Spisula Subtruncata (Da Costa, 1778) Within an Annual Cycle

Journal of Experimental Marine Biology and Ecology 301 (2004) 141–157 www.elsevier.com/locate/jembe

Variation of the physiological energetics of the bivalve Spisula subtruncata (da Costa, 1778) within an annual cycle

J.L. Rueda*, A.C. Smaal

Centre for Shellfish Research, Netherlands Institute for Fisheries Research (RIVO), P.O. Box 77, 4400 AB Yerseke, The Netherlands Received 24 April 2003; received in revised form 30 August 2003; accepted 30 September 2003

Abstract

Spisula subtruncata is an infaunal filter-feeding bivalve, which lives in shallow sandy bottoms (2–20 m depth) from Norway to the Atlantic coasts of Morocco, including the Mediterranean Sea. Considering that fisheries of this species have become an important economic resource in some European countries (e.g. The Netherlands), it is of great interest to know the seasonal variation in its physiological energetics. For this purpose, individuals of S. subtruncata were collected and maintained under ambient temperature and seawater conditions of Dutch coastal waters. Physiological processes related to the acquisition and utilisation of energy (e.g. clearance rate [CR], absorption and oxygen uptake) were measured under ambient conditions of the period March 1999 to February 2000. Mean annual clearance and respiration rates (RR) were 0.99 l hÀ1 and 0.23 À1 ml O2 h for a standard individual of 250 mg. Values for both clearance and respiration rate were high during spring and summer and low during autumn and winter. Stepwise multiple regression analyses indicated a significant relationship of the clearance rate with temperature and particulate organic matter (POM), whereas respiration rate was significantly related to temperature, absorption rate (AR) of the animals and their reproductive condition. Absorption efficiency (AE) of the food was significantly related to food quality. Scope for growth (SFG) of S. subtruncata, as well as flesh weight of the animals, was high in summer and low in winter. D 2003 Elsevier B.V. All rights reserved.

Keywords: Absorption; Bivalve; Consumption; Filtration; Respiration; Scope for growth

* Corresponding author. Departamento de Biologia Animal, Facultad de Ciencias, Campus de Teatinos, Universidad de Ma´laga, s/n, Ma´laga 29071, Spain. Tel./fax: +34-952-370938. E-mail address: [email protected] (J.L. Rueda).

1. Introduction

Spisula subtruncata is a common bivalve along the Dutch coast, living in shallow soft bottoms, mainly between 2 and 20 m depth. Its habitat represents a dynamic environment, with variations in the concentration of suspended particulate matter (seston), food quantity and quality and temperature. In a non-seasonal study, physiological response of this bivalve was shown to depend on seston quantity and quality (Rueda and Smaal, 2002). As a response to the environmental variations, the physiological energetics of S. subtruncata may fluctuate throughout the year. Seasonal interactions of environmental variables with the filtration activity and metabolism of bivalves have been well documented in different species. Clearance and respiration rates (RR) of cockles (Cerastoderma edule Linne´, 1758) from the Dutch coast were influenced by temperature and reproductive condition during natural seasonal cycles (Smaal et al., 1997). As food availability is higher in spring and summer, the net result was a maximum scope for growth (SFG) in these seasons of the year. In the common mussel (Mytilus edulis Linne´, 1758), no relation has been observed between seasonal variations of clearance rate (CR) with temperature (Smaal et al., 1997). Seasonal variations of the respiration rate of mussels and cockles were related to a combination of temperature and reproductive condition (Smaal et al., 1997; Iglesias and Navarro, 1991). Monthly respiration and excretion rates of the mussel (Mytilus galloprovincialis Lamarck, 1819), within an annual cycle, were influenced only by the quantity and quality of food as shown for the Ria de Arosa (northwestern Spain) (Babarro et al., 2000). Little information about feeding and ecophysiology of the bivalve S. subtruncata is available (Møhlenberg and Riisga˚rd, 1979; Kiørboe and Møhlenberg, 1981; Møhlen- berg and Kiørboe, 1981). Recently, growth rates of this species have been measured in populations in the North Sea over a 4-year period (Degraer et al., 1998). In this case, intra-annual fluctuations of the individual weight were recorded, with higher growth rates in spring and summer when compared to the cold seasons (autumn and winter). In the present study, we address the natural seasonal variation in physiological energetics of S. subtruncata over a year. We test the hypothesis that internal regulation of the physiological processes acts as a strategy to optimize energy gains, in a dynamic habitat, in terms of food supply and quality.

