© 2005 International Council for the Exploration of the Sea
Energy storage and utilization in relation to the reproductive cycle in the razor clam Ensis arcuatus (Jeffreys, 1865)
a Centro de Investigacións Mariñas, Consellería de Pesca e Asuntos Marítimos Xunta de Galicia, Aptdo n° 13, 36620 Vilanova de Arousa, Pontevedra, Spain
b Facultade de Ciencias do Mar, Universidade de Vigo Campus Lagoas-Marcosende 36200 Vigo, Spain
*Correspondence to S. Darriba: tel: +34 986500155; fax: +34 986506788. e-mail: sdarriba{at}cimacoron.org.
Seasonal changes in condition indices and biochemical components of the digestive gland, anterior adductor muscle, foot, and gonad of Ensis arcuatus (Jeffreys, 1865) were analysed from February 1998 to June 1999 in relation to environmental conditions and reproductive events. During summer, E. arcuatus accumulated lipids, particularly triacylglycerols, in the digestive gland and glycogen in the anterior adductor muscle and foot while the gonad was in sexual rest. Phytoplankton blooms caused by the upwelling of cold waters, rich in nutrients, from offshore are responsible for the high availability of food during reserve storage. In autumn, when gametogenesis started, oceanographic conditions changed to a situation with low temperature throughout the water column because of the vertical mixing, and food became scarce. At that point in time, the energy requirements for basal metabolism and the reproductive process were provided by the mobilization of triacylglycerols and glycogen stored in the digestive gland and muscle tissues, respectively. The pattern exhibited by E. arcuatus based on the accumulation of reserves in summer and the subsequent mobilization during gonadal development seems to follow a conservative pattern.
Keywords: carbohydrates, chlorophyll, condition indices, Ensis arcuatus, gametogenesis, temperature, total proteins, triacylglycerols
Received 5 February 2004; accepted 20 February 2005.
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Introduction |
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Seasonal variations in the metabolic activities of marine bivalves reflect the complex interactions between the available food, environmental conditions, and reproductive activity. This is demonstrated in several papers on different species of the families Mytilidae (Waldock and Holland, 1979; Emmett et al., 1987; Jaramillo and Navarro, 1995), Ostreidae (Mann, 1979; Fernández-Castro and De Vido-De Mattio, 1987; Ruiz, 1992; Ruiz et al., 1992a, b; Rosique et al., 1995), Cardiidae (Newell and Bayne, 1980; Navarro et al., 1989), Veneridae (Adachi, 1979; Beninger and Lucas, 1984), Pectinidae (Taylor and Venn, 1979; Barber and Blake, 1981; Lubet et al., 1987a, b; Epp et al., 1988; Pazos, 1993; Román et al., 1996; Pazos et al., 1996, 1997), and Arcidae (Galap et al., 1997).
The energy required to carry out the gametogenic process can be covered by using directly ingested food or the previous storage of reserves in the gonad or other tissues (Ansell, 1974; Gabbott, 1975; Barber and Blake, 1983; Pazos et al., 1997). Reproduction, on the other hand, is influenced by environmental conditions (Barber and Blake, 1981; MacDonald and Thompson, 1985) and by genetic characteristics (Barber and Blake, 1991) that determine differences between species and between populations of the same species.
In marine invertebrates, the energy storage and gonad development cycles may occur simultaneously or they may be clearly separated (Bayne, 1976). The relationship between the two cycles defines the reproductive pattern of the species. So, in a conservative pattern, gametogenesis takes place at the expense of energy stored in some body tissues, while in an opportunistic pattern gametogenesis occurs when the availability of food in the water is plentiful enough to support the energy required by the process.
Ensis arcuatus is the most important species of Solenidae in Spain and its commercial value has increased recently. In contrast to other bivalve molluscs, there are only few publications on the Solenidae family despite the economic interest in this species. The objective of the work was to study energy storage in different body tissues of E. arcuatus, its relation to the gametogenic cycle, and to describe the reproductive pattern of the species.
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Material and methods |
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Measurements of body parts and histological analysis
Twenty adult specimens of Ensis arcuatus were collected, approximately every 2 weeks, from the natural bed of the Cíes Islands (Ría de Vigo – Northwest Spain) (Figure 1) from February 1998 to June 1999. Samples were taken by professional "apnea" divers who harvest this resource on a daily basis. After collection, the samples were transported to the laboratory covered with damp cloths and placed in a portable fridge to be processed after 24 h.
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Each specimen was measured to record the following parameters: total length, total weight, shell dry weight, anterior adductor muscle fresh weight, foot fresh weight, and gonad fresh weight.
