Biochemical composition and energy content of the mussel Mytilus galloprovincialis of subtidal and rocky shore origin: Influence of environmental variables and source of mussel seed

Composición bioquímica y contenido energético del mejillón Mytilus galloprovincialis de origen submareal e intermareal: Influencia de las variables ambientales y de su origen

L. Freites¹, M.J. Fernández-Reiriz^1* and U. Labarta¹

¹ Consejo Superior de Investigaciones Científicas Instituto de Investigaciones Marinas Eduardo Cabello 6 36208 Vigo, Spain. *E-mail: mjreiriz@iim.csic.es

Recibido en marzo de 2003;
aceptado en junio de 2003.

Abstract

The energy content and biochemical composition of seed of the mussel Mytilus galloprovincialis of two distinct origins (subtidal and rocky shore) were compared after both groups were transferred to the water column (subtidal habitat) in the Arousa Ria, NW Spain. The experimental period comprised a total of 228 days. The relative percentages of carbohydrates, glycogen and lipids were significantly higher in the subtidal mussels in the first 15 days, whereas the proteins were significantly higher in the rocky shore mussels during the first 36 days. A multiple regression analysis referred to the same time period showed that the origin of the mussel seed influenced significantly the variance observed in the energy values of all biochemical components (measured in terms of kJ mg^-1). In the period between days 50 and 110, mussel origin only showed a significant effect on the lipid energy content, but in contrast to the previous period, the content was greater in the rocky shore mussels. The multiple regression analysis showed that after 125 days of culture, mussel origin did not influence the variance observed in the energy values of all biochemical components, whereas the ratio chlorophyll a/particulate organic material, temperature and total particulate material influenced significantly the variance observed in the different energy values of all biochemical components. Changes observed in the energy content of carbohydrates and lipids throughout the different periods of this study can be related to catabolism-anabolism processes linked mainly to the availability of phytoplanktonic food. Moreover, with regard to lipids, our results suggest that changes were also associated with reproductive activity.

Key words: biochemical composition, energy content, Mytilus galloprovincialis, mussel seed, Arousa Ria.

Resumen

Se compararon el contenido energético y la composición bioquímica de semillas del mejillón Mytilus galloprovincialis de dos orígenes distintos (submareal e intermareal), una vez que ambas semillas fueron colocadas en la columna de agua (hábitat submareal) en la Ría de Arousa, Noroeste de España. El periodo experimental comprendió un total de 228 días. Los porcentajes relativos de glucógeno y lípidos, además del de carbohidratos, fueron significativamente más altos en los mejillones de origen submareal, durante los primeros 8 y 15 días, respectivamente, mientras que en los de origen intermareal, las proteínas fueron significativamente más altas en los primeros 36 días. Durante este mismo periodo, el análisis de regresión múltiple mostró que el origen de los mejillones influyó de significativamente en la varianza observada en los valores energéticos de los diferentes componentes bioquímicos, medidos en términos de kJ mg^-1. Subsecuentemente, entre el día 50 y el 110, el origen de los mejillones sigue influyendo de manera significativa en el contenido de lípidos, pero en contraste con el periodo anterior, el contenido energético de los mismos fue mayor en los mejillones de origen intermareal. A partir de los 125 días, el análisis de regresión múltiple mostró que el origen de los mejillones no influye en la varianza observada en los equivalentes energéticos de todos los componentes bioquímicos, mientras que la razón clorofila a/MPO, la temperatura y la MPT influyeron significativamente en la varianza observada en los diferentes valores energéticos de todos los componentes bioquímicos. Los cambios observados en la energía contenida en carbohidratos y lípidos, a través de los diferentes periodos de este estudio, pueden estar relacionados con los procesos de anabolismo-catabolismo, ligados a la disponibilidad del alimento fitoplanctónico. Además, con respecto a los lípidos, nuestros resultados sugieren que los cambios observados en los mismos pudieron ser influenciados por la actividad reproductiva.