2. Material and methods

2.1. Collection of animals and maintenance

From March 1999 to March 2000, mature individuals of S. subtruncata were dredged monthly from populations in the southern coast of Texel Island (North of The Nether- lands) (Fig. 1). Animals were transported to a field laboratory in the Oosterschelde (SW Netherlands) (Fig. 1), where they were held in a tank with sandy substrate and supplied with seawater. The experiments were performed within the same week of collection of J.L. Rueda, A.C. Smaal / J. Exp. Mar. Biol. Ecol. 301 (2004) 141–157 143

Fig. 1. Location of Texel island (sampling site) and the Oosterschelde (laboratory site). the animals. Shell length was measured to the nearest 0.5 mm. Individuals with a shell length around 25 mm were selected for the physiological experiments. Shell and flesh of the bivalves were dried for 48 h at 70 jC and weighed for dry weight (DW) and ashed for 4 h at 520 jC in order to calculate the ash-free dry weight (AFDW). Condition index (CI) was calculated as the percentage of dry weight of flesh compared to dry weight of shell (CI = DW flesh Â 100/DW shell). A total number of 445 individuals were measured; 280 just after collection from the field and 165 after the physiological experiments. Data on the reproductive condition of the clams was derived from macroscopic observations of the gonad and according to the previous observations of Le Pennec (1980).

2.2. Diet characterization

Seawater containing the diet was mixed in a container and pumped to eight grazing- respiration chambers. The water was collected at high tide from the Oosterschelde, which is an unpolluted estuary in the southwest of The Netherlands. Particle concentration in the 144 J.L. Rueda, A.C. Smaal / J. Exp. Mar. Biol. Ecol. 301 (2004) 141–157 water samples of each experiment was monitored with a Coulter Counter, fitted with a 100-Am tube. The number of particles in all experiments ranged between 10,000 and 15,000 mlÀ1, except in the experiment of June 1999 which had a high concentration (20,000–30,000 particles mlÀ1) of the Haptophyta Phaeocystis sp. In each experiment, three water samples were taken during the experimental period. For weight determinations of total particulate matter (TPM) and particulate organic matter (POM), water samples of 1 l were filtered onto pre-ashed (450 jC for 4 h) and weighed GFC filters, rinsed with seawater-isotonic ammonium formate and dried at 80 jC for 24 h. The dry weight of retained material gave the TPM and the weight loss on ignition at 450 jC for 4 h gave the POM. The organic content of suspended matter as percentage is used in terms of a food quality parameter and was computed as: Q=(POM Â 100)/TPM. The chlorophyll a content (Chl a) was estimated by filtering 1 l of water and extracting with acetone 90% during 18 h in darkness. The absorbance of the sample was measured using a spectrometer LS30 at 440 and 670 nm before and after adding three drops of HCl 0.3 M.

