Energy Storage and Utilization in Relation to the Reproductive Cycle in the Razor Clam Ensis Arcuatus (Jeﬀreys, 1865)

ICES Journal of Marine Science, 62: 886e896 (2005) doi:10.1016/j.icesjms.2005.02.010

Energy storage and utilization in relation to the reproductive cycle in the razor clam Ensis arcuatus (Jeﬀreys, 1865)

Susana Darriba, Fuencisla San Juan, and Alejandro Guerra Downloaded from https://academic.oup.com/icesjms/article/62/5/886/856445 by guest on 29 September 2021

Darriba, S., San Juan, F., and Guerra, A. 2005. Energy storage and utilization in relation to the reproductive cycle in the razor clam Ensis arcuatus (Jeﬀreys, 1865). e ICES Journal of Marine Science, 62: 886e896.

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. Ó 2005 International Council for the Exploration of the Sea. Published by Elsevier Ltd. All rights reserved. Keywords: carbohydrates, chlorophyll, condition indices, Ensis arcuatus, gametogenesis, temperature, total proteins, triacylglycerols. Received 5 February 2004; accepted 20 February 2005. S. Darriba, and A. Guerra: Centro de Investigaciońs Marinãs, Consellerıá de Pesca e Asuntos Marı´timos, Xunta de Galicia, Aptdo n( 13, 36620 Vilanova de Arousa, Pontevedra, Spain. F. San Juan: Facultade de Ciencias do Mar, Universidade de Vigo, Campus Lagoas- Marcosende 36200 Vigo, Spain. Correspondence to S. Darriba: tel:C34 986500155; fax: C34 986506788; e-mail: [email protected].

Introduction (Ansell, 1974; Gabbott, 1975; Barber and Blake, 1983; Pazos et al., 1997). Reproduction, on the other hand, is Seasonal variations in the metabolic activities of marine influenced by environmental conditions (Barber and Blake, bivalves reflect the complex interactions between the 1981; MacDonald and Thompson, 1985) and by genetic available food, environmental conditions, and reproductive characteristics (Barber and Blake, 1991) that determine activity. This is demonstrated in several papers on different differences between species and between populations of the species of the families Mytilidae (Waldock and Holland, same species. 1979; Emmett et al., 1987; Jaramillo and Navarro, 1995), In marine invertebrates, the energy storage and gonad Ostreidae (Mann, 1979; Fernańdez-Castro and De Vido-De development cycles may occur simultaneously or they may Mattio, 1987; Ruiz, 1992; Ruiz et al., 1992a, b; Rosique be clearly separated (Bayne, 1976). The relationship between et al., 1995), Cardiidae (Newell and Bayne, 1980; Navarro the two cycles defines the reproductive pattern of the species. et al., 1989), Veneridae (Adachi, 1979; Beninger and So, in a conservative pattern, gametogenesis takes place at Lucas, 1984), Pectinidae (Taylor and Venn, 1979; Barber the expense of energy stored in some body tissues, while in an and Blake, 1981; Lubet et al., 1987a, b; Epp et al., 1988; opportunistic pattern gametogenesis occurs when the avail- Pazos, 1993; Romań et al., 1996; Pazos et al., 1996, 1997), ability of food in the water is plentiful enough to support the and Arcidae (Galap et al., 1997). energy required by the process. The energy required to carry out the gametogenic process Ensis arcuatus is the most important species of Solenidae can be covered by using directly ingested food or the in Spain and its commercial value has increased recently. In previous storage of reserves in the gonad or other tissues contrast to other bivalve molluscs, there are only few

1054-3139/$30.00 Ó 2005 International Council for the Exploration of the Sea. Published by Elsevier Ltd. All rights reserved. Energy storage and utilization in Ensis arcuatus 887 publications on the Solenidae family despite the economic the biochemical analyses of total proteins, total lipids, interest in this species. The objective of the work was to triacylglycerols, glycogen, and free glucose. study energy storage in different body tissues of Total proteins were determined according to Lowry et al. E. arcuatus, its relation to the gametogenic cycle, and to (1951). The samples had been previously deproteinized by describe the reproductive pattern of the species. 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 Material and methods a bath at 40(C for 1 h, then diluted with distilled water for Measurements of body parts the determination. Downloaded from https://academic.oup.com/icesjms/article/62/5/886/856445 by guest on 29 September 2021 and histological analysis Lipid extraction was done according to the method of Folch et al. (1957), as modified by Beninger and Lucas Twenty adult specimens of Ensis arcuatus were collected, (1984). The quantification of total lipids was determined approximately every 2 weeks, from the natural bed of the using spectrophotometric methods in relation to Tri- Cıés Islands (Rıá de Vigo e Northwest Spain) (Figure 1) estearine, after acid hydrolysis, according to Marsh and from February 1998 to June 1999. Samples were taken by Weinstein (1966). professional ‘‘apnea’’ divers who harvest this resource on For the quantification of triacylglycerols the method of a daily basis. After collection, the samples were transported Wahlefeld (1974) was followed and consisted of the to the laboratory covered with damp cloths and placed in enzymatic hydrolysis of triacylglycerols with the subsequent a portable fridge to be processed after 24 h. determination of liberated glycerol by colorimetry (kit Each specimen was measured to record the following Triglycerides GPO-PAP 701912 e Boehringer Mannheim). parameters: total length, total weight, shell dry weight, Carbohydrates (glycogen and free glucose) were ana- anterior adductor muscle fresh weight, foot fresh weight, lysed by the method of Keppler and Decker (1984), and gonad fresh weight. modified by Crespo (1989), after the deproteinization of the Dissection of the tissues was carried out in vivo, each sample by precipitation with PCA as described earlier. The tissue being placed on a filter paper to drain the excess method was based on enzymatic hydrolysis of glycogen of water, then weighed (fresh weight). Next, they were with the amyloglucosidase enzyme. Next, the glucose was immediately frozen in liquid nitrogen and stored at ÿ80(C oxidized by glucose-oxidase to gluconate and hydrogen until analysis. The shells were weighed after 3 h at 60(C. peroxide, which reacts with phenol to produce quinonimine, Condition indices of each dissected tissue were calculated and which is detectable by spectrophotometry (kit glucose using the