thus, the ability to rapidly quantify and differentiate particles in a fluid environment makes the flow-cytometer an ideal tool to study particle clearance in filter-feeding bivalves. The application of flow-cytometry to analyzing natural seston is still limited. Laboratory studies have shown that bivalve particle retention efficiency (thus clearance ability) was related to particle size (Møhlenberg and Riisgård, 1978; Palmer and Williams, 1980; Riisgård, 1988). In addition, particle selectivity based upon food quality has been well documented (Shumway et al., 1985; MacDonald and Ward, 1994; James et al., 2001). Thus, in nature bivalves can be expected to have differential impacts on individual particle types and sizes comprising the seston. Very few studies, however, have quantified particle clearance based upon size and type in nature. We report here particle clearance rates of captive populations of the economically-important bivalve species, the northern bay scallop Argopecten irradians irradians, under semi-natural conditions. Scallops were kept in large (10 m by 1.2 m) raceway tanks with flow-through, untreated seawater, and a number of measurements were made for 2 weeks. This approach permitted undisturbed and acclimated scallops to be exposed to natural variations in seston abundance, composition,

and particle size distribution, as well as natural variations in other chemical and physical properties. A flow-cytometric technique was developed to quantify and group particles based upon size and chlorophyll a content. The flow-cytometric approach allowed for rapid measurement of particle clearance on different seston components simultaneously. 2. Materials and methods 2.1. Experimental settings Experiments were carried out in a raceway tank system described by Rhodes and Widman (1980). The raceway tanks are 10 m long, 1.2 m wide, 0.42 m tall in size and are made of black fiberglass. Seawater is pumped directly to the tanks from Milford Harbor (Milford, CT, USA) through a PVC piping system. A baffle is set up at the inflow section to accomplish mixing of the inflow water before it flows to the rest of the tank. The height of the water is determined by an outflow standpipe at the opposite end of the tank from the inflow section. In this experiment, the inflow sections of tanks were slightly elevated (ca. 1°) to encourage water flow. The water depths near the inflow were 19.8 ± 1.4 cm (mean ± SE), and depths near the outflow were 28± 1.0 cm (mean ± SE). The overall mean water depth for all of the tanks was 24.2 ± 1.1 cm (mean ± SE). The average water flow for all tanks during the experiment was 43.37 L min− 1, with mean for individual tanks ranging from 40.65 to 46.84 L min− 1. The mean velocity of water flow in the tanks was 12.22 cm min− 1. Eight raceway tanks were set up for the experiment. Two tanks were used as control tanks without any scallops. Three tanks each had 1 L packed volume of scallops (about 526 ml displacement volume or 311 animals) as a low-density scallop treatment. The remaining 3 tanks each had 4 L packed volume of scallops (about 2104 ml displacement volume or about 1153 animals) as a high-density scallop treatment. The lowdensity treatment was intended to maintain scallops in an environment free of stress from over-crowding. The high-density treatment subjected scallops in more intra-species competition for food while still allowing for good growth. The two density treatments were determined based upon the reported results of scallop growth in the same raceway tanks at a similar flow rate (Rhodes and Widman, 1980). Although scallops are capable of some mobility to escape predators, they often attach with byssal threads to the sides of tanks or stay at the bottom. Thus, adequate mixing of water in the tank is important in exposing animals to a replenished particle field. Mixing of water was confirmed by tracing fluorescent dye introduced into a tank. One liter of seawater containing 27 g of fluorescein was poured into the input section of the tank along with the regular flow. Immediately after the introduction of fluorescein, water samples were taken synoptically from 16 stations at multiple depths throughout the tank from inflow to outflow. Samples were collected every 2 to 10 min until no fluorescein was detected in the tank. Fluorescein concentrations were quantified in a Turner Designs Fluorometer (Turner Designs, Sunnyvale, CA, USA) calibrated with known concentrations of fluorescein. The flow pattern in the tank was visualized by plotting the relative concentrations of fluorescein in the tank in a time series. The fluorescein concentrations near the bottom and side wall of the tank were interpolated based upon the boundary effect described by Best and Leeder (1993) and the nearest measured concentrations. Matlab software (The MathWorks, Inc., Natick, MA) was used to constructing 3D images of fluorescein in the tank at each sampling time. The dye experiment showed that the tank was overall well-mixed from surface to bottom. 2.2. Scallops Northern bay scallops Argopecten irradians irradians used in this experiment were from one cohort spawned at the Milford Laboratory hatchery in March 2006. Scallops were 4 months old with a mean