2.3. Experimental design and measurement of physiological rates

Experiments were conducted with single animals in grazing-respiration chambers with an average volume of 296 ml. For all measurements, eight chambers were used, of which seven were filled with one individual and one served as a control without an animal. Generally, three series of experimental measurements were executed during each month. Each clam was placed in a chamber with a water flow adjusted to have a maximum reduction of particle concentration of not more than 30% of the outflow. Flow rates through each chamber were similar and were determined by simultaneously collecting and measuring the volume of water collected from each outflow. Clearance rate (CR: l hÀ1) was estimated by measuring the removal of suspended particles from the water flowing through the chambers containing individuals. A coulter counter fitted with a 100-Am orifice tube was used to determine the difference between the particle concentration in the outflow from the control chamber (C1) and the outflow from each experimental chamber containing an individual (C2). CR was calculated as flow rate multiplied by (C1 À C2)/C2. Mean clearance rates were calculated in each experiment based on several measurements (three to five) of seven clams. Measurements were done for a period of 2–3 h after an acclimation period of 4 h with the experimental diet. Filtration rate (FR: mg hÀ1) represents the TPM (mg lÀ1), which is filtered from the suspension, and it was calculated as FR = CR ÂTPM and filtration rate of particulate organic matter (OFR: mg hÀ1) as OFR = CR Â POM. All experiments were under pseudofaeces threshold, except the June 1999 experiment. Rejection rates of pseudofaeces were generally equal to 0 and ingestion rates of total (IR: mg hÀ1) and organic (OIR: mg hÀ1) particulate matter were estimated as IR = FR and OIR = OFR, respectively. Faeces produced by each individual were collected using a Pasteur pipette and deposited on pre-ashed and pre-weighed GFC filters. Inorganic and organic mass of faeces were determined by using similar methods as those described for seawater samples (see Section 2.2). Absorption efficiency (AE) was calculated according to Conover (1966) J.L. Rueda, A.C. Smaal / J. Exp. Mar. Biol. Ecol. 301 (2004) 141–157 145 as AE=( f À e)/(1À e) Â f, where f is the ratio ash-free dry weight/dry weight of food and e is the ratio ash-free dry weight/dry weight of faeces. Absorption rate (AR) was calculated as AR = AE Â OFR. À1 Respiration rate (RR: ml O2 h ) were measured after maintaining the clams under the same environmental conditions of the clearance rate measurements. Oxygen concentration in water samples was measured by Winkler titriation and, in some experiments, the linear decrease of the oxygen tension was also measured for a period of 2–3 h and tested at intervals of 10 min by polarographic electrodes. The water flow to the chamber was stopped and samples of water were collected at the start and at the end of the period of measurement. The respiration rate was calculated as RR=([O2]to À [O2]t1) Â À1 (Vchamber À Vanimal)/(t1 À t0). Where RR = the rate of consumption in mg O2 h ,[O2] = the oxygen concentration at the start (to) and at the end (t1) of the lineal decline, V = the volume of chamber and animal in liters and t = the difference in time expressed in hours. Measurement of the decline of oxygen concentration were also taken in a parallel control chamber without animals. The concentration of O2 is finally expressed in ml O2 which is mg O2 divided by 1.482 (Widdows, 1985).

2.4. Scope for growth

The SFG (JÀ1 hÀ1 250 mgÀ1 AFDW) was calculated as SFG = AE Â consumption À respiration, where consumption was equal to CR Â POM Â 23.5 and respiration was RR Â 14.5. Scope for growth was calculated for each monthly series of experiments. Using the derived model, seasonal SFG recalculations were performed from monthly environmental data during the period April 1999–March 2000 (Table 1).These

Table 1 Monthly values of seawater parameters in (1) the experimental diets and (2) average values from locations around Texel island (Dutch Wadden Sea) for the period between March 1999 and March 2000 Month Experiment Texel T TPM POM Chl a Q T TPM POM Chl a Q (jC) (mg lÀ 1) (mg lÀ 1) (AglÀ 1) (%) (jC) (mg lÀ 1) (mg lÀ 1) (AglÀ 1) (%) March 1999 9 6.9 F 0.1 1.3 F 0.1 1.5 F 0.2 19.0 – – – – – April 1999 10 7.3 F 0.0 1.3 F 0.1 4.5 F 0.3 17.5 9 10.1 F 4.4 3.2 F 0.5 22.2 F 2.5 31.7 May 1999 14 6.9 F 0.0 2.8 F 0.0 7.6 F 0.5 39.9 12 8.8 F 2.5 2.1 F 0.3 5.5 F 1.6 24.3 June 1999 16 10.4 F 0.5 5.1 F 0.2 13.1 F 1.1 49.5 16 5.6 F 2.9 1.6 F 0.4 11.6 F 4.7 29.1 July 1999 18 7.5 F 0.3 1.8 F 0.1 5.7 F 0.3 23.6 17 3.5 F 0.6 1.3 F 0.2 7.5 F 1.1 38.6 August 1999 20 9.6 F 0.1 1.9 F 0.2 1.5 F 0.1 20.4 19 9.3 F 7.5 1.6 F 0.6 5.7 F 3.6 17.7 September 1999 19 8.9 F 0.1 2.1 F 0.1 2.2 F 0.1 23.5 17 7.6 F 2.8 1.3 F 0.3 6.2 F 1.8 17.5 October 1999 8 8.6 F 0.1 2.5 F 0.2 5.5 F 0.4 28.5 13 21.3 F 7.5 1.9 F 0.5 4.5 F 1.2 12.5 November 1999 7 9.0 F 0.2 1.3 F 0.1 1.0 F 0.0 14.4 10 35 F 12.1 2.6 F 0.8 2.8 F 0.5 7.4 December 1999 – – – – – 7 32.4 F 13.7 2.4 F 0.9 2.6 F 0.8 7.4 January 2000 5 7.1 F 0.1 1.0 F 0.0 1.8 F 0.1 14.5 5 26.3 F 6.9 2.3 F 0.4 2.1 F 0.4 8.7 February 2000 7 6.9 F 0.2 1.3 F 0.1 2.3 F 0.2 18.9 4 46.9 F 11.7 3.1 F 0.7 4.5 F 0.8 6.6 March 2000 – – – – – 6 3.8 F 1.1 0.8 F 0.1 5.5 F 1.7 22.2 Temperature (T), TPM (mean F standard error), POM (mean F standard error), chlorophyll a (Chl a) (mean F standard error) and food quality (Q). 146 J.L. Rueda, A.C. Smaal / J. Exp. Mar. Biol. Ecol. 301 (2004) 141–157 environmental data represent the average data of water samples of the coasts close to the collection of the animals, around the island of Texel, where large populations of S. subtruncata occur. The environmental data were provided by the National Institute for Coastal and Marine Management (RIKZ, The Netherlands).

