Energetic physiology of the Caribbean scallops Argopecten nucleus and Nodipecten nodosus fed with different microalgal diets

Aquaculture 270 (2007) 299 – 311 www.elsevier.com/locate/aqua-online

Energetic physiology of the Caribbean scallops Argopecten nucleus and Nodipecten nodosus fed with different microalgal diets L.A. Velasco ⁎ Instituto de Investigaciones Tropicales (INTROPIC), Universidad del Magdalena, Carrera 2 No 18-27, Taganga, Santa Marta, Colombia Received 3 May 2006; received in revised form 30 March 2007; accepted 3 April 2007

Abstract The Caribbean scallops Argopecten nucleus and Nodipecten nodosus are currently being placed into mass culture in Colombia. The limited availability of wild seed upon which to base these cultures has promoted research into the development of artificial production of this seed in hatcheries. In support of this effort, we studied the effects of different diets on the physiology of the two scallop species in order to determine the optimal feeding regimes for maintenance of adult specimens in the laboratory. Seven monoalgal diets were tested, including Chaetoceros calcitrans, Chaetoceros muelleri, Isochrysis galbana (Ig), Nannochloris oculata (No), Phaeodactylum tricornutum (Pt), Tetraselmis chui (Tc) and Tetraselmis tetrahele (Tt). Four mixed diets were also tested, including I. galbana + C. calcitrans (Ig + Cc), I. galbana + N. oculata (Ig + No), I. galbana + T. tetrahele (Ig + Tt), and I. galbana + lipid emulsion of docosahexanoic acid DHA (Ig + lip). All the dietary trials were carried out under uniform conditions of temperature (25 °C), salinity (36‰) and algal concentration (0.45 mg L− 1). Physiological variables measured in association with each diet included feeding rates (clearance, ingestion and absorption), oxygen consumption and ammonium excretion rates as well as their scope for growth. The results showed that the best scope for growth for both scallops was obtained with diet Ig since this diet induced the highest feeding rates, accompanied by the lowest oxygen consumption and ammonium excretion. The feeding rates and scope for growth of A. nucleus were greater than those of N. nodosus for the majority of the diets, which was attributed to a higher rate of water pumping by the former species. Greater capacity for branchial food retention by A. nucleus was discarded as a possibility since N. nodosus had a greater branchial surface area per unit dry weight than A. nucleus. In spite of these differences, the oxygen consumption and the excretion rates were similar between the two scallops which suggested that A. nucleus was more efficient in its use of oxygen and retention of body proteins for physiological functioning. Mixed diets or addition of DHA did not permit increases in scope for growth in either of the scallops over that observed using monoalgal diet Ig, which suggest that biologically and economically this diet is optimal for the feeding of adult scallops in the laboratory. © 2007 Elsevier B.V. All rights reserved. Keywords: Scallop; Broodstock maintenance; Microalgae diets; Physiology; Scope for growth; Argopecten nucleus; Nodipecten nodosus; Caribbean Colombia

1. Introduction Food quality is one of the most important factors which affect the growth and maturation in scallops (Heasman ⁎ Tel./fax: +57 5 4219133. E-mail address: [email protected]. 0044-8486/$ - see front matter © 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.aquaculture.2007.04.005

et al., 1994, 1996; Laing and Psimopoulous, 1998; Bayne and Newell, 1983; Utting and Millican, 1998). According to Epifanio (1979), the presence or absence of certain microalgal species in a diet is more important than its overall content of proteins, lipids, carbohydrates, or amino acids. The best results in experimentation with growth and maturation of scallops have been obtained

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when using diets composed of mixtures of living microalgae (Epifanio, 1979; Navarro and Thompson, 1995; Albentosa et al., 1997; Milke et al., 2004), which are: (a) in the exponential growth phase (Ryan et al., 1999), (b) with diameters between 1 and 15 μm for juvenile and adult specimens (Møhlenberg and Riisgård, 1978) and up to 7 or 8 μm for larvae (Alix et al., 1996); and (c) which are easily digested (Hildreth, 1980; Williams, 1981; Crosby et al., 1989, Alix et al., 1996). It has also been established that the development of the gonad can be increased and the development of larvae promoted by the addition of microalgae rich in polyunsaturated essential fatty acids such as eicosapentanoic (EPA; 20:5n − 3), docosahexanoic (DHA; 22:6n − 3), and arachidonic (AA; 20: 4n − 6) acids (Soudant et al., 1996; Wikfors et al., 1996; Samain et al., 1997; Utting and Millican, 1997; Krupski et al., 1998; Martínez et al., 2000) as well as some lipidic or vitamin supplements (Tian et al., 1993; Coutteau et al., 1996; Navarro et al., 2000). Direct determination of the effectiveness of given microalgae and supplements which permit maximization of maturation and growth require numerous longterm experiments which place important demands on physical facilities, time, labor, and economic resources. The use of physiological measurements is an alternative method for estimating comparative value among diets, with greater simplicity and in less time, as well as providing more information on the factors responsible of the organism's responses (Widdows, 1985a). The scope for growth is an index very precise and sensible to stress conditions when the measurements are made carefully (Widdows, 1985a; Grant and Cranford, 1991) which is positively correlated with the bivalve growth rate (Bayne et al., 1979; Riisgård and Randlov, 1981) and gonadic ripeness (MacDonald and Bourne, 1987; Navarro et al., 2000). Various studies of physiological responses related to different types of microalgae have been carried out with filter feeding bivalves, including Mytilus edulis (Møhlenberg and Riisgård, 1978; Hildreth, 1980; Ward et al., 2003), Pecten opercularis, Musculus niger, Venerupsis pullastra (Møhlenberg and Riisgård, 1978), Crassostrea virginica (Romberger and Epifanio, 1981; Shumway et al., 1985; Riisgård, 1988; Ward et al., 2003) Ostrea edulis, Ensis directus, Mya arenaria, Arctica islandica, Placopecten magellanicus (Shumway et al., 1985), Geukensia demissa, Spisula solidísima, Mercenaria mercenaria (Riisgård, 1988), Argopecten purpuratus (Díaz and Martínez, 1992), Ruditapes decussatus (Albentosa et al., 1996), Argopecten ventricosus (Lora-Vilchis and Maeda-Martínez, 1997), Pecten maximus (Laing, 2004) and Pteria sterna (Martínez-Fernán-