Introduction

Parallel studies comparing the biochemical composition of mussel specimens originating from suspended culture in the water column and from the rocky shore intertidal zone are notably lacking. Rodhouse et al. (1984) compared the growth, dry weight and reproductive cycle of Mytilus edulis originating from both suspended culture at 5 m depth and from the rocky shore zone (13% air exposure). Under these conditions, the mussels from the suspended culture depended primarily on food of phytoplanktonic origin, and during autumn, their dry weight decreased and they did not show gametogenic development as a consequence of the decrease in phytoplanktonic food. In contrast, during this same autumnal period, the rocky shore mussels utilized a mixture of food dominated by detritus (particulate organic matter), which supplied them with sufficient energy to satisfy basic metabolic requirements as well as gametogenic development.

Other studies have shown that differences in the biochemical composition of mussel populations located in zones with distinct environmental conditions were due to qualitative and/ or quantitative differences in the availability of food of phy-toplanktonic origin (Pérez-Camacho et al., 1995; Fernández-Reiriz et al., 1996; Okumus and Stirling, 1998). With regards to natural populations of mussels of the genus Mytilus spp., annual fluctuations in the different components of the biochemical composition have been related to environmental parameters and the reproductive cycle (Pieters et al., 1979, 1980; Zurburg et al., 1979; De Moreno et al., 1980; Kluytmans et al., 1980; Zandee et al., 1980; Bressan and Marin, 1985; Fernández-Reiriz et al., 1996; Okumus and Stirling, 1998). Accordingly, these studies agreed with the theory established by Bayne (1976), whereby after feeding, a series of metabolic processes come into play, from which the energy obtained from the food could be initially accumulated as reserve tissue and thereafter destined for gametogenesis and/or used in periods of low food availability. This would give as a result a biochemical cycle and, consequently, a reproductive cycle.

An additional aspect that greatly affected the biochemical composition of the intertidal rocky shore mussels is the frequent periods of air exposure, at which times the bivalves were denied a food source. Consequently, the resulting effect would be similar to starvation (Hummel et al., 1989). Bivalves subjected to conditions of nutritive stress have displayed a decrease in glycogen (Bayne, 1973; Gabbott and Bayne, 1973; Riley, 1976). Apart from carbohydrates, specimens of the oyster Crassostrea gigas subjected to food stress over a relatively long period (405 days), showed a decrease of 44% and 23% in proteins and lipids, respectively (Whyte et al., 1990).

More recently, Pérez-Camacho et al. (1995) compared growth of mussel seed obtained from different intertidal zones of the Galician coast and from seed collectors placed on raft culture. This study indicated that seed origin had a strong influence on mussel growth, since seeds from collectors showed greater growth rates than those of the rocky shore. This difference was attributed to some aspects of the initial state of the collector seed, such as initial greater condition index and previous adaptation to culture conditions under permanent immersion.

The present work is part of a larger research that aims to elucidate what causes the differences observed in the performance of both groups of mussel seed. Two studies have been published about the changes that occurred in the different lipid classes (Freites et al., 2002a) and fatty acid profiles (Freites et al. , 2002b) of juvenile populations of subtidal and rocky shore origin of Mytilus galloprovincialis. This study describes the changes that occurred in the biochemical composition (proteins, lipids and carbohydrates) and energetic content of these two seed groups after being transferred to the same habitat (subtidal) in the Arousa Ria, NW Spain.

Material and methods

The experimental period extended from 27 November 1995 to 3 July 1996. Individuals used in this study were taken from the rocky shore zone of the Arousa Ria, Spain, and from collector ropes (subtidal environment) suspended from a mussel raft, installed in the same ria. Both groups of seed were from the previous spring-summer spawning and therefore belonged to the same year class. Additionally, the sampling locations for both populations were 2 km away from each other. At the outset, no significant differences in size and initial mass of the two mussels groups were noted (ANOVA, P > 0.05). The location on the raft, rope density (1.6 kg m^-1) and depth of culture (1.5-5.0 m) were identical for both seed groups.