2.5. Size standardization of physiological rates

Once the physiological measurements were completed, shell length of each individual was recorded to the nearest 0.1 mm as well as the ash-free dry weight of the soft tissues (weight after drying the soft tissues at 80 jC during 48 h weight after calcinations at 520 jC during 4 h). Physiological rates were standardized to an equivalent of 250 mg ash-free dry tissue of S. subtruncata in order to avoid variation of the rates due to changes in the monthly weight of the animals. In this case, the following function was used: Ys = Ye* b (standard weight/We) , where Ys = physiological rate of a standard-sized animal, Ye = un- corrected physiological rate, We = ash-free dry weight (mg) of the experimental animal and b = the weight power established for clearance and respiration rates of S. subtruncata.In this case, the b coefficient used for CR recalculations was 0.58 and for RR was 0.7 (Rueda et al., in prep).

2.6. Data analysis

Multiple stepwise regression analysis was used to determine the effect of environmental parameters of the diet and of internal parameters on the variation of the monthly physiological rates. Data of the rates used in the regression analysis were log trans- formed. Multiple regression analyses were executed with forward steps, where at each step a parameter was included in the test and backward steps where at each step a parameter was excluded according to a high p-value ( p>0.01). Results were similar when using both procedures. All statistical analyses were executed with the SYSTAT statistical software package version 9.0 for windows.

3. Results

3.1. Diets

Values of seawater parameters measured in the experiments and in the location of collection of the animals (average values from sampling sites around Texel island) are presented in Table 1. Seawater temperature in the experiments varied between 5 jC (January 2000) and 20 jC (August 1999). The TPM reached values between 6.9 and 9.6 mg lÀ1, except in the experiments during June 1999, which contained a high amount of algal cells from the species Phaeocystis sp. In August 1999, the high TPM value was due to resuspension of fine sediments after storm. POM concentrations varied from 1 to 5 mg lÀ1 and chlorophyll a concentrations from 1.0 to 13.1 AglÀ1. Both high POM and chlorophyll a values were measured during the experiments performed in June 1999. The quality of the seston varied from 14% to 49% organic material. J.L. Rueda, A.C. Smaal / J. Exp. Mar. Biol. Ecol. 301 (2004) 141–157 147

3.2. Animals

Dry weight of the shell for an standard individual of 25 mm was similar during the studied period with values around 1.4 g/individual (Fig. 2). Values of percentage of organic material in the shell were between 3.5% and 4%. Seasonal changes in the DW and AFDW of the animals was registered with lower values in winter months than in summer months (Fig. 3). The reproductive period was from April to June 1999, shown by the presence of specimens with active (coloured) gonads amounting to at least half of the visceral mass. From July to March, the gonads of the individuals were macroscopi- cally determined as non-active (non-coloured). The weight increased between March and April, mainly related to a fast development of the gonad. The decrease due to gamete release took place during June and July when a lower number of animals with active gonads were observed. In the winter months of January and February a decrease of AFDW was in the range of 20–40% of the average AFDW compared to values from July to November. Dry weight of the flesh of the animal represents between 5% and 10% of the total dry weight of the individual (flesh + shell). The total fresh weight of 25 mm individuals followed the same seasonal pattern as for DW and AFDW, with an average value of 2.5 g in winter and higher values, around 3.2 g, in spring and summer. The amount of fresh flesh weight represents 25–30% of the total fresh weight based on average values for every month.