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shell height of 19.3 ± 0.39 mm (mean ± SE) when the experiment was conducted. 2.3. Measurements and data collection The experiment was run for 2 weeks. Flow rates for each tank were measured at least once daily at the outflow. Daily measurements of inflow water included temperature, salinity, DO (dissolved oxygen), and pH. Temperature, salinity and DO were measured by a handheld device (YSI 85). pH of inflow water was measured by a Mettler Toledo MA 235 pH/Ion Analyzer. Inflow water was also checked daily in the light microscope for harmful and unusual algal species. TSS (total suspended solids), Chlorophyll a, and size-fractioned particles were measured daily in water from the inflow of one tank (assuming all of the tanks had the same inflow water) and outflow of each tank. Particle quantification was done by a flow-cytometric method (below). TSS measurement followed a standard, gravimetric procedure. In brief, 200 ml water was passed through a pre-weighed GF/F glass fiber filter (0.7 μm pore size). Filters were stored at − 20 °C until analyses. Filters were dried at 103–105 °C for at least 1 h and then cooled in a desiccator and weighed again. For chlorophyll a analysis, 200 ml water was passed through a GF/F glass fiber filter and stored at − 20 °C until analyses. Upon analyses, filters were sonicated in 90% acetone solution with a Tekmar Sonic Disruptor, at output control of 7% duty cycle = 80% for 2 min. The sonicated solutions were then kept refrigerated overnight (20 h) to allow for pigment extraction. Samples were then centrifuged at 8000 × g for 10 min, and the supernatant was drawn for chlorophyll a determination using a Turner Designs Fluorometer pre-calibrated with a chlorophyll a standard from Turner Designs Inc. (Sunnyvale, California, USA). Sedimentation rates were determined for all tanks. The sedimentation in the control tanks represents the natural settling of particles while sedimentation in the scallop treatment tanks included settling of both natural particles and biodeposites from scallops. To determine the sedimentation rates, 12 sediment traps were placed in each tank, 3 evenly across the tank in each of the 4 sections of the tank. The 3 traps near the inflow of the tank were 50 ml glass beakers, anticipating the greatest settling near the inflow. The other 9 traps were glass Petri dishes with a diameter of 5.0 cm. The bottoms of the traps were glued to the tanks so they would not be turned over by currents or scallops. At the end of the experiment, sediment was collected from each trap and stored in −80 °C until analyses. Sediment dry weight was measured according to (Steeby et al., 2004). In short, sediment was dried at 103 °C for 24 h before it was weighed. The net removal of particulate materials (PM) from the water by scallops in each tank was estimated by assuming that PM of the inflow equals PM of the outflow plus PM settled in the tank, plus PM removed by scallops.

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signal for chlorophyll a, a mixture of 3 cultured algal strains with a wide size range was bleached with about 1% sodium hypochlorite (A-1 Bleach, James Austin Company, Mars, PA). The 3 strains were all from the Milford Laboratory Microalgal Culture Collection: 580, Chlorella autotrophica, PLY 429, Tetraselmis chui, and Gymno F, Akashiwo sanguinea (originally Gymnodinium splendens). FL3 signals from these bleached algae were assumed to result from refraction and reflection of the blue laser light. Thus, a plot of FL3 vs. FSC (related to cell size) was used to determine if a particle of a given size contains chlorophyll: particles with FL3 above the plotted line were categorized as chlorophyll a-containing particles (phytoplankton); those below the line were categorized as detritus (Fig. 1B). Particles were also grouped into 4 size categories, <2 µm, 2– 5 µm, 5–20 µm and >20 µm, based upon FSC (Fig. 1B). Particle size ranges were determined by the plot of FL3 vs. FSC using the three algal cultures (Fig. 1A). Sizes of the algal cells were determined microscopically with a calibrated ocular micrometer. Fig. 1B shows particles in a water sample divided into 8 groups, based upon size and chlorophyll a content. 2.5. Clearance rate (CR) calculation CR was calculated with an equation modified from Petersen et al. (2004) in which the flow-through chamber method was described. The main difference between our flow-through system (tanks) and Petersen et al. (2004) was that their system had much smaller volume of water (only 319 ml, but ours was approximately 2.7× 106 ml). The residence time for our system was also much longer (average of 62 min vs. 2 min). In our experimental design, we corrected for the particles settling by