Dissection of the tissues was carried out in vivo, each tissue being placed on a filter paper to drain the excess of water, then weighed (fresh weight). Next, they were immediately frozen in liquid nitrogen and stored at –80°C until analysis. The shells were weighed after 3 h at 60°C.
Condition indices of each dissected tissue were calculated using the formula tissue fresh weight/shell dry weight.
Each specimen was examined histologically to determine sex and the gametogenic stage according to the scale defined for E. arcuatus by Darriba et al. (2004), based on seven stages: (0) sexual rest, (I) start of gametogenesis, (II) advanced gametogenesis, (IIIA) ripe, (IIIB) start of spawning, (IIIC) restoration, and (IV) exhaustion.
Biochemical analyses
For each sample, we pooled tissues of specimens of the same sex and gametogenic stage. The use of pooled tissue of many individuals to determine the average biochemical composition was recommended by Giese (1966) and Giese et al. (1967).
Each pool obtained was homogenized, at 0–4°C, in a buffer (40 mM Tris–HCl buffer, pH 7, 0.12 M KCl) with a 1:2 ratio (weight:volume) for the gonad and digestive gland and 1:4 ratio for muscle tissues (anterior adductor muscle and foot). Different homogenizing techniques were used: soft tissues (gonad and digestive gland) were homogenized at 2000–2500 rpm with a Tri-R homogenizer (Tri-R Stir-R model 43), and hard tissues (anterior adductor muscle and foot) were homogenized at 15000 rpm with a Tempest IQ squared homogenizer (Virtix). The homogenate obtained was frozen at –80°C in 1 ml aliquots for the biochemical analyses of total proteins, total lipids, triacylglycerols, glycogen, and free glucose.
Total proteins were determined according to Lowry et al. (1951). The samples had been previously deproteinized by dilution in 12% perchloric acid (PCA), at a 1:4.5 ratio (w:v) for soft tissues and at a 1:7.5 ratio for hard tissues. Proteins were collected in the precipitate after centrifugation at 7800 rpm, and resuspended with 10 ml of 1 N NaOH in a bath at 40°C for 1 h, then diluted with distilled water for the determination.
Lipid extraction was done according to the method of Folch et al. (1957), as modified by Beninger and Lucas (1984). The quantification of total lipids was determined using spectrophotometric methods in relation to Triestearine, after acid hydrolysis, according to Marsh and Weinstein (1966).
For the quantification of triacylglycerols the method of Wahlefeld (1974) was followed and consisted of the enzymatic hydrolysis of triacylglycerols with the subsequent determination of liberated glycerol by colorimetry (kit Triglycerides GPO-PAP 701912 – Boehringer Mannheim).
Carbohydrates (glycogen and free glucose) were analysed by the method of Keppler and Decker (1984), modified by Crespo (1989), after the deproteinization of the sample by precipitation with PCA as described earlier. The method was based on enzymatic hydrolysis of glycogen with the amyloglucosidase enzyme. Next, the glucose was oxidized by glucose-oxidase to gluconate and hydrogen peroxide, which reacts with phenol to produce quinonimine, and which is detectable by spectrophotometry (kit glucose GOD-PAP/Trinder – Spinreact).
All determinations were done in triplicate, applying the average value to the graphic representations.
Environmental parameters
The Centro de Control do Medio Mariño (CCMM) recorded temperature and chlorophyll a concentration of the water column at a point located in the Ría de Vigo (42°13'75''N 8°52'20''W) on a weekly basis. Values referred to depth ranges of 0–5, 5–10, and 10–15 m. Temperature was measured using a CTD temperature sensor and chlorophyll by spectrofluorometry.
Statistical analysis
Statistical analyses were performed using the statistical package SPSS for Windows, version 9.0.1, applying the Mann–Whitney U-test.
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Results |
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Environmental parameters
Figure 2a shows temperature data in three depth ranges during the sampling period. From January to May 1998 temperature was similar at all depths, ranging between 14°C and 15°C, which would indicate a vertical mixing of the water column. In May the water was colder in the 10–15 m range than at the surface. This situation re-occurred in July and August after a warming of the water column in the first half of June. The surface temperature was > 15°C after the middle of May and the highest temperatures were recorded at the end of June and beginning of September. From the middle of October onwards, vertical mixing re-occurred and the temperature of the water column decreased progressively, reaching values < 13°C at the end of the year. During the first 4 months of 1999 the vertical mixing of water continued, and from May onwards, the temperature was higher on the surface, as in the previous year.