3.3. Physiological rates

CR values for a standardised animal of 250 mg AFDW are expressed as average and standard error per month (Fig. 4). CR values were low in winter months with

Fig. 2. DW (g) (white bars) and percentage of organic matter of shell (% OM shell) (black bars) of 25-mm shell length individuals of S. subtruncata collected during the period March 1999–February 2000. Mean + standard deviation. 148 J.L. Rueda, A.C. Smaal / J. Exp. Mar. Biol. Ecol. 301 (2004) 141–157

Fig. 3. DW (mg) (white bars) and AFDW (mg) (black bars) of tissues of S. subtruncata during the period March 1999–February 2000. Mean + standard deviation. Presence of coloured gonads indicated as active gonads. average values of 0.55 l hÀ1 and maximum values were recorded in summer months, around 1.5 l hÀ1 per individual. In June 1999, a reduction of the CR was recorded, together with high concentrations of the algal cell Phaeocystis sp. Multiple regression analysis showed a significant positive relationship of log CR with temperature and a significant negative relationship with the particulate organic matter concentration (Table 2). The relationship of CR with POM displayed an asymptotic logarithmic function.

Fig. 4. Monthly CR (l hÀ1) of standardised individuals of S. subtruncata (250 mg AFDW) (bars) related to temperature (jC) (line). Mean + standard deviation. J.L. Rueda, A.C. Smaal / J. Exp. Mar. Biol. Ecol. 301 (2004) 141–157 149

Table 2 Stepwise multiple regression analyses of log physiological rates (clearance and respiration rates) of S. subtruncata (standardized to 250 mg AFDW) and of absorption efficiency with: TPM, POM, food quality (Q), chlorophyll a concentration (Chl a), temperature (T), reproductive condition (RC) and interaction of T with RC (T Â RC) Terms Coefficient S.E. FNr2 P Clearance rate Retained 198 0.628 < 0.001 Constant À 0.184 0.031 T 0.029 0.002 151.918 < 0.001 POM À 0.099 0.009 111.492 < 0.001 Rejected Chl a TPM Q RC T Â RC

Respiration rate Retained 202 0.643 < 0.001 Constant À 1.091 0.047 T 0.046 0.003 184.310 < 0.001 POM À 0.217 0.028 59.054 < 0.001 Chl a 0.045 0.009 26.587 < 0.001 RC 0.257 0.067 14.852 < 0.001 Rejected TPM Q T Â RC

Absorption efficiency Retained 39 0.520 < 0.001 Constant 0.046 0.049 Q 1.278 0.203 39.726 < 0.001 Rejected RC POM Chl a TPM T T Â RC

No significant relationship was found with total particulate matter and food quality. The effect of the reproductive condition of the animal (active from April to June) was also considered in the multiple regression analysis; however, no significant relationship was registered with the clearance rate. The annual average clearance rate was 0.99 l hÀ1 for an individual of 250 mg AFDW. RR for standard animals followed a seasonal pattern as for CR, with minimum À1 values (0.08 ml O2 h for an individual of 250 mg AFDW) in the winter season and À1 maximum values in spring and summer (between 0.20 and 0.40 ml O2 h ) (Fig. 5). 150 J.L. Rueda, A.C. Smaal / J. Exp. Mar. Biol. Ecol. 301 (2004) 141–157

À1 Fig. 5. Monthly RR (ml O2 h ) of standardised individuals of S. subtruncata (250 mg AFDW) (bars) related to temperature (jC) (line). Mean + standard deviation.

Multiple regression analysis showed that respiration rates were positively correlated to external variables such as temperature, chlorophyll a and negatively correlated to POM (Table 2). A second multiple regression analysis showed that RR was highly correlated with internal variables such as the AR and the reproduction condition (CR) of the clams (Table 3). RR values had a positive lineal relationship with temperature, when not taking into account the data of individuals with gonads in active state (April to June) (Fig. 6). RR values in active gonad states were higher compared to the relationship of temperature and RR in individuals with non-active gonad states. À 1 The mean annual respiration rate was 0.23 ml O2 h for an individual of 250 mg AFDW. AE varied between 20% and 50%. In a multiple regression analysis, food quality was the only environmental parameter significantly related to absorption efficiency (Table 2).