2.4. Particle differentiation and quantification by flow-cytometer Water samples from inflows and outflows of tanks were collected and pre-screened through a 180-µm nylon sieve to remove large particles. Water samples were then fixed with 4% (v/v) formaldehyde. Upon analyses, 50 ml water samples were concentrated to 2 ml by centrifugation at 1000 ×g for 30 min. Next, 10–20 µl of a known count (2.8 × 106 ml− 1) of yellow-fluorescent, plastic microbeads (Polyscience, Inc., Warrington, PA) were added to 0.5 ml of concentrated water sample. Water samples were then analyzed by a FACScan flowcytometer (B–D BioSciences, San Jose, CA) equipped with a 488-nm laser. The flow-cytometer was adjusted to obtain quantitative data on FL3 (fluorescence at 650 nm related to chlorophyll a), FL1 (fluorescence at 530 nm to record the beads), FSC (forward scatter, which is related to particle size) for each particle that passes the laser interrogation point. Prior to the experiment, a calibration method was developed to categorize particles as “phytoplankton” or “detritus,” based upon chlorophyll a content. To determine how much FL3 is a meaningful

Fig. 1. Flow-cytometer biplots of size (X-axis) and chlorophyll fluorescence (Y-axis) of suspended particles to demonstrate criteria used to discriminate size classes of particles (as indicated) and consider them to be microalgal or not (divided by the dotted line). A. Cultured phytoplankton with microscopically-determined sizes. B. Particles in one of the inflow water samples during the experiment.

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gravity by setting up control tanks with no scallops. Thus, the following was the equation used: CR = ð1 = nÞ⁎ðCoutc −Couts Þ = ððCin + Couts Þ = 2Þ⁎Q

ð1Þ

Where CR is the clearance rate, n the number of animals in the tank, Cout-c the outflow concentration of the control tanks, Cout-s the outflow of the scallop treatment tanks, Cin the concentration of the inflow (same for all tanks) and Q the flow rate. The differences in the particle concentration in the inflow and outflow of scallop tanks (Cin − Cout-s) are attributable to uptake by scallops and gravity settling, or Cin − Cout-s = Cuptake + Csettling. Thus the difference attributable to scallop uptake can be quantified as: Cuptake = ðCin −Couts Þ−Csettling The settling portion can be quantified from the control tanks wherein no scallop uptake takes place, or Csettling = (Cin − Cout-c), thus Cuptake = Coutc −Couts In Eq. (1), the concentration to which scallops were exposed was approximated by the means of inflow and outflow particle counts ((Cin + Cout-s)/2). Petersen et al. (2004) used the outflow count for the count to which experimental bivalves were exposed. Because our tank system had a longer residence time, it was more accurate to use the means of inflow and outflow counts to represent the particles to which scallops were exposed. Weight-specific clearance rates were determined using the allometric equation (Shumway et al., 2003): b

CRw = ðWs = We Þ CR Where CRw is the weight-specific clearance rate (clearance rate for an animal of standard weight), Ws the standard weight of scallop, We the observed weight of animals, b the weight exponent for the physiological rate of 0.75 for bay scallops with a standard weight of 1 g (Bricelj and Kuenstner, 1989) and CR the measured clearance rate. Individual scallop body weight was estimated based upon the shell height and the relationship between the shell height and weight established using the bay scallops from the same geographic population of similar sizes and kept under similar conditions: W = 0:143 × H

2:877

2

ðn = 36; R = 0:80Þ

Where W is the total dry weight in mg, and H the shell height in mm.

Table 1 Scallops in different treatment tanks at the end of the experiment. Total body Growth rate TreatmentDisplacement Total # of Mean shell tank number volume (ml) live scallops height ± SE dry weight (μm d-1) (g) (mm) Lowdensity-1 Lowdensity-2 Lowdensity-3 Higndensity-1 Highdensity-2 Highdensity-3