dez et al., 2004). These studies showed that the physiological rates varied according to the type of microalgae, food supplement and species of bivalve. Argopecten nucleus and Nodipecten nodosus are two scallop species common in the Caribbean Sea bordering Colombia. Culture of these scallops and production of their “seed” under hatchery conditions have come under investigation based on positive results which were produced in preliminary studies on their growth, production, and markets (Urban, 1999). These species are epibenthic filter feeders, which are functional hermaphrodites, inhabiting bottoms between 10 and 100 m in depth. A. nucleus is a species of moderate size (∼50 mm) and free-living, while N. nodosus is a large species (∼150 mm) which lives attached to hard substrates. No natural banks of these species have been recorded in the Caribbean off Colombia, and the only large populations are those maintained in culture, produced from the scarce wild seed obtained in artificial collectors (Urban, 1999). Since the lack of naturally occurring seed prevents major development of commercial cultures of these scallops, attempts are now being made in Colombia to produce the necessary seed under hatchery conditions. The present study is part on an effort to determine the optimal laboratory conditions for maintaining adults of A. nucleus and N. nodosus, some of whose physiological variables were evaluated during experimental feeding with different monospecific and mixed microalgal diets. These variables included particle clearance rates, ingestion rates, absorption rates, oxygen consumption, ammonium excretion, absorption efficiency and scope for growth. 2. Materials and methods 2.1. Collection and maintenance of test individuals Each month, about 30 specimens of A. nucleus (length 44 ± 4.9 mm and dry tissue weight 1.25 ± 0.4 g) and N. nodosus (length 71 ± 11 mm and dry tissue weight 2.45 ± 1.33 g) were obtained from at the bivalve culture station at Neguanje Bay (Lat. 11°20′ N., Long. 74.05′ W.), in the Tayrona National Natural Park (PNNT), Colombia. The sea in this region has water temperatures between 22 and 30 °C, salinities between 33 and 37‰ and seston concentrations between 0.2 and 4.7 mg L− 1. Organic content of the seston varies between 15 and 60% (Urban, 1999). The scallops were transported in humid conditions to the Moluscos y Microalgas Laboratory of the Universidad de Magdalena, Taganga (Lat. 11° 16′ N, Long. 74° 11′ W) where their shells were cleaned of encrustations and sediments and each

L.A. Velasco / Aquaculture 270 (2007) 299–311

individual was marked for identification. Acclimation to laboratory conditions was achieved by holding the scallops in an aerated 250 L seawater tank at a temperature of 25 °C and 36‰ salinity for at least a week. Scallops were fed with microalgal suspension of laboratory-cultured Isochrysis galbana and Chaetoceros calcitrans (1:1) at a rate of 3% (dry biomass) of their dry body weight daily. 2.2. Experimental design Eleven dietary treatments were used in the feeding experiments which included pure cultures of 1) Isochrysis aff. galbana (Green) (Ig), 2) C. calcitrans (Paulsen) (Cc), 3) Chaetoceros muelleri (Lemmermann) (Cm), 4) Tetraselmis chui (Tc), 5) Tetraselmis tetrahele (G.S. West) (Tt), 6) Nannochloris oculata (No) and 7) Phaeodactylum tricornutum (Bohlin) (Pt), with four mixed diets including, 1) I. galbana + C. calcitrans (Ig + Cc), 2) I. galbana + N. oculata (Ig + No), 3) I. galbana + T. tetrahele (Ig + Tt) and 4) 70% I. galbana + 30% lipid emulsion of EmDHA (Ig + lip). Each experimental diet was administered under conditions of constant temperature (25 °C), salinity (36‰), and concentration (a number of cells equivalent to 0.45 mg L− 1 of dry weight). This concentration was the highest at which none of the scallops reject cells as pseudofeces. Each treatment was applied to 7 different scallops of both species selected haphazardly and was run in a different day for a total of 15 h, which included 12 h of acclimation to the diet and 3 h of physiological feeding measurements. The experiment was made in 3 months, applying 3 or 4 treatments each month. The microalgae used to prepare the experimental diets was cultured in Guillard F/2 medium (1974) and used in the exponential phase. Samples of microalgae cultures was analyzed in terms of cell diameter and number of cells by microscopy (Table 1); energetic content using an adiabatic calorimeter (IKA-WORKS