Table 3 Stepwise multiple regression analysis of respiration rates of S. subtruncata (standardized to 250 mg AFDW) with internal variables of the clams: reproductive condition (RC), IR, AR and interaction of IR and AR with RC (IR Â RC, AR Â RC) Terms Coefficient S.E. FNr2 P Retained 39 0.900 < 0.001 Constant 0.058 0.029 AR 0.185 0.037 25.140 < 0.005 RC 0.553 0.133 17.222 < 0.005 AR Â RC À 0.429 0.128 11.263 < 0.05 Rejected IR IR Â RC J.L. Rueda, A.C. Smaal / J. Exp. Mar. Biol. Ecol. 301 (2004) 141–157 151

À1 Fig. 6. Mean RR (ml O2 h ) of standardised individuals of S. subtruncata (250 mg AFDW) not presenting active gonads (open circles) related with temperature (jC). N = 17, r2 = 0.93, p < 0.01.

3.4. Scope for growth

High SFG values resulted from May to September with a decrease (25%) in autumn and winter months (Fig. 7). Another series of SFG values were recalculated by using the models of the multiple regression analyses and average environmental data from coastal locations around Texel island (Table 1). In this case, a seasonality in SFG was also clear, with higher values in spring and summer months in comparison to winter values (Fig. 8).

Fig. 7. Energy budget of S. subtruncata (J hÀ1 250 mgÀ1 AFDW) per month based on experimental conditions. FR (white bar), AR (shaded bar), RR (negative) and production rate or SFG (dashed bar) are presented per month. 152 J.L. Rueda, A.C. Smaal / J. Exp. Mar. Biol. Ecol. 301 (2004) 141–157

Fig. 8. Seasonal variation of the recalculated scope for growth of S. subtruncata (J hÀ1 250 mgÀ1 AFDW) (bars), using environmental data of coastal locations from Texel island (Dutch Wadden Sea) (Table 1), and the condition index of individuals of S. subtruncata (circles) collected in the same area and period of time. Spring 1999 (Sp), summer 1999 (Su), autumn 1999 (Au) and winter 2000 (W). Mean F standard deviation.

Moreover, seasonal changes of SFG were correlated to seasonal variation of the condition index of individuals collected from the same area (N =12, r2 = 0.673, p < 0.01).

4. Discussion

4.1. Diets and food consumption

S. subtruncata lives in a subtidal and dynamic environment with variations related to the quantity and quality of seston, both on a small (e.g. storm events) and large time scale (e.g. phytoplankton blooms), and with variations of temperature, normally on a large time scale (e.g. season). Temperature and chlorophyll a values from the experiments were similar to field conditions, but TPM and POM were lower in our experiments. Experi- mental TPM concentrations were, generally, below 10 mg lÀ 1, which sometimes may not reflect ambient winter conditions (Table 1). This may decrease the SFG values obtained in the recalculations using the environmental data from locations, where individuals of S. subtruncata were collected. In the experiments of May and June, blooms of phytoplankton occurred in the water containing a large proportion of Phaeocystis sp. cells. The presence of blooms of this species during spring occurs yearly along the Dutch coast (Bakker et al., 1994).Itis known that these colonies reduce the clearance rate of bivalve filter feeders (e.g. blue mussel M. edulis)(Smaal and Twisk, 1997; Smaal et al., 1997), and it may also explain the decrease in the CR values during the experiments of May and June 1999. Moreover, a negative significant relationship between CR and POM (this study) or Chl a (Rueda et al., unpublished data) has also been found in this bivalve. Negative effects of POM in J.L. Rueda, A.C. Smaal / J. Exp. Mar. Biol. Ecol. 301 (2004) 141–157 153 clearance rates of bivalves have also been found in previous studies on physiological regulation of temperate bivalves in non-seasonal studies (Iglesias et al., 1996; Navarro et al., 1994). Møhlenberg and Kiørboe (1981) also found a negative relation between the CR of S. subtruncata and the chlorophyll a concentration of the diet. In recent studies, CR has been found to vary in a unimodal relationship over changes on seston concentrations when including a wide range of seston concentrations, with low CR values at very low and very high seston concentrations (Hawkins et al., 1999,2001). According to that, the negative relationship found in our study represents the downside part of the asymptotic relation found when including higher seston concentrations (Rueda et al., unpublished data). In a previous study (Rueda and Smaal, 2002), regulatory mechanisms of the ingestion rate in S. subtruncata were determined by food composition, with reductions of clearance rate as a mechanism under high quality values of organic matter, and the production of pseudofaeces as a mechanism under low ratios of POM. These mechanisms are therefore similar to those observed in other filter feeding bivalves (e.g. C. edule) (Iglesias et al., 1996; Navarro et al., 1992). Reductions in the clearance rate at high food concentrations (POM) may represent an adaptation of bivalves to gain energy efficiently by reducing the metabolic costs of pumping. An increase of the CR in these situations (e.g. high concentration of algae and detritus) could affect the filtration efficiency (e.g. gills blocking), avoiding rejection of pseudofaeces and hindering the selection of food particles. In S. subtruncata, a positive significant correlation was found between absorption efficiency of food and food quality. The same mechanism has been observed in other temperate and tropical bivalve species (Navarro et al., 1994; Iglesias et al., 1996; Navarro and Widdows, 1997; Wong and Cheung, 1999). This increment of the absorption efficiency at high food qualities has been described in C. edule and related to endogenous factors during the digestive processes (Willows, 1992; Navarro and Iglesias, 1993; Ibarrola et al., 2000).InS. subtruncata, an increase of absorption efficiency could represent an efficient mechanism to maximise the consumption when high food quantity/quality is available, since clearance rate is normally reduced in these conditions.