620

288

24.1 ± 0.58

389.61

400

736

274

24.0 ± 0.52

366.26

475

684

274

23.9 ± 0.62

361.89

383

2750

1014

22.2 ± 0.57

1083.08

242

2735

984

23.4 ± 0.62

1222.92

342

2893

1000

24.1 ± 0.57

1352.80

400

low-density treatment, the final mean shell height was 24.0 ± 0.57 mm from the beginning of 19.3 ± 0.39 mm. The high-density treatment yielded a slightly slower growth compared to the lowdensity treatment, with the final shell height of 23.2 ± 0.59 mm, although the difference was not statistically significant. 3.2. Characteristics of inflow water Temperature, salinity, and dissolved oxygen in the inflow water showed little fluctuation during the 13-day experiment from August 3, 2006 to August 16, 2006. Temperature ranged from 22.5 °C to 24.4 °C (23.3 ± 0.5 °C). During the experimental period, no significant rainfall occurred, and salinity was almost constant ranging from 26.2 to 27.0 (26.4 ± 0.4). Dissolved oxygen in the inflow water ranged from 4.02 to 5.40 mg L− 1 (4.86 ± 0.39), a range wherein aquatic organisms would not experience oxygen stress. No harmful algae were observed in the water during the experiment. Natural seston containing both phytoplankton and detritus varied greatly during the 13-day experiment (Figs. 2–4). Total suspended solids ranged from 4 to 18 mg L− 1 (Fig. 3). The mean chlorophyll a concentration was 2.93 ± 1.61 μg L− 1.There was a peak in chlorophyll a (about 7 μg L− 1) in the second day of experiment (Fig. 2). During most of the days in the experiment, the chlorophyll a level was between 1 and 2.5 μg L− 1. The most abundant phytoplankton size category in the inflow water was in the smallest size group of <2 μm, with a mean count of 1.86 ± 1.19 × 103 cells ml− 1 (Fig. 4A). The next most abundant phytoplankton size category was the largest, >20 μm, with a mean

2.6. Statistical analyses The main objective of this experiment was to quantify particle clearance by scallops in a semi-natural setting, at the population level, with a flow-cytometric approach. Two scallop density treatments were applied. Daily measurements of clearance rate for 13 days reflected variations in environmental parameters. Clearance rates for the different sizes and types of particles, and between scallop-density treatments, were tested by MANOVA. Once a significant difference was found, a subsequent multiple range test was done to distinguish specific contrasts. All statistical analyses were done with the computer software StatGraphics plus (originally developed by Manugistics, Inc, Maryland, now marketed by StatPoint, Inc,Virginia, USA). 3. Results 3.1. Scallop growth The scallops grew significantly during the experiment in both lowdensity and high-density treatments (p < 0.001) (Table 1). For the

Fig. 2. Mean chlorophyll a in inflow water and outflow water of different treatment tanks during the experiment. Measurements were made near 7:15 am in the morning when the letter a was added after the dates. All other measurements were made near 1:15 pm.

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5 μm, 5–20 μm and >20 μm, were 2.96 ± 3.81× 104 ml− 1, 1.29 ± 1.49 × 104 ml− 1 and 4.42 ± 0.43 × 103 ml− 1, respectively. The ratio of phytoplankton to detritus in the inflow water increased as the sizes of particles increased (Fig. 4c), with means of 0.0013, 0.0060, 0.0153 and 0.121 for <2 μm, 2–5 μm, 5–20 μm, and >20 μm, respectively, during the 13-day experiment. The particles in the largest size group, >20 μm, had a remarkably higher ratio of phytoplankton to detritus compared to the other three size groups. 3.4. Clearance rates of individual particle sizes

Fig. 3. Mean total suspended solids in inflow water and outflow water of different treatment tanks during the experiment.

count of 339 ± 231 cells ml− 1. The two medium-sized groups of phytoplankton had the lowest counts of 100 to 200 cells ml− 1. In contrast to phytoplankton, counts of detrital particles in the inflow water increased as the sizes of particles decreased (Fig. 4B). Detrital particles in the size group of <2 μm were present in the water in millions per ml. The mean counts for the other three size groups, 2–