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Inc.); total particulate matter (TPM) and the organic content (POM) following the gravimetric method described by Strickland and Parsons (1972). The docosahexanoic acid emulsion (DHA; Caers et al., 1999) was obtained from the Artemia Reference Center in Belgium. The experimental diets were prepared into two vigorously aerated tanks (2000 L) adding appropriate volumes of microalgae cultures to 1 μm microfiltered seawater, previously knowing the number of cells in 1 mg of dry weight and the density of the microalgae culture. These diets were administered using a flowthrough system with 16 chambers (0.8 L for A. nucleus and 1.6 L for N. nodosus), designed following Riisgård (1977). A constant flow (150 ± 10 mL min− 1) of the experimental diet was directed by gravity from a distributing tank (2000 L) into each chamber using silicone tubes (∅ = 5 mm) provided of control valves; 14 chambers were used for individual bivalves (7 individuals of each species) and 2 chambers contained empty valves which served as controls. The flow rate was selected in order to obtain a difference of 20 to 40% in cell numbers between chambers with bivalves and control chambers, at these cell retention values the pumping rate is independent of the water flow rate and the recirculation of water in the palial cavity is avoided (Navarro and Thompson, 1996). A constant level of experimental diets was maintained in the distributing tank by means of an overflow and a continuous pumping of experimental diet from another tank (2000 L). Valve opening by test specimens was continually observed and individuals which failed to open normally were eliminated from the experiment. 2.3. Determination of the physiological variables Feeding physiological variables were determined by the biodeposition method described by Iglesias et al. (1998), as validated by Navarro and Velasco (2003)

Table 1 Characteristics of microalgae used in the preparation of test diets fed to A. nucleus and N. nodosus during measurements of their physiological variables Division

Species

Number of cells/dry wt. (×106 cel mg− 1)

Organic content (%)

Size range (μm)

Energy content (J mg− 1)

Characteristics

Bacillariophyta (Diatoms)

C. calcitrans (Cc) C. muelleri (Cm) P. tricornutum (Pt) T. chui (Tc) T. tetrahele (Tt) N. oculata (No) I. galbana (Ig)

21.7 30.3 20.9 2.1 9.1 30.6 5.1

77.3 69.4 79.2 67 67 49.2 74.8

3–7 7–10 10–12 12–15 10–12 2–3 3–6

12.08 12.06 14.39 16.03 16.11 19.84 11.40

Immotile, setae

Chlorophyta (Green algae)

Haptophyta (Flagellates)

Motile, 4 flagellae Immotile Motile, 2 flagellae

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using the ‘flow-through chamber method’ described by Riisgård (2001). Experimental diet was sampled (4 L) from the outflow of both control chambers every hour. At the end of the 12 h of acclimation all of the deposited material was eliminated from the chambers and feces of each animal were quantitatively collected every hour using Pasteur pipettes. The mass and organic content of experimental diet and feces samples were estimated using the same gravimetric method used for the microalgae. The feeding physiological variables of each animal were determined using the equations described by Iglesias et al. (1998). Clearance rate (CR): is the volume of water completely cleared of particles per unit time.

specimens. Then two water samples were taken from each experimental chamber to determine oxygen consumption rates and rates of excretion. Oxygen consumption was never measured at ambient oxygen tension lower than 70% saturation. Oxygen concentration was estimated following the Winkler method as modified by Carritt and Carpenter (Strickland and Parsons, 1972) and ammonia excretion was determined by the phenolhypochlorite method (Widdows, 1985b). Scope for growth (SFG) is a physiological index of energy balance to estimate production (growth +reproduction) by an individual animal. It was calculated from the equation given by Widdows (1985a) after converting all the physiological rates to energy equivalents:

CR ðL h1 Þ ¼ IIR=PIM

SFG ð Jh1 Þ ¼ A  ðR þ U Þ

ð1Þ

where IIR is the inorganic ingestion rate (mg h− 1) and PIM is the particulate inorganic matter (mg L− 1). Ingestion (IR) rate: represents the amount of particulate material removed and ingested from the water per unit time. Inorgani cingestion rate

IIR ð mgh1 Þ ¼ IER

Organic ingestion rate OIR ð mgh1 Þ ¼ IIRTðPOM=PIMÞ Total ingestion rate

IR ð mgh1 Þ ¼ IIR þ OIR

ð6Þ

where: A = energy absorbed (J h− 1) = AR mg h− 1 × energy content of each microalgae J m (Table 1), R = oxygen consumption (J h− 1) = OC mLO2 × 20.08 J (Gnaiger, 1983), U = ammonium excretion (J h− 1) = UR mg NH4– N h− 1 × 24.8 J (Elliot and Davison, 1975).

ð2Þ 2.4. Branchial area ð3Þ ð4Þ

where: IIR = inorganic ingestion rate; IER = rate of production of feces inorganic matter, POM = particulate organic matter (mg L − 1 ) and PIM = particulate inorganic matter (mg L− 1). Efficiency (AE) and rate of absorption (AR): represent material ingested which is absorbed per unit time. AE ð%Þ ¼ AR=OIR ⁎ 100

ð4Þ

AR ð mgh1 Þ ¼ OIR  OER

ð5Þ

where: OER = rate of production of feces organic matter (mg h− 1). Oxygen consumption (OCR: mL O2 h − 1 ) and ammonium excretion (UR: μg NH4–N h− 1) of all the scallops utilized in the feeding treatments were determined on just fed animals (3 min) by placing them in individual chambers (0.8 and 3 L for A. nucleus and N. nodosus, respectively) after rinsing the chambers with a 25% HCl and filling with b 1 μm filtered and totally saturated of oxygen seawater. Chambers were sealed and incubated for 2 h at the same temperature at which they were fed, alongside a control chamber devoid of

Fifteen specimens of A. nucleus (40 + 1 mm) and 11 of N. nodosus (83.1 + 8 mm) were dissected to obtain the branchia. These organs from individual specimens were individually spread on a flat surface and photographed, and their surface areas (BA) were then determined using a Scion image program v. 3.0b. The soft tissues were dried at 70 °C for 48 h, and then individually weighed. 2.5. Standardization of variables The physiological rates and branchial area were converted to a standard individual of 1 g dry tissue weight and with a macroscopic gonadal stage of I. For this, the soft tissues were dried at 70 °C for 48 h, and then individually weighed. Standardization employed the equation of Bayne et al. (1987). Yts ¼ ð1 g=WeÞb1  Ye