Environmental variables

Seston and corresponding fractions, TPM (Total Particulate Material) = POM (Particulate Organic Material) + PIM (Particulate Inorganic Material), and particulate volume (mm³) were recorded (6 sub-samples) at the same interval depth of the mussel seeds under study. The seston samples were filtered with previously washed Whatman GF/C filters (3 times with 10 mL distilled water) and dried (muffle 450°C/4 h). The filtered material was washed with a solution of ammonium formate (0.5 M) to eliminate salts and dried (110°C/24 h). The organic component (POM) was determined gravimetrically after sample combustion (muffle 450°C/4 h). The particulate volume was determined with a Coulter Counter Multisizer II. The indicators of nutritional quality Q₁ (POM/TPM), Q₂ (POM/particle volume) and the ratio chl-a/POM were subsequently calculated. The data characterizing the seston phytoplankton component, expressed as concentration of chl-a, temperature (T) and salinity, were provided by the Marine Environment Quality Control Centre at the Ministry of Fisheries, Shell Fisheries and Aquaculture of the Galician Regional Government (Xunta de Galicia). Chlorophyll a was calculated from fluorescence data.

Treatment of samples

For each survey, three subsets of mussels (n = 3), each comprising 30 individuals, were taken at random from both mussel groups, thus making a total of 90 individuals per mussel group. Each subset came from a different rope, for both mussel groups. The soft tissues of the individuals of each sub-sample were separated, freeze-dried at -70°C under a vacuum of 0.018 mbar, and stored at -70°C. Tissues were pulverized using a model 6 Fritsch Pulverisette, and homogenized with water in an ultrasonic vibrator Sonifier 250.

Biochemical composition

Quantification of proteins was carried out following the methodology described by Lowry et al. (1951), after hydrolysis of the sample with NaOH 0.5 N at 30°C for 24 h. Bovine albumin (Sigma) was used as a standard and underwent the same hydrolysis treatment.

Carbohydrates were measured by the phenolsulphur reaction as proposed by Strickland and Parsons (1968), with glucose as a standard. The same method was also used to determine glycogen after precipitation with 100% ethanol.

Lipids were first extracted with chloroform:methanol (1:2), and after centrifugation (3246 G), the precipitate was again extracted with chloroform:methanol (2:1). Both supernatants were then washed with chloroform:methanol:water (8:4:3), as described previously by Fernández-Reiriz et al. (1998). The solvents contain 0.05% butylated hydroxytoluene (BHT). Total lipids were determined gravimetrically by evaporating the solvent of 200 µL of purified extract onto pre-weighed aluminium plates on a slide warmer (60-80°C) (Rouser et al., 1967).

Statistical analysis

Differences in the biochemical components between subtidal and rocky shore mussel seeds were analyzed by a oneway analysis of variance (ANOVA). Relative percentages of biochemical components were previously arcsine transformed (Zar, 1984), and the Bartlett test of homogeneity of variance was applied to the data.

To study the influence of environmental parameters on the variation of the energy equivalents of the different biochemical components, in collector and rocky shore mussel seeds, a multivariate Stepwise regression was performed. In this analysis, the source of mussel seed "origin" factor is attributed to a qualitative factor (dummy), in such a way that the collector mussels are assigned a value of 0 (zero) and the rocky shore mussels a value of 1 (one). The significance level employed for the regression analysis was 95% (Zar, 1984).

Results

Environmental variables

With the advance of winter, a sustained decrease in water temperature was observed until reaching a minimum of 12.5°C (fig. 1a). Thereafter, a sustained increase of temperature was noted during spring, until peaking in June (16.3°C). Chlorophyll a presented relatively low values during the winter (fig. 1b), with minimal concentrations (0.61 ng L^-1); however, chlorophyll a increases shortly thereafter, reaching maximum values during spring (3.71 ng L^-1). With regards to seston, between late November and early February (fig. 1c) a series of fluctuations occurred, with maximum values for TPM, POM and PIM in early January (2.56, 1.00 and 1.29 ng L^-1, respectively). Following these increased concentrations, two new increments in TPM and POM were observed in February (1.34 and 0.57 mg L^-1, respectively) and in April (1.38 and 0.64 mg L^-1, respectively). Similar events to those described for seston also occur in the particulate volume (fig. 1d), with an emphatic peak in April (1.66 mm³). Accordingly, three peaks can be observed in the particulate volume, the first corresponding to the seston maximum at the start of January, and the following two peaks corresponding to those observed in chlorophyll a during spring. Regarding food quality, two clear periods can be discerned in Q2 (POM/particle volume) (fig. 1e). The first interval between late November and mid-February (winter) was characterized by values generally above 0.6, and the second in spring, had values generally below 0.6. These two clearly defined intervals are reflected in the changing chlorophyll a/POM ratio (fig. 1f).