Clearance rates of all 4 size groups of phytoplankton and detritus varied daily for scallops in both low- and high-density treatment tanks, as indicated by the large variance bars (Fig. 6). For scallops in both density-treatment tanks, CRs on phytoplankton in the medium-to-large size range of 5–20 μm were the highest among all of the 4 size groups, followed by the > 20 μm size group. CR on the smallest-sized phytoplankton was the lowest. MANOVA indicated that differences in CR between different size groups were significant (p < 0.01). A subsequent multiple range test was used to identify the differences between specific pairs of size groups. For scallops in low-density tanks, CR of phytoplankton in the 5–20 μm size category was significantly higher than that of other size categories (Table 2a); whereas, CRs of the other three size groups did not exhibit significant differences. For scallops in high-density tanks, CR on the smallest phytoplankton (<2 μm) was significantly lower than CRs of sizes 5–20 μm and >20 μm (Table 2b). Mean CR of 5–20 μm phytoplankton was also significantly different from that of <2 μm and 2–5 μm phytoplankton (Table 2b). Clearance of detritus increased with particle size except for the largest size group (Fig. 6); MANOVA indicated that differences were significant for both low- and high-density treatments (p < 0.01). Multiple range tests indicated that CR on the smallest-sized detritus was significantly lower than CRs of other size groups in both low- and high-density treatments, except for the 2–5 μm size category in the high-density treatment (Table 2c and d). 3.5. Clearance rates of particle types (phytoplankton or detritus) Clearance rates of phytoplankton were significantly greater than those of detritus for particles >20 μm, in the low-density treatment Table 2 Results of the multiple range tests showing the difference in the clearance rates of particles in different size categories. Size category

Counts

Mean

Homogenous groups

a. For the clearance of scallops on phytoplankton in low-density tanks. ANOVA p = 0.0006 < 2 µm 11 0.95 a 2–5 µm 11 1.21 a 5–20 µm 10 3.90 b > 20 µm 11 1.65 a b. For the clearance of scallops on phytoplankton in high-density tanks. ANOVA p = 0.0006 < 2 µm 11 0.32 a 2–5 µm 11 0.60 ab 5–20 µm 10 1.62 c > 20 µm 11 1.35 bc c. For the clearance of scallops on detritus in low-density tanks. ANOVA p = 0.0003 < 2 µm 11 0.24 a 2–5 µm 11 2.35 b 5–20 µm 10 3.46 b > 20 µm 11 3.90 b d. For the clearance of scallops on detritus in high-density tanks. ANOVA p = 0.0003 < 2 µm 11 0.16 a 2–5 µm 11 0.83 ab 5–20 µm 10 1.50 bc > 20 µm 11 1.84 c Fig. 4. Inflow water phytoplankton (A), detritus (B) and the ratio of phytoplankton to detritus (C) during the experiment.

Different letters in the last column denote significant difference between groups. Method used: 95% LSD.

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Table 3 Differences in clearance rates of phytoplankton and detritus, results from MANOVA. Size category Low-density <2 µm 2–5 µm 5–20 µm >20 µm High-density <2 µm 2–5 µm 5–20 µm >20 µm a

df

p-value

10 10 9 10

0.1048 0.1254 0.5565 0.0116a

10 10 9 10

0.1764 0.3926 0.5563 0.1100

Next to p-value indicates a significant difference.

(MANOVA, p < 0.05) (Table 3). There was a tendency for higher CRs of phytoplankton than detritus for <2 μm at low scallop density and >20 μm at high scallop density. No difference was found for other size contrasts. 3.6. Clearance rate and scallop density Scallops kept in the low-density tanks consistently exhibited higher CR than those in the high-density tanks (Figs. 5 and 6). The mean CR of total chlorophyll a for scallops in the low-density tanks was over three times that of scallops in high-density tanks, with means of 1.97 ± 0.24 and 0.48 ± 0.08 L h− 1 gDW− 1 (mean ± SE), respectively. The difference between the CR in the two density treatments was highly significant (p < 0.001). When particles were partitioned into a total of 2 × 4 (size and type) groups, CRs for the low-density treatment were consistently higher than those for the high-density treatment, regardless of particle types and sizes (Fig. 6, Table 4). MANOVA tests revealed that most of the variation in CR was from scallop density rather than other variables associated with ambient conditions. Of the 8 particle groups, all but one exhibited a significant difference in CR between the density treatments (Table 4). 3.7. Sedimentation The natural settling of particulate materials (PM) in the experimental tanks, as represented by the sedimentation of control tanks, was 9.01 ± 2.11 g m− 2 d− 1. The sedimentation rates in the scallop-containing tanks were 13.10 ± 1.24 g m− 2 d− 1 and 20.31 ± 7.11 g m− 2 d− 1, for the low- and high-density tanks, respectively. The weight-specific sedimentation from the scallops (biodeposition rate) was significantly higher in the low-density tanks (0.175 ± 0.05 g day− 1 gDW− 1) than that of the high-density tanks (0.155 ± 0.103 g day− 1 gDW− 1). Scallops in low-density treatment tanks had significantly higher net removal rate

Fig. 5. Variation of weight-specific clearance rate of total chlorophyll a for scallops in the low and high density tanks. Vertical bars represent standard error.