4.2. Reproductive condition

Gametogenesis was registered in our specimens by the presence of active coloured gonads from April to June 1999, with an increment of the somatic weight in comparison to winter measurements. The gonads from different individuals displayed two colour patterns: (1) dark pink or red in females and (2) yellow to orange in males. This was confirmed after microscopic observations of reproductive cells of different coloured gonads. Degraer et al. (1998) observed the presence of a weight peak in individuals from the Belgian coasts in the months of April and May during 2 consecutive years, as a result of developed gonads. Le Pennec (1980) studied the development of the gonad in S. subtruncata, and observed the presence of 100% active cells in the gonad during May and June with a creamy orange male gonad as well as a pink female gonad. This sex dimorphism based on coloration pattern has been observed in other bivalve species such as different species of the genus Donax in the coasts off the south of Spain (Tirado and 154 J.L. Rueda, A.C. Smaal / J. Exp. Mar. Biol. Ecol. 301 (2004) 141–157

Salas, 1998).InS. subtruncata, the large SFG values obtained in experimental conditions during spring may represent a significant increase of the energy available for reproductive processes.

4.3. Metabolism losses

Oxygen consumption of S. subtruncata presented a seasonal trend (Fig. 5), with an À1 À1 annual average value of 0.61 ml O2 h g , which is similar to a previous non- seasonal study for this species (Møhlenberg and Kiørboe, 1981) or for observations during natural seasonal cycles of the cockle C. edule (Smaal et al., 1997). Both temperature and reproductive condition are important factors influencing the respira- tory processes of S. subtruncata. During the reproductive peak (April–June), RR was sometimes doubled when comparing these values to the relation between RR of non- reproductive stages and temperature (Fig. 6). Previous studies on natural seasonal cycles of bivalves from temperate waters have registered increases of RR during the reproductive processes (e.g. gametogenesis, spawning) (Newell and Bayne, 1980; Iglesias and Navarro, 1991; Smaal et al., 1997; Urrutia et al., 1999). However, CR of clams were not influenced by reproductive processes in our study as found in other bivalve species during natural seasonal cycles (Smaal et al., 1997). An increase in the production of gonadal tissues of S. subtruncata may also represent an increase of the metabolic rate of the cells and, therefore, a higher respiration rate associated to these processes. These energetic costs are balanced by the high food availability during spring, maximizing the consumption. Newell and Thompson (1984) associated reductions in the CR with the spawning processes of M. edulis which is not in concordance with S. subtruncata. In accordance with Widdows (1978), the reproductive cycle exerts a major influence on the respiration and excretion rates of bivalves, while the other process such as filtration remain relatively independent of the reproductive processes. Respiration rates were positively influenced by chlorophyll a (phytoplankton) and negatively related to POM (particle concentration of both detritus and phytoplankton). High POM concentrations result in an indirect decrease of the respiration rate because of the direct reduction in the pumping activity of S. subtruncata (N =9,r2 = 0.951, p < 0.001: excluding data during gametogenesis). However, at high chlorophyll a levels, an increase in RR occurs due to the absorptive processes (Table 3) and, therefore, an indirect positive relationship was found between RR and chlorophyll a concentrations (Table 2). Babarro et al (2000) registered a seasonal variation of oxygen consumption in mussels M. galloprovincialis from Rı´a de Arousa (NW Spain) related to changes in temperature, food quality and chlorophyll a concentrations. In this study under natural seasonal conditions, respiration rates increased at high food availability. Navarro and Thompson (1996) observed a similar relation between RR and food available in the bivalve Modiolus modiolus. Both studies discussed that an increase of RR at high food quality may occur due to an increase in the activity of the digestive processes, as it was found in the cockle C. edule (Ibarrola et al., 2000). In the latter case, an increase of the absorption of food at high food quality represented a higher activity of the digestive glands, thus a higher respiration rate of the cells during the digestive processes. J.L. Rueda, A.C. Smaal / J. Exp. Mar. Biol. Ecol. 301 (2004) 141–157 155