Biochemical composition of mussels of both origins

With regards to percentage organic material in the individual mussels of subtidal and rocky shore origin (table 1), no significant differences were observed (ANOVA, P > 0.05) throughout the experimental period. The relative percentages of carbohydrates, glycogen and lipids were significantly higher in the subtidal mussels (ANOVA, P < 0.05, 0.001 and 0.05, respectively), whereas proteins were significantly higher in the rocky shore mussels (ANOVA, P < 0.01). These differences were maintained for the first 8 days (glycogen and lipids), 15 days (carbohydrates), and 36 days (proteins) after initiation of the experiment. From day 50 to the end of the experiment, no significant differences were observed (ANOVA, P > 0.05) in the relative percentages of any of the aforementioned biochemical components (table 1).

The biochemical composition, expressed in terms of energetic contents (kJ mg^-1), allows equations to be calculated that relate the changes that occur in proteins, carbohydrates, lipids and total organic material (fig. 2a-d) of both groups of mussels throughout the experimental culture. The best fit of protein and carbohydrate energy content (fig. 2a-b) for the mussel groups is a quadratic equation (second degree polynomial), whereas the energy from lipids and total organic material is fitted by a third degree polynomial relationship (fig. 2c-d).

For the detailed analysis of the variability in energy content of the different biochemical components and total organic material, taking into account the data of the environmental variables, the experimental period was divided into three periods: winter, transitional and spring-summer. The same temporal criterion was employed for the multiple regression analysis (Stepwise) described below, with the aim of elucidating the influence of environmental parameters and source of mussel seed (origin) on the evolution of the energy content generated from the various biochemical components.

Winter period (relatively low phytoplanktonic food availability)

This period corresponds to the interval between the initial sampling (day 0) and the 5th sampling (day 36). An increment of the protein energy contribution of 2.52 and 1.78 kJ mg^-1 (21.99% and 14.01%) was observed in both the subtidal and rocky shore mussels, respectively (table 2). In contrast, during this same period, there was a deficit in carbohydrate energy of -1.40 and -1.04 kJ mg^-1 (-42.05% and -40.84%), and in lipid energy of -2.06 and -1.36 kJ mg^-1 (-34.80% and -27.10%) for the subtidal and rocky shore mussels, respectively. In this way, the parallel loss observed in the carbohydrate and lipid energy was greater than the contribution observed from proteins. Consequently, a total energy loss of the organic material was observed of -0.93 and -0.61 kJ mg^-1 (-4.49% and -3.00%) for the subtidal and rocky shore mussels, respectively (table 2).

Transitional period (average phytoplanktonic food availability)

This period corresponds to the interval between the 6th sampling (day 50) and the 10th sampling (day 110, table 2). In contrast to the winter situation, there was a loss of -1.63 and -2.46 kJ mg^-1 (-11.79% and -17.20%) of the energy contributed by the proteins of the subtidal and rocky shore mussels, respectively. On the other hand, a gain of 1.24 and 1.59 kJ mg^-1 (71.13% and 119.65%) was observed in the energy obtained from carbohydrates, and up to 0.64 and 1.55 kJ mg^-1 (13.95% and 34.75%) from the lipids, respectively. Accordingly, the energy storage in these latter compounds is greater in this period than the observed loss in proteins, which results in an increase in the energy storage in the organic material of 0.25 and 0.67 kJ mg^-1 (1.23% and 3.34%), respectively.