Fig. 6. Mean weight-specific clearance rates of phytoplankton and detritus for the 4 size groups for the low- and high-density treatment. Vertical bars represent standard errors.

of PM from water column (0.621 ± 0.485 g day− 1 gDW− 1) comparing to scallops in high-density treatment tanks (0.209 ± 0.066 g day− 1 gDW− 1). 4. Discussion The present study demonstrated that flow-cytometric analysis of natural seston can provide new insights into the filter-feeding activities of bivalve shellfish, and these activities may contribute to water clarity in nature. Our application of flow-cytometry to the study of particle clearance by scallops in a semi-natural setting revealed some fundamental characteristics of scallop feeding behavior previously observed only in more-controlled, (mostly) laboratory studies, such as size-dependent clearance rates, particle selectivity, and particle densitydependent clearance rates. It is encouraging to note that the weight specific clearance rates measured in the present study agreed remarkably well with previous closely-related southern bay scallop, Argopecten irradians concentricus, which was 2–3 l h− 1 gWW− 1 (KirbySmith, 1970). The specific flow-cytometric application used in the present study allowed for rapid measurements of particle clearance rate on different seston components simultaneously. Differentiating phytoplankton from detritus and determining the size ranges of particles in a natural seston assemblage were two main advantages of our procedure. In addition to the details of information yielded, the efficiency is also remarkable, with approximately 1 min per sample of processing time once the samples are taken. Other published methods for achieving similar particle details (both types and sizes) involved tedious sizefraction filtering and subsequent weighing of particles and measurement of chlorophyll (Hawkins et al., 2001; Capriulo et al., 2002). Protozoa such as heterotrophic flagellates, ciliates, and rotiers, can be ingested and assimilated by filter-feeding bivalves, particularly in systems where the microbial food web is dominated by picoplankton and micro- and meso-zooplankton (Dupuy et al., 1999; Wong et al., 2003a,b). Caution should be taken when applying this technique to

Y. Li et al. / Aquaculture 296 (2009) 237–245

243

Table 4 Variation in clearance rates contrasting scallop density in the tank or natural variability in environmental parameters such as seston composition, water chemistry and physics, results of MANOVA.

Phytoplankton, <2 µm Phytoplankton, 2−5 µm Phytoplankton, 5–20 µm Phytoplankton, >20 µm Detritus, <2 µm Detritus, 2–5 µm Detritus, 5–20 µm Detritus, >20 µm

Source of variation

df

MS

F

p

CR (mean ± SE) (L h− 1 g− 1 ) low-density

CR (mean ± SE) (L h− 1 g− 1 ) high-density

Scallop density Environmental variability Scallop density Environmental variability Scallop density Environmental variability Scallop density Environmental variability Scallop density Environmental variability Scallop density Environmental variability Scallop density Environmental variability Scallop density Environmental variability

1 10 1 10 1 10 1 10 1 10 1 10 1 10 1 10

17.13 6.52 15.04 6.69 75.41 12.66 5.93 3.35 2.85 1.79 34.90 4.69 66.70 7.515 74.19 13.49

3.69 1.33 5.20 2.32 7.99 1.34 1.68 0.99 2.77 1.83 11.17 1.50 10.18 1.14 7.48 1.36

0.057 0.191 0.026 0.023 0.007 0.225 0.187 0.460 0.102 0.078 0.002 0.165 0.002 0.349 0.008 0.222