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Figure 2b shows the concentration of chlorophyll a in the water column during the same period. From January to the beginning of May 1998, concentrations of chlorophyll a were very low, except in February. From May to the beginning of November, the highest values of the year alternated with low, values. From November 1998 to May 1999 values were very low, and from May 1999 onwards the highest values were obtained (maximum values for the 2 years), and these alternated with low values.
Condition indices
Figure 3 shows the seasonal evolution of the condition indices (left) and the average of the condition indices per gametogenic stage (right). A similar pattern can be observed between sexes.
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The gonadal condition index (GCI; Figure 3a) showed minimum values, close to zero, from July to November 1998 (stages 0 and I), increasing to maximum values (stages IIIA and IIIB) from February to June 1999. The first half of the 2 years studied showed consecutive maxima interspersed with lower values because of drops in the index (stage IIIC).
In 1998 the digestive gland condition index (DCI; Figure 3b) had two maxima – not very high – in March and April. From May to July the variation in the DCI was not statistically significant (p > 0.05; Mann–Whitney U), but it reached its highest values in summer. During autumn the DCI declined gradually to a minimum level reached at the beginning of 1999. The first months of 1999 showed a similar pattern as in 1998. The highest DCI values, with statistical significance, occurred when the population was in sexual rest (stage 0), and the lowest values when the population was mature and at the stage of gamete release (stages IIIA and IIIB, respectively).
The anterior adductor muscle condition index (MCI; Figure 3c) and the foot condition index (FCI; Figure 3d) showed an annual trend, with the lowest values during the first half of 1998 and 1999, then a subsequent rise to highest values in the second half of each year. Compared with the gametogenic stages there was a significant drop in the MCI and FCI when the population was mature (stage IIIA).
On comparing the environmental parameters analysed (Figure 2), we observed that, in the period of minimum GCI values and maximum values of the other indices (July–November; Figure 3), the concentration of chlorophyll a was high and the water column had high temperatures on the surface and low temperatures at depth (Figure 2).
Biochemical contents
Figure 4 shows the gonadal biochemical contents of those samples that had a sufficient amount of tissue to be subject to analysis. Total proteins were the most important component in quantitative terms (Figure 4c), varying between 75 and 150 mg g–1, with no statistically significant differences between sexes (p > 0.05, Mann–Whitney U-test). Lipid contents were significantly higher in females, in both total lipids and triacylglycerols (Figure 4b) (p = 0.000, Mann–Whitney U-test).
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Figure 5 shows the biochemical contents of the digestive gland during the sampling period and in relation to the gametogenic stages. Lipids were the most important component in quantitative terms (Figure 5a). During the first half of 1998 and 1999, the total lipid concentration was less than 250 mg g–1, but it was higher from July 1998 to December 1998. Triacylglycerols (Figure 5b) represented 73.7% of total lipids in males and 69.7% in females. Thus, they were the most important lipids in the digestive gland in terms of quantity, showing statistical significance between the sexes (p < 0.005, Mann–Whitney U-test). Total proteins (Figure 5c) were the second most important biochemical component in terms of quantity, showing no seasonal pattern. Glycogen and glucose were minor components. As regards glycogen, there were two annual minima (February and May 1998, and February 1999) and a period of maximum concentration in summer. In relation to the gametogenic stages, the decrease in lipids and glycogen from the early stages to ripeness (IIIA) was noteworthy (Figure 5a, b, d).
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The anterior adductor muscle and the foot were composed mainly of proteins (Figures 6c, 7c). Lipids were found in very low concentrations, less than in the other tissues (Figures 6a, 7a), principally the triacylglycerols (Figures 6b, 7b). Glycogen concentrations were higher than in the other tissues and the seasonal pattern was similar to that of the digestive gland, with two annual minima and a peak in summer (Figures 6d, 7d). As far as the gametogenic stages were concerned, there was a major decrease from the sexual rest (stage 0) to the mature stage (stage IIIA).
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Compared with the environmental parameters analysed, we observed that, in the period of high lipid concentrations, particularly triacylglycerols in the digestive gland (Figure 5a, b) and glycogen in the anterior adductor muscle (Figure 6d) and the foot (Figure 7d), the concentration of chlorophyll a was high, with high surface temperatures and lower temperatures at depth (Figure 2).
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Discussion |
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By combining the results of the reproductive cycle study of Ensis arcuatus, the biomass and the biochemical contents of different body parts, and environmental conditions, we were able to evaluate the reproductive pattern. Analyses of different body parts instead of the whole specimen are very important in this context (Giese, 1969).
The study of the reproductive cycle was based on the gonadal condition index (GCI), which proved to be highly efficient in distinguishing between the different stages of gametogenic development. The cycle of E. arcuatus was annual and comprised a rest phase during summer, followed by a rapid and intense process of gametogenesis beginning at the start of autumn, leading to successive spawning and restoration in winter and spring (Darriba et al., 2004).