ð7Þ

Yms ¼ ð1=EÞb2  Yts

ð8Þ

where Yts = physiological rate of a specimen of standard size (1 g), Ye = uncorrected rate, We = weight of the experimental individual, b1 = dependence of the physiological rate on the size of the individual, Yms =

L.A. Velasco / Aquaculture 270 (2007) 299–311 Table 2 A. nucleus and N. nodosus. Regression equations between weight (W), maturity (M), physiological variables (ER: feces ejection rate, OCR: oxygen consumption rate, UR: excretion rate) and branchial area (BA) Equation

r2

pb

A. nucleus ER = 0.5105e0.9838W ER = 0.4958e−0.6702M OCR = 0.071e1.5216W OCR = 0.3345e0.4284M UR = 22.768e1.1569W UR = 75.995e0.1067M BA = 10.5219 ⁎ W 0.516526

0.6616 0.5262 0.5450 0.3271 0.6387 0.0697 0.8705

0.0080 0.0001 0.0070 0.0070 0.0040 0.2300 0.0001

N. nodosus ER = 0.3493e0.4111W ER = 0.4948e−0.4384M OCR = 0.3343e0.3133W OCR = 0.235e0.5211M UR = 65.455e0.1978W UR = 165.08e−0.0163M BA = 13.5708 ⁎ W 0.701248

0.6343 0.5791 0.4562 0.7722 0.3632 0.0037 0.9571

0.0200 0.0006 0.0300 0.0002 0.0900 0.8500 0.0001

physiological rate of an individual in a standard state of maturity (1), E = stage of maturity of the experimental individual (between 1 and 4) and b2 = dependence of the physiological rates on the stage of maturity of the individuals. Values for “b” which were used for each physiological rate were determined based on exponential regressions among the physiological measurements, the dry weights of 14 specimens of varied size and the

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macroscopic gonadic stage of 21 animals of different degree of maturity. Values of b1 and/or b2 for the feces ejection rates (ER), oxygen consumption rates (OCR), excretion rates (UR), and branchial areas (BA) for A. nucleus and N. nodosus correspond to the exponents of the equations of the Table 2. 2.6. Statistical analyses Tests for normality and homocedasticity were carried out on the physiological variables measured individually. Clearance and excretion rates were Ln transformed, ingestion and absorption rates were transformed to square roots, and rank transformation was applied to absorption efficiency, oxygen consumption rate and scope for growth. Two-way analysis of variance (ANOVA) was carried out to establish the effect of the diet, scallop species and its interaction on each of the physiological variables. In physiological variables where were detected some influence of the diet, was applied a one-way ANOVA using a Bonferroni multiple range test to determine the specific differences between treatments. When scallop species and the interaction between this factor and the diet were detected, several one way ANOVA's were performed comparing both species on each diet to detect significant differences. Spearman correlation tests were used to establish the existence of associations between the feeding rates and the characteristics of the microalgae (size, density, organic or energetic content), as

Fig. 1. A. nucleus and N. nodosus. Physiological rates measured during feeding with different microalgal diets. A. Clearance rate (CR), B. ingestion rate (IR). I. galbana (Ig), C. calcitrans (Cc), C. muelleri (Cm), T. chui (Tc), T. tetrahele (Tt), N. oculata (No), P. tricornutum (Pt), lipid emulsion EmDHA (lip). Values are means ± S.E. The microalgae with the same case letters belong to a homogeneous group identified by the Bonferroni multiple range test. Significant differences existed among the groups (p b 0.05).

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Table 3 A. nucleus and N. nodosus. Factorial ANOVA's between diets, species of scallops and its interactions for each physiological variable Physiological variable

Source

df

F

p

Log(CR)

sp Diet sp ⁎ diet sp Diet sp ⁎ diet sp Diet sp ⁎ diet sp Diet sp ⁎ diet sp Diet sp ⁎ diet sp Diet sp ⁎ diet sp Diet sp ⁎ diet

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

131.27 34.73 2.98 90.39 22.71 3.49 252.39 391.58 13.41 59.07 26.67 3.09 11.82 5.01 1.78 10.35 12.14 4.38 44.40 13.04 2.34

0.0000 0.0000 0.0025 0.0000 0.0000 0.0006 0.0000 0.0000 0.0000 0.0000 0.0000 0.0019 0.0009 0.0000 0.0349 0.0018 0.0000 0.0000 0.0000 0.0000 0.0162

SQRT(IR)

Rank(AE)

SQRT(AR)

Rank(OCR)

Ln(ER)

Rank(SFG)

well as between some of the physiological rates and the scope for growth. Finally, an ANCOVA was carried out to establish the existence of differences between the branchial areas of the two scallop species having the weight as covariate. All the statistical analyses were carried out using Statgraphics-plus 5.0® software and an α of 0.05 in all determinations of significance.