Spring-summer period (relatively high phytoplanktonic food availability)

Influence of mussel origin and environmental parameters on energy content evolution

Winter period

The Stepwise multiple regression analysis applied to the winter period, from late November to early January (table 3), showed that the TPM contribution explained the greatest percentage of the variance observed in the energy content of the proteins and carbohydrates (57.2% and 43.7%), whereas chlorophyll a/POM contributed to explain the greatest percentage of lipids and organic material (61.8% and 59.2%, respectively). With the exception of the energy contained within the organic material, the "origin" term increased the explanation of variance of the protein, carbohydrate and lipid energy equivalents up to 78.1%, 85.8% and 79.0%, respectively, with a negative coefficient for the energy content of carbohydrates and lipids. This is evidence of the higher energy content of subtidal mussels.

Transitional period

During the period between mid-January and mid-March, TPM alone explained more than 40% of the variance of the protein, carbohydrate, lipid and organic material energy content of both mussel groups (table 4). Furthermore, chlorophyll a/POM participated in the explanation of the variance in energy content in the lipids and organic material, raising them to 55.9% and 50.5%, respectively. Additionally, the mussel origin continued to participate in the explanation of the variance observed in lipid energy content and subsequently for the organic material during this second period. In both cases the coefficients are positive, thus illustrating the higher energy contents of the rocky shore mussels.

Spring-summer period

Between the end of March and the beginning of July (spring, table 5), TPM was the parameter contributing the greatest percentage explanation of the variability in energy content of the carbohydrates, lipids and organic material (31.8% and 35.0%, respectively). Conversely, temperature attained the greatest percentage explanation of the variance in protein energy equivalents (41.2%). Furthermore, temperature displayed a negative coefficient and is thus evidence of an inverse relationship between this parameter and protein energy. Moreover, chlorophyll a/POM also participated in the explanation of the variance in the energy contained within the proteins, carbohydrates, lipids and organic material in both mussel groups, raising the respective explanations to 79.2%, 78.5%, 55.8% and 55.8% (table 5).

The correlation between the environmental parameters, mussel origin and energy values of proteins, carbohydrates, lipids and organic material described above were all significant (P < 0.05) (tables 3, 4, 5).

The results from the present study show that at the start of the experiment the subtidal and rocky shore mussels presented significant differences in relative percentage (% organic material) of all the integrals of the biochemical composition studied (proteins, carbohydrates, glycogen and lipids). These differences were maintained at least over the first eight days and concur with previous studies, which show that the biochemical composition of the bivalves clearly reflects the conditions of the habitat where they develop (Whyte et al., 1990; Napolitano et al., 1992; Fernández-Reiriz et al., 1996; Okumus and Stirling, 1998).

The rationale for the differences between mussel groups is probably the contrasting conditions of mussel seed development. Frequent periods of air exposure exercise a greater influence on the energy reserves of rocky shore mussels due to the absence of food. Accordingly, a decrease in tissue weight has been observed in mussel specimens subjected to periods of starvation as a result of catabolism of other energy reserves such as lipids and proteins (Hummel et al., 1989). Furthermore, during periods of air exposure, rocky shore mussels do not have access to the dissolved oxygen in seawater, at which times they rely on anaerobic metabolism (glycolysis) as the principal source of energy (De Zwaan and Mathieu, 1992). The upshot is a decrease in glycogen reserves, a phenomenon observed elsewhere in other species of bivalve molluscs affected by air exposure (Gäde, 1975; Bayne et al., 1982; Gabbott, 1983; Hummel et al., 1989).

In view of the fact that no data were obtained on the reproductive cycle of the mussel seed in the present study, variations observed in the biochemical composition cannot be related directly with this aspect. Notwithstanding, some insight can be gained based on three studies carried out in the Galician rias related to the reproductive cycle of this species, namely, a description of the gametogenic cycle and energy reserves by Ferrán et al. (1990), and detailed studies of the gametogenic cycle described by Villalba (1995) and Cáceres-Martínez and Figueras (1998). Hence, the observed increase in energy equivalents of protein from November to January (winter) could be interpreted as a process of active gametogenesis, given that Villalba (1995) and Cáceres-Martínez and Figueras (1998) observed a high percentage of specimens in this stage of the reproductive cycle in the Arousa and Vigo Rias. In contrast, the decrease in energy contribution of carbohydrate and lipid reserves would probably be related to the low availability of phytoplanktonic food from November to January (see fig. 1b). The latter agrees with the results of Zandee et al. (1980) and Okumus and Stirling (1998), who also noted a decrease in carbohydrate and lipid content during winter in the mussel M. edulis.