Condition indices of somatic tissues showed an inverse relationship with those of the gonad owing to the accumulation of nutrients in somatic tissues in summer while the gonad was at sexual rest. The high concentrations of chlorophyll a in summer resulted in a great availability of food, allowing the accumulation of nutrients owing to the excess of food ingested, as observed in Argopecten irradians concentricus (Barber and Blake, 1981).
Biochemical analysis of the somatic tissues revealed that the increment in summer biomass was due to the accumulation of lipids, particularly triacylglycerols (reserve lipids), in the digestive gland and glycogen in the anterior adductor muscle and the foot.
The concentrations of chlorophyll a in the water column were directly related to the temperature. The upwelling of cold water rich in nutrients from offshore in spring and summer and the level of light at the same time gave rise to the proliferation of phytoplankton blooms. Consequently, the environmental conditions affected the reserve cycle.
The absence of reserve cells in the gonad of E. arcuatus (Darriba et al., 2004) suggested that mobilization of nutrients from other tissues was necessary to provide energy for gametogenesis, molecules for multiple cell divisions, and reserves for gametes, especially oocytes.
The female gonad had a higher concentration of lipids, triacylglycerols in particular, probably owing to the accumulation of reserves in the oocytes and the absence of these reserves in the spermatocytes. In fact, total lipids and organic matter are considered good indicators of oocyte quality and larva viability (Massapina et al., 1999).
The high concentration of total proteins in gonads of both sexes may be explained by the presence of structural proteins in gametes of both sexes, and also reserve proteins in the female oocytes. These results were similar to the descriptions by other authors, who considered proteins to be the principal component of bivalve oocytes, along with lipids (Holland, 1978; Gabbott, 1983).
The digestive gland has been known as a site of nutrient storage in bivalves for several decades (Nakazima, 1956; Owen, 1966; Bayne et al., 1976). An inverse relationship between the size of the digestive gland and the development of the gonad was found in some species, suggesting the transfer of nutrients from the digestive gland to the gonad (Giese, 1969). This was confirmed using 14C as a marker (Sastry and Blake, 1971; Voogt, 1972; Vassallo, 1973).
In E. arcuatus, the digestive gland appears to act as an important reserve storage site for lipids, which were transferred to the gonad during gamete development, as the triacylglycerol concentration decreased while gonadal biomass increased before spawning. Furthermore, the relationship between digestive gland biochemical content and the gametogenic stages clearly showed a decrease in lipids from the sexual rest to the ripe stage.
A similar situation was observed with glycogen in the analysed muscle tissues (anterior adductor muscle and foot), as well as in the digestive gland. When the availability of food was high in summer, glycogen was stored, but it was mobilized when gamete development started in autumn. These glycogen reserves may allow the synthesis of lipids in the gametes, as reported by several authors (Voogt, 1972; Vassallo, 1973; Gabbott, 1975; Waldock and Holland, 1979; Beninger and Lucas, 1984; Lubet, 1996), in addition to maintaining the energy requirements of gametogenesis.
Storage of lipids in the digestive gland and glycogen in the adductor muscle, during periods when food availability exceeds the metabolic demand, and which are subsequently utilized in energy-requiring processes including gametogenesis, has been described for several different scallops (Taylor and Venn, 1979; Barber and Blake, 1981; Lubet et al., 1987b; Román et al., 1996, Pazos et al., 1997). However, in E. arcuatus the foot also appears to act as a storage site for glycogen, with a high quantity of reserves, because of its size.
Gametogenesis is a process that requires energy. In females there is a demand for lipids, especially triacylglycerols, evidenced by the numerous lipid droplets in the yolk and the low quantities of glycogen. In spermatogenesis, there is a demand for energy required by the multiplication of cells and a need for structural lipids for the membranes.
In conclusion, storage of reserves in E. arcuatus took place during summer when the upwelling phenomenon occurred, leading to phytoplankton blooms and consequently high food availability. This process produced a surplus of ingested nutrients, which were accumulated as reserve lipids in the digestive gland and as glycogen in the anterior adductor muscle and the foot. In autumn, when gametogenesis started, oceanographic conditions changed and food became scarce, so gonadal development occurred at the expense of the reserves stored in the somatic tissues, as discussed earlier. Lastly, the reproduction of E. arcuatus may be considered to follow a conservative pattern.
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Acknowledgements |
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This research was supported by the Junta Asesora Nacional de Cultivos Marinos (JACUMAR). We thank the Centro de Control do Medio Mariño for providing temperature and chlorophyll a data.
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References |
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