3. Results 3.1. Clearance rate Clearance rates ranged between 1.02 and 12.25 L h− 1 in A. nucleus and between 0.26 and 6.44 L h− 1 in N. nodosus Fig. 1(A). The two-way ANOVA showed that the diet, species of scallop and their interactions affect significantly the clearance rate (Table 3). The multiple range test showed that the clearance rates of A. nucleus were significantly higher with Ig than with Cm, Tc, Ig+ lip and Ig +No, but it was statistically similar to that obtained with the other diets (Fig. 1A). The values of N. nodosus were significantly higher with Cc in comparison with Cm, No, Tc, Ig + No, Ig + Cc and Ig + lip, but it was similar to the obtained with Ig, Ig + Tt, Pt and Tt (Fig. 1A). Significantly lower clearance rates were observed with Tc in both scallop species. The clearance rate of A. nucleus was significantly higher than that of N. nodosus (Table 3) except on diets of Cc, and Ig + Tt where no significant differences were found (p N 0.05). There was no significant correlation between the means of the clearance rates and the characteristics of the microalgae offered to the scallops (density, size, organic and energetic contents) (n = 11, p N 0.05). 3.2. Ingestion rate The ingestion rates varied between 0.47 and 4.78 L h − 1 in A. nucleus and between 0.1 and 1.84 L h − 1 in N. nodosus (Fig. 1B). The diet, specie of scallop and

Fig. 2. A. nucleus and N. nodosus. Physiological rates measured during feeding with different microalgal diets. A. Absorption efficiency (AE) and B. absorption rate (AR). I. galbana (Ig), C. calcitrans (Cc), C. muelleri (Cm), T. chui (Tc), T. tetrahele (Tt), N. oculata (No), P. tricornutum (Pt), lipid emulsion EmDHA (lip). Values are means ± S.E. The microalgae with the same case letters belong to a homogeneous group identified by the Bonferroni multiple range test. Significant differences existed among the groups (p b 0.05).

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Fig. 3. A. nucleus and N. nodosus. A. Oxygen consumption rate (OCR) and B. Excretion rate (UR) with different microalgal diets. I. galbana (Ig), C. calcitrans (Cc), C. muelleri (Cm), T. chui (Tc), T. tetrahele (Tt), N. oculata (No), P. tricornutum (Pt), lipid emulsion EmDHA (lip). Values are means ± S.E. The microalgae with the same case letters belong to a homogeneous group identified by the Bonferroni multiple range test. Significant differences existed among the groups (p b 0.05).

its interactions affect significantly the ingestion rate (Table 3). The multiple range analysis showed that statistically higher ingestion rates of A. nucleus were obtained with Ig than with the remainder diets excepting No and Ig + Tt with which values were statistically similar (Fig. 1B). Ingestion rates of N. nodosus fed with Ig + Tt were similar to those obtained with Ig and Cc but significantly higher than the other diets. A. nucleus had ingestion rates which were significantly higher than those of N. nodosus (Table 3), except when using diets Cc and Ig + Tt, with which no significant differences were noted between the scallop species (p N 0.05). No significant correlation was observed between the means of the ingestion rates and the characteristics of the microalgae in the diets (density, size, organic and energetic contents) (n = 11, p N 0.05).

3.3. Absorption efficiency The absorption efficiency of A. nucleus varied between 40 and 96.2% while that of N. nodosus was between − 48 and 95.3% (Fig. 2A). The two-way ANOVA showed that the diet, species of scallop and their interactions affect significantly the absorption efficiency (Table 3). Based on the multiple range test the absorption efficiency of A. nucleus was significantly greater with the Ig diet and lower with the Cm, Pt, No and Tc diets. In N. nodosus significantly higher values were obtained with Ig diet, and lower values with Tc, No and Cm (Fig. 2A). Absorption efficiencies of A. nucleus were significantly higher than those of N. nodosus (Table 3) except with the Pt and Cm diets, where there were no significant inter-specific differences (p N 0.05). No significant correlations were

Fig. 4. A. nucleus and N. nodosus. Scope for growth (SFG) with different microalgal diets. I. galbana (Ig), C. calcitrans (Cc), C. muelleri (Cm), T. chui (Tc), T. tetrahele (Tt), N. oculata (No), P. tricornutum (Pt), lipid emulsion EmDHA (lip). Values are means± S.E. The microalgae with the same case letters belong to a homogeneous group identified by the Bonferroni multiple range test. Significant differences existed among the groups (p b 0.05).

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observed between the means of absorption efficiencies and individual characteristics of the microalgae used in the diets administered (density, size, organic and energetic contents) (n = 11, p N 0.05). 3.4. Absorption rate The absorption rates varied between 0.13 and 3.42 mg h− 1 in A. nucleus and between − 0.06 and 1.24 mg h− 1 in N. nodosus (Fig. 2B). The diet, specie of scallop and its interactions affect significantly the absorption rate (Table 3). Highest values of absorption rate were obtained with the Ig diet in A. nucleus and with Ig and Ig + Tt in N. nodosus (Fig. 2B). The absorption rate for A. nucleus was significantly higher than that of N. nodosus (Table 3), except when feeding with diets Cc, Pt and Ig + Tt where no significant differences were observed (p N 0.05). 3.5. Oxygen consumption rate Oxygen consumption ranged between 0.37 and 1.77 mL O2 h− 1 in A. nucleus and between 0.74 and 1.41 mL O2 h− 1 in N. nodosus (Fig. 3A). In N. nodosus the rate of oxygen consumption was not significantly affected by the type of diet administered (df = 61, F = 1.38, p = 0.2179). This was in contrast to A. nucleus, which had a significantly higher rate of oxygen consumption when fed with diets Tt and No than when was fed with Tc, Cc and Ig (Fig. 3A). The oxygen consumption of N. nodosus was statistically higher to that of A. nucleus only when fed with Cc and Ig + lip (Table 3) with the other diets values were similar (p N 0.05). 3.6. Excretion rate The rate of ammonium production by A. nucleus varied between 84.9 and 856.1 μg NH3–H h− 1 while in N. nodosus it varied between 99.7 and 1,011.3 μg NH3– H h−1 (Fig. 3B). The diet, specie of scallop and its interactions affect significantly the excretion rate (Table 3). The multiple range tests showed that the excretion rate of A. nucleus fed with the microalgae Cc was significantly higher than with the other treatments excepting No diet (Fig. 3B). In N. nodosus the excretion rate obtained with diets Tc and Cc were statistically higher than the remaining diets less Ig + Cc and No, which were similar (Fig. 3B). The excretion rates were statistically similar between the two scallop species (p N 0.05) except when fed with Ig, No and Tc (Table 3).