0.47 ± 0.22

0.16 ± 0.07

0.60 ± 0.20

0.30 ± 0.08

1.93 ± 0.32

0.80 ± 0.25

0.82 ± 0.18

0.67 ± 0.19

0.12 ± 0.05

0.08 ± 0.02

1.16 ± 0.27

0.41 ± 0.09

1.71 ± 0.35

0.74 ± 0.16

1.93 ± 0.40

0.91 ± 0.20

such systems as this method does not discriminate these protozoa from detrital particles. The technique described here is appropriate, however, for most coastal marine systems, such as Milford Harbor, at times when the plankton community is dominated by micro-sized, autotrophic algae and meso- and macro-zooplankton. It is well known that bivalves, including scallops, are able to actively select high-quality food through pre-ingestive (selective rejection of particles in pseudofeces) or post-ingestive (by rapid gut passage) selection (Kiørboe and Møhlenberg, 1981; MacDonald and Ward, 1994; Shumway et al., 1997; Ward et al., 1997; Bacon et al., 1998; Ward et al., 1998; Pales Espinosa et al., 2007). When seston is dominated by low-quality particles, however, selection ability has been shown to decrease (Bacon et al., 1998). A high rate of rejection may overwhelm the capacity of the selection system and result in non-selection or negative selection for high-quality particles (MacDonald and Ward, 1994). Our results showed that, under the natural seston conditions in Milford Harbor during the study, scallops did not select phytoplankton over detritus for small to medium sized particles (1–20 μm); for larger particles, CR of detritus tended to be higher than of phytoplankton. There are two possible reasons that no active selection of phytoplankton was observed in our experiment. First, there was, indeed, an overwhelmingly-larger number of detrital particles than phytoplankton present in the water, particularly in the smallest size range. Thus, the ability to select may have been overwhelmed, as suggested by MacDonald and Ward (1994). This latter study showed that selection efficiency in the scallop Placopecten magellanicus decreased when the ratio of high-quality to low-quality particles (chlorophyll vs. non-chlorophyll) fell below 1 µg chlorophyll a per mg seston dry weight. During most of our experiment, this ratio was in the range of 0.2 µg chlorophyll a per mg seston dry weight. Secondly, some of the large flagellates present frequently in high abundance, such as Euglena spp., may be able to escape from the feeding currents; therefore, the clearance of large-sized phytoplankton may have been inhibited by the non-passive nature of these large phytoplankton particles. Dionisio Piere et al. (2004) reported that mussels had a higher clearance rate on phytoplankton over detritus when exposed to natural seston from a lake, but no information regarding relative composition of phytoplankton and detritus in the seston was provided in this study. The current experiment clearly demonstrated that clearance rates of natural seston were size-dependent. For both phytoplankton and detritus, the lowest clearance rates were on the smallest size category (<2 μm). The maximum CR on phytoplankton was for the size category 5–20 μm. The phytoplankton CR decrease as the size increased above 20 μm was addressed above. In contrast to phytoplankton, CR on detritus did not decrease as the particle size increased. The particle size-

dependent CRs observed with the natural seston from Milford Harbor corresponded well with previous laboratory studies showing that particle retention efficiency in scallops increases with particle size until maximum efficiency was reached (Møhlenberg and Riisgård, 1978; Palmer and Williams, 1980; Riisgård, 1988). Most scallop species were able to retain particles larger than 3–4 μm with 100% efficiency (Møhlenberg and Riisgård, 1978; Riisgård, 1988). The Northern bay scallop was one of the few scallop species that reached maximum retention efficiency only above 5–7 μm (Palmer and Williams, 1980). The observed difference in CR from the two scallop-density treatments may be a response to the availability of particles in the tanks; lower clearance rate in the high-density tank could be a consequence of seston depletion. In the high-density treatment, scallops near the input (upstream) of the tank most-likely cleared more particles than those in the low-density treatment tanks, leaving fewer particles available for those near the downstream ends of the tanks. This interpretation is supported by the fact that there were far fewer particles in the outflows of high-density tanks than those of low-density tanks (Figs. 2 and 3). Responses of scallops to changes in availability of food supply have been well documented (MacDonald et al., 2006). Numerous laboratory studies showed that scallop CR decreases with an increase in food availability when food-particle densities are fairly high, generally above 1 × 106 cells ml− 1, depending on the species and life stage of scallop (Palmer and Williams, 1980; Cahalan et al., 1989; Lu and Blake, 1996). Particle densities in these laboratory studies were much higher than those of natural waters, and in our experiment wherein phytoplankton abundances were generally below 1000 cells ml− 1. Hawkins et al. (2001) showed a very flexible response of CR to natural particle availability at a wide range of particle densities. These authors demonstrated that, at naturallyoccurring, low particle densities, CR increased with increasing particle density until a maximum CR was reached. The CR then decreased with an increase in particle density. Our results are in agreement with those of Hawkins et al. (2001), as particle densities in our study were in the low particle-density range. Similar responses were common in blue mussels, Mytilus edulis. At relatively low particle densities (from 6 × 103 to 4 × 104 particles ml− 1), exhalant siphon area increased (indicating increased pumping rate) with increasing particle densities (Newell et al., 2001). When the ambient concentrations of chlorophyll a were high (exceeding 10 μg l− 1), however, M. edulis reduced CR by partially closing valves (Møhlenberg, 1999). The similar growth of scallops in the high- and low-density tanks indicated no food limitation in the high-density tanks during the experiment, although particle (including food) depletion in highdensity tanks apparently occurred, as indicated by the lower weightspecific CR. Rhodes and Widman (1980) reported a slight reduction in