4.4. Scope for growth and tissue growth

Seasonal changes in the weight of S. subtruncata displayed a significant relationship with the recalculated SFG values obtained using the environmental data from its habitat. The weight of individuals in spring months almost doubled the winter values. This same trend was found for the SFG values. Degraer et al. (1998) studied the growth of individuals of S. subtruncata in the Belgian coasts during a period of 4 years. They showed a seasonal growth pattern with weight gain from spring to summer and loss of weight during the winter season. This seasonal trend is also reflected in the SFG values from the laboratory experiments and the recalculations using environmental data (Figs. 7 and 8). Seasonal fluctuations in the weight between June and November could be related to the release of the gametes, together with changes in the somatic and storage production. This fluctuation in growth sometimes is marked in the shell by the presence of intra-annual growth rings (Richardson, 1988). The winter weight loss of S. subtruncata is within a similar range (20–40%) as those observed for other bivalve species, such as Macoma balthica, Scrobicularia plana, Mya arenaria or C. edule in the Dutch Wadden Sea (Zwarts, 1991). A seasonal pattern in the scope for growth was clear in S. subtruncata, with higher values in spring and summer months and lower values during autumn and winter. Increased respiration rates were an effect of reproduction, superimposed on the temperature effect (Fig. 6), which increased the metabolic losses of S. subtruncata. However, consumption remains high during gametogenesis due to the increase of the available food in their habitat, resulting in a high SFG. Gametogenesis during the spring bloom of phytoplankton apparently profits from the increase of available food in this period. A similar matching was found in other bivalves, such as Spisula solida with a gametogenesis during late winter and spring in the South of Portugal (Gaspar and Monteiro, 1999).

4.5. Concluding remarks

Changes in the physiology and somatic/gametic development of S. subtruncata are influenced by the seasonal variation of its environment. Seasonality in consumption and respiration of the clams occurs in such a way that during autumn and winter S. subtruncata remains in a dormant stage by reducing the metabolic losses and the pumping activity. This activity reduction results in low SFG values with winter body weight losses and the absence of growth. During spring and summer, the clams compensate for low levels of food by vigorous pumping (and thus respiring) and at high levels of food by increasing the absorption of food and reducing both the pumping capacity and metabolic losses related to clearance processes. During reproduction, its metabolic losses are increased as a result of increasing respiration rates. However, reproduction occurs when the available food reaches maximum levels in its habitat. This results in a positive scope for growth due to the high absorption of the food.

Acknowledgements The authors are grateful to Joke Kesteloo and Johan Craeymeersch, from Netherlands Institute for Fisheries research (RIVO-The Netherlands), for their support at different 156 J.L. Rueda, A.C. Smaal / J. Exp. Mar. Biol. Ecol. 301 (2004) 141–157 stages of this research. Thanks to Gilles Wattel from National Institute for Coastal and Marine Management (RIKZ, The Netherlands) for providing the environmental data off the Dutch Wadden Sea. Herman Hummel and Roelof Bogaards from Netherlands Institute for Estuarine and Marine Ecology (NIOO-CEMO, The Netherlands) provided us laboratory facilities. We also thank the valuable comments made by Dr. Sandra Shumway and one anonymous referee. This research has been supported by a Marie-Curie training research grant of the European Commission, within the project SIMCERE (FAIR GT97- 4525). [SS]

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