3.7. Scope for growth The scope for growth varied between − 27.5 and 25.1 J h− 1 in A. nucleus and between − 38.7 and − 6.7 J h− 1 in N. nodosus (Fig. 4). The diet, species of scallop and its interactions affect significantly the scope for growth (Table 3). The multiple range tests showed that the scope for growth was significantly higher with the Ig diet than with the other diets excepting Ig + lip, Ig + Tt and Tc in A. nucleus and Ig + Tt, Pt, Ig + No and Ig + lip in N. nodosus, where values were similar to the Ig ones (Fig. 4). The scope for growth of A. nucleus with Ig, Tc and Ig + lip, was significantly greater than that of N. nodosus (Table 3) with the rest of the diets values were similar (p N 0.05). The scope for growth was positively correlated with the absorption rate (n = 117, r N 0.9900, p b 0.0001), negatively correlated with ammonia excretion rate (n = 117, r b − 0.2422, p b 0.05) and not correlated with oxygen consumption rate (n = 117, p N 0.05). 3.8. Branchial area The branchial surface of A. nucleus per unit dry weight of specimen was 10.45 ± 0.65 mm2 and that of N. nodosus was 14.35 ± 0.76 mm2. The ANCOVA showed significant between-species differences in branchial area, with that of N. nodosus being higher (df = 1, F = 11.55, p = 0.0026). 4. Discussion 4.1. Feeding rates The highest clearance rates of A. nucleus and N. nodosus obtained with Ig and Cc, respectively, indicates that these microalgae induce the increase of the pumping rate of the water toward the branchial cavity. Increasing clearance rates with certain microalgae has been observed in other bivalve species. P. magellanicus fed with C. calcitrans demonstrated greater clearance rates than when fed with I. galbana (Grant and Cranford, 1991) and P. maximus show higher clearance rates when fed monoalgal diets of Pavlova lutheri and C. calcitrans, and lower with N. oculata (Laing, 2004). The chemical composition and presence of certain algal metabolites may activate the clearance rates, as has been observed in P. magellanicus in the presence of extracts from C. muelleri (Ward et al., 1992), and in A. purpuratus when supplemented with DHA (Navarro et al., 2000). In A. nucleus and N. nodosus it is possible that algal metabolites of I. galbana and

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C. calcitrans acted as activators of the clearance rate as in other scallops, but the presence of DHA in our tests did not increase the clearance rates of these species. The highest ingestion rates did not completely coincide with the highest clearance rates in the two scallop species studied. In A. nucleus, of the seven diets which induced the highest clearance rates (Ig, Tt, Cc, No, Pt, Ig+ Tt and Ig + Cc), only three (Ig, No and Ig + Tt), were within the group of diets with the highest ingestion rates. In N. nodosus, of the five diets which induced the highest clearance rates (Cc, Ig, Ig + Tt, Pt and Tt), only three (Ig + Tt, Ig and Cc) coincided with the highest ingestion rates. This suggest that the different microalgal species were not retained with the same efficiency, the cells possessed characteristics which either promoted or impeded the probability of being retained on the branchiae and transferred to the feeding channels. Scallops as P. magellanicus preferentially retain large and less dense particles (Brillant and MacDonald, 2000a); could discriminate between cells of different compositions (Brillant and MacDonald, 2000b) and between living and non-living cells (Brillant and MacDonald, 2003). The absence of a relation between the ingestion rate of the scallops with size, density, motility, presence of setae, energetic content and organic content of the microalgae allowed the supposition that there was interaction between these microalgae characteristics masking their individual effects on the retention efficiency, or that this variable was affected by other microalgal characteristics not measured in this study. Lower rates of ingestion were obtained with the Ig + No and Ig + Cc mixed diets, than with these algae when presented individually, suggesting the occurrence of antagonistic effects when mixing these microalgae. In contrast, the Ig + Tt mixed diet produced similar values to the Ig diet, and greater values than the Tt diet, observing an averaging effect compared with the two diets administered independently. Mixtures of microalgae can produce positive, negative, or intermediate effects, depending on the microalgal species employed. Similar intermediate effects have been described for P. maximus fed with mixed diets of P. lutheri with other microalgae such as Rhinomonas reticulata, Tetraselmis suecica, I. galbana, Chaetoceros ceratosporum and N. oculata (Laing, 2004). The low ingestion rates observed with mixed diets may be explained by preferential retention of one of the microalgae, as observed in O. edulis which preferentially clears the dinoflagellate Prorocentrum minimum compared with similarly sized cells of P. tricornutum and the flagellate Chroomonas salina (Shumway et al., 1985). The absorption efficiency obtained with the monoalgal diet Ig was the highest observed, and indicated that