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growth with the initial scallop density of 5 l per tank (similar to our high-density treatment) with a slightly higher flow rate (more food available). The Rhodes and Widman (1980) experiment lasted for over two months, much longer than the present study of 13 days. Thus, a reduction in growth in high-density tanks may occur over a longer period of time. The lower biodeposition rate (per DW scallop) in high-density tanks, coupled with lower CR and good growth, indicated that scallops were able to utilized food efficiently at high-density. A practical implication is that, when animals are grown at high-density, such as in an aquaculture setting, the amount of biodeposits generated on a per biomass basis would be lower than under lower density. The fundamental ecosystem services of bivalve shellfish populations are largely attributed to the fact that animals are able to filter suspended particles from the overlying water. Quantitative information on particle clearance, thus bioenergetics, is one of the key parameters needed to understand the effects of bivalve aquaculture on harvestable yield from a given location (Prins et al., 1997) and impacts on the ecosystems in which bivalve farms are located, including phytoplankton dynamics (Alpine and Cloern, 1992; Prins et al., 1997; Boegman et al., 2008) and water quality (e.g., Qualls et al., 2007; Boegman et al., 2008). Thus, information on seston removal by filter-feeding bivalves has broad implication for water-quality management associated with bivalve restoration projects and aquaculture activities (e.g., Thayer et al., 2005; Fulford et al., 2007). With the high efficiency and detail achieved by our flow-cytometric method, it is possible to obtain such information on more detailed spatial and temporal scales. More recently, this method has been applied successfully to quantifying particle removal by a commercial oyster nursery system in a natural, coastal environment (Li et al., 2009). Improved knowledge of bivalve shellfish interactions with natural particle assemblages, including phytoplankton communities, and how bivalve filtration influences pelagic–benthic coupling, will help guide decisions about the ecosystem services realized by manipulation of shellfish populations in coastal waters, whether through aquaculture or restoration activities. Acknowledgements We are grateful for the following individuals who helped with the project: the Milford Laboratory staff for participating in dye experiment, Barry Smith of the Milford Laboratory for leveling the tank, for the dye experiment, the Milford Laboratory maintenance staff for keeping the raceway tanks in working order, Jennifer Alex of the Milford Laboratory for assisting in flow cytometer analyses and providing algal cultures, James Manning of the Woods Hole Laboratory of NOAA Fisheries for assisting in Matlab software. Constructive comments by Roger Newell and an anonymous reviewer are greatly appreciated. References Alpine, A.E., Cloern, J.E., 1992. Trophic interactions and direct physical effects control phytoplankton biomass and production in an estuary. Limnol. Oceanogr. 37, 946–955. Bacon, G.S., MacDonald, B.A., Ward, J.E., 1998. Physiological responses of infaunal (Mya arenaria) and epifaunal (Placopecten magellanicus) bivalves to variations in the concentration and quality of suspended particles Ι. Feeding activity and selection. J. Exp. Mar. Biol. Ecol. 219, 105–125. Best, J.L., Leeder, M.R., 1993. Drag reduction in turbulent muddy seawater flows and some sedimentary consequences. Sedimentology 40, 1129–1137. Boegman, L., Loewen, M.R., Culver, D.A., Hamblin, P.F., Charlton, M.N., 2008. Spatialdynamic modeling of algal biomass in Lake Erie: relative impacts of dreissenid mussels and nutrient loads. J. Environ. Eng.-Asce 134, 456–468. Bricelj, V.M., Kuenstner, S.H., 1989. Effects of the “brown tide” on the feeding, physiology and growth of juvenile and adult bay scallops and mussels. In: Casper, E.M., Bricelj, V.M. (Eds.), Novel Phytoplankton Blooms: Causes and Impacts of Recurrent Brown Tides and Other Unusual Blooms. Springer-Verlag, Berlin. 491–509 pp. Cahalan, J.A., Siddall, S.E., Luckenbach, M.W., 1989. Effects of flow velocity, food concentration and particle flux on growth rates of juvenile bay scallops Argopecten irradians. Mar. Ecol. Prog. Ser. 129, 45–60.

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