The rise in energy generated from carbohydrates and lipids over the transitional period between winter and spring is probably due to increasing availability of phytoplanktonic food (see fig. 1b). Furthermore, in view of the fact that Villalba (1995) and Cáceres-Martínez and Figueras (1998) observed a high percentage of specimens with their gametes maturing between January and April, a parallel process of accumulation of lipid reserves in the gametes cannot be ruled out. Accumulation of lipid reserves in the mussel M. galloprovincialis has been recorded previously by Lubet et al. (1983, 1986) during oocycte maturation.

From April onwards a simultaneous decrease in energy content in protein and lipid was observed in both mussel groups. These decreases in both components suggest the spawning process; in the same period of the year, Villalba (1995) and Cáceres-Martínez and Figueras (1998) observed an increase in the percentage of specimens of M. galloprovincialis in a post-spawning state in the Arousa and Vigo Rias, respectively. Also, considerable decreases in lipid content resulting from spawning were also observed in natural populations of the mussels Mytilus platensis (De Moreno et al., 1980), M. edulis (Zandee et al., 1980) and M. galloprovincialis (Bressan and Marin, 1985; Da Ros et al., 1985; Ferrán et al., 1990); in the oysters Crassostrea virginica (Chu et al., 1990) and C. gigas (Berthelin et al., 2000); in the pectinids Pecten maximus (Besnard et al., 1989; Pazos et al., 1997) and Argopecten purpuratus (Martínez, 1991); and in the clam Glycymeris glycymeris (Galap et al. , 1997).

Furthermore, a decrease in protein following spawning has been observed in the bivalves M. edulis (Zandee et al., 1980), M. galloprovincialis (Bressan and Marin, 1985), Argopecten irradians irradians (Epp et al., 1988), Ostrea edulis (Ruiz et al., 1992) and Crassostrea iridescens (Páez-Osuna et al., 1993).

With respect to the influence of the various environmental parameters and seed origin on the trends of the protein, carbohydrate, lipid and organic material energy values, several interesting features can be highlighted. From the start of the experiment to the beginning of January (days 1-37), the multiple regression analysis showed that seed origin had a significant influence on the variance observed in the energy content of the proteins, carbohydrates and lipids. It is worth emphasizing that the energy contribution of carbohydrates and lipids was lower in the rocky shore mussels (negative coefficient). This agrees with studies in which a decrease of carbohydrate and/or lipid reserves in rocky shore mussels subject to frequent periods of air exposure (Bayne et al., 1982; Gabbott, 1983; De Zwaan and Mathieu, 1992) was observed. Moreover, the mussel origin term continued to participate significantly in the explanation of the variance of the lipid energy content during the transitional period (days 50-110). In this case, however, the coefficient was positive, thus demonstrating that mussels of rocky shore origin were able to accumulate more lipid energy than subtidal mussels.

This regression model changes after spring because the mussel origin did not participate in the explanation of the variance of any of the different integrals of the biochemical compositions. However, at this point, TPM and chlorophyll a/ POM together explained percentages of variance greater than 38.0%, 38.5%, 55.8% and 55.8% in the protein, carbohydrate, lipid and organic material energy contribution, respectively. The positive coefficients suggest that energy acquisition in this period depends on these environmental parameters. Similar increments in total energy as a result of the high availability of food of phytoplanktonic origin have been observed in the oyster Ostrea puelchana (Fernández and de Vido, 1987) and O. edulis (Ruiz et al., 1992).

The authors would like to thank Ana Ayala, Beatriz González, Lourdes Nieto and Sonia Villar for their technical assistance in the biochemical analyses; Juan Maneiro (Centro de Control de la Calidad del Medio Marino de la Consejería de Pesca, Marisqueo y Acuicultura, Xunta de Galicia) for the data of the environmental variables; and the crew of the José María Navaz from the Instituto Español de Oceanografía. This study was financed by the project CICYT REN2001-0501/MAR. Luis Freites was supported by a grant from the Consejo Nacional de Investigaciones Científicas y Tecnológicas (CONICIT), Venezuela.

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