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this microalgae contained components of higher digestibility than those of the other species tested. I. galbana have no cell walls (Laing and Utting, 1980), which may facilitate actions of digestive enzymes on their internal components. Results with other bivalves have suggested that Ig is highly digestible, as with A. purpuratus (Navarro et al., 2000), juvenile clams (Laing et al., 1987), A. ventricosus larvae (Lora-Vilchis and MaedaMartínez, 1997) and P. sterna larvae (MartínezFernández et al., 2004). The absorption efficiencies of A. nucleus and N. nodosus obtained with mixed diets gave intermediate values than did the same algae when presented independently. It is probable that in these cases there was preferential absorption of Ig over other accompanying microalgae as has been shown to occur in Crassostrea gigas when fed with a mixed diet of C. salina, P. minimum and P. tricornutum, in which the flagellate C. salina is preferentially absorbed (Shumway et al., 1985). This preferential absorption may be due to a capacity for postingestive selection of particles within the stomach as it has been demonstrated in Pecten magellanicus, where larger, less dense particles are retained longer within the stomach (Brillant and MacDonald, 2000a). The low absorption efficiencies shown with Tc, No and Cm in Argopecen nucleus and N. nodosus indicate that these microalgae contain components which are digested with difficulty and/or possess thick cell walls resistant to enzymatic attack. Similar low absorption efficiencies have been described for some of these microalgae in other molluscan species, for example, T. suecica remains unaltered after passing through the digestive tract of M. edulis (Hildreth, 1980) and N. oculata is poorly digested by larvae of A. ventricosus (Lora-Vilchis and Maeda-Martínez, 1997). The negative absorption efficiency obtained with the Tc diet in N. nodosus suggested in addition to the fact that this microalgae was not efficiently absorbed, important fecal metabolic losses occurred. It has been stated that these losses represent a constant proportion of ingested material which increases with increase in the ingestion rate (Navarro et al., 1994). In the present study, however, the ingestion rate with this diet was one of the lowest observed and thus it is not probable that an increase in abrasion of material in the digestive tract occurred under these conditions. It is possible that enzymes produced in the digestion of this microalga were not able to act, and that they were lost along with the fecal matter of N. nodosus. The higher values of the feeding rates of A. nucleus compared with those of N. nodosus may be due to a greater capacity in A. nucleus for retaining particles on

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the branchiae, or to a greater pumping rate for water through the pallial cavity. These two scallops, similarly to other scallops, have branchiae of the heterorhabdic, folded-filibranch type (Morton, 1983), which are capable of transporting retained particles to the mouth by way of three dorsal and two ventral feeding channels (Beninger et al., 1991). In spite of their similarity in structure and functioning of the branchiae, the branchial area A. nucleus is less than that of N. nodosus, which suggests that N. nodosus has a greater capacity for branchial particle retention. In view of this, the interspecific differences in clearance and ingestion rates may be due to differences in the velocity of water pumped through the pallial cavity. Ward et al. (2003) showed that particles are transported on the branchiae at different speeds, depending on the quality of the food and the species of bivalve. They attribute this to differences in the beating of the cilia, number of active cilia, or type of mucus produced on the surface of the branchiae. On the other hand, the greater absorption efficiency in A. nucleus when compared with N. nodosus suggests that the former species has greater variability and/or activity with regard to its digestive enzymes.

to use protein reserves stored in its tissues, thus increasing the production of nitrogenous waste, or 2) the microalgae contained high levels of proteins and their metabolism provoked an increase in the release of nitrogenous waste. Considering that the Tc diet were absorbed with low efficiency, it is probable that this case were due to the first condition (1) cited above. When using the Cc diet, the scallop species had high absorption efficiencies, and in agreement with a parallel study underway concurrently to the present research, it was determined that the protein content of this microalgae was relatively high (40%; Carrera, 2005). Therefore in this case it was probable that the second case (2) cited above was in effect. In spite of the higher rate of absorption observed in A. nucleus compared with N. nodosus, its excretion rates were similar, which suggest that N. nodosus used proteins as an energy source at a higher rate than did A. nucleus, and was less efficient in retention of the proteins absorbed. This phenomenon was observed when comparing the absorption rates and excretion by Mulinia edulis and Mytilus chilensis fed on the same diets (Velasco and Navarro, 2003).

4.2. Oxygen consumption rate

4.4. Scope for growth

The rate of oxygen consumption by A. nucleus was independent of the type of algal diet, as observed in A. purpuratus offered various diets (Navarro et al., 2000). Higher oxygen consumption by A. nucleus when it was fed with Tt and No diets than when was fed with Tc, Cc and Ig. Is possible that Tt and No stored energy in complex chemical bonds which required a high metabolic expenditure for their utilization. Tetraselmis sp. contains polymeric complexes made up of proteins and polysaccharides, with a low content of monooligosaccharides (Whyte, 1987). Although A. nucleus had greater feeding activity than N. nodosus, it did not have a greater energy requirement, except when fed diets of Cc and Ig + lip, which suggested that A. nucleus was more efficient in its use of oxygen in carrying out physiological functions than was N. nodosus.

The negative potential growth values obtained using most of the diets tested can be attributed to the small quantity of energy acquired from the reduced amounts of food available, and which were below the threshold of pseudofeces production. Feeding of other epibenthic bivalves such as M. edulis at microalgal concentrations below the threshold of pseudofeces production (2.7 mg L− 1) may indeed permit the existence of positive scope for growths (Velasco and Navarro, 2002, 2003). Although A. nucleus and N. nodosus live in environments having low suspended particle concentrations (between 0.2 and 4.7 mg L− 1) and have a threshold of pseudofeces production which is relatively low (0.45 mg L− 1), they require food concentrations higher than this value in order to recover enough energy for growth and reproduction. An exception to this was when A. nucleus was fed I. galbana or a mixture of this species with T. tetrahele or the DHA lipid emulsion. The greater scope for growth obtained with the Ig diet than the majority of the diets in both scallops indicated that consumption of I. galbana improve the physiological condition in a short term and possibly the growth and gonadic ripeness in a long term, if there is not an important effect on the acclimation to the other diets tested which increases the efficiency of the feeding and metabolic processes. I. galbana contains a high content of docosahexanoic acid (DHA) and a low

4.3. Excretion rate The excretion rates of the scallops studied was affected by the microalgal diet offered, which was in contrast to results obtained in the study of A. purpuratus (Navarro et al., 2000). The high excretion rates obtained with Cc in A. nucleus and with Tc and Cc in N. nodosus may be related to: 1) the energy provided by these microalgae was insufficient, and the scallop was forced

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content of eicosapentanoic acid (EPA) (Helm and Laing, 1987; Volkman et al., 1989), while other microalgae similar to those used in the present diets have shown an inverse pattern, as in the cases of C. calcitrans (Helm and Laing, 1987), Nannochloris atomus (Volkman et al., 1989), T. chui (Volkman et al., 1989), T. suecica (Volkman et al., 1989; Caers et al., 1997) and C. muelleri (Volkman et al., 1989; Milke et al., 2004). According to Lane (1989) most bivalves require diets containing the essential fatty acids such as EPA and DHA, nevertheless, the results of the present study suggested that A. nucleus and N. nodosus do not require high values of EPA, but needs DHA. The high correlation between the scope for growth and the absorption rate showed that the potential growth responses related to the various diets was based mainly on the absorption rate rather than oxygen consumption or ammonia excretion rates. With some diets, however, the losses of energy through oxygen consumption and excretion affected the scope for growth, as in the cases of the Ig + lip, Tc, Pt and Ig + No diets, where in spite of the fact that the absorption rate was relatively low, the scope for growth was high due to the compensating effect of the reduced losses due to oxygen consumption and/or excretion. Studies carried out on other species of bivalves with different type of diets have also encountered scope for growths which were mainly regulated by the absorption rate (Navarro et al., 2000; Grant and Cranford, 1991). Nevertheless, the specific responses to given microalgal species or lipid supplements may vary, as observed with A. purpuratus which showed a significantly higher scope for growth when fed a diet composed of I. galbana + EmDHA, when compared with that obtained using the microalgae alone (Navarro et al., 2000). Also, P. magellanicus showed a better scope for growth when fed with C. calcitrans rather than I. galbana (Grant and Cranford, 1991). Studies carried out directly on bivalve growth and survival parameters have obtained results similar to those obtained in the present study. Diets including I. galbana have produced comparatively higher growth in C. virginica and M. mercenaria (Epifanio, 1979; Romberger and Epifanio, 1981), and in R. decussatus (Albentosa et al., 1997). Reduced growth rates have been found for diets including T. chui in C. virginica and M. mercenaria (Epifanio, 1979), C. muelleri in P. magellanicus (Milke et al., 2004) and with P. tricornutum in R. decussatus (Albentosa et al., 1996). Similarly, the inclusion of lipid emulsions (EmDHA) in the microalgal diet of larvae of A. purpuratus did not improve their growth or survival (Uriarte et al., 2003). In other cases, contrasting results have been obtained, as in the growth of A. purpuratus on

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addition of EmDHA to a monoalgal diet of Dunaliella tertiolecta (Nevejan et al., 2003). The similarity between the scope for growth of A. nucleus and N. nodosus in most of the treatments was due to the responses in oxygen consumption and excretion by these scallops. This indicates that the scallops competed for the same diet with equal advantage except when fed with the Ig, Tc or Ig + lip diets, where A. nucleus had a physiological advantage over N. nodosus. In conclusion, greater scope for growth of A. nucleus and N. nodosus were obtained with the I. galbana monoalgal diet, which were due to the high feeding rates plus the low losses of energy indicated by the oxygen consumption and excretion results. The scallops fed with the other microalgae showed lower scope for growth, due to lower clearance, ingestion, and/or absorption rates, and the higher consumption of oxygen and/or excretion rates. The mixed microalgal diets or addition of the EmDHA lipid emulsion to the diets did not improve the scope for growth of A. nucleus and N. nodosus over those obtained with the Ig diet, which could be the optimal diet for use in the laboratory based on its cost of production and biological efficiency. Acknowledgements The author gratefully acknowledges W. Barbosa, J. Barros and the staff of the Moluscos y Microalgas Laboratory of the Universidad del Magdalena (UM) for their help during the experiments and data analysis. Thanks are also to ASOPLAM, UNIDAD DE PARQUES NACIONALES and INVEMAR for providing the experimental animals. This study was financed by the UM and research projects COLCIENCIAS-SENA 1117-09-12394 and the International Foundation for Science (IFS) A/3363-1. References Albentosa, M., Pérez-Camacho, A., Labarta, U., Fernandez-Reiriz, M.J., 1996. Evaluation of live microalgal diets for the seed culture of Ruditapes decussatus using physiological and biochemical parameters. Aquaculture 148 (1), 11–23. Albentosa, M., Pérez-Camacho, A., Labarta, U., Fernandez-Reiriz, M.J., 1997. Evaluation of freeze-dried microalgal diets for the seed culture of Ruditapes decussatus using physiological and biochemical parameters. Aquaculture 154, 305–321. Alix, J.H., Dixon, M.S., Smith, B.C., Wikfors, H., 1996. Scallop larval feeding experiments: some surprises and unanswered questions. J. Shellfish Res. 15 (2), 451. Bayne, B.L., Newell, R.C., 1983. Physiological energetics of marine molluscs. In: de Saleuddin, A.S.M., Wilbur, K.M. (Eds.), The Mollusca. . Physiology, Part 1, vol. 4. Academic Press, New York, pp. 407–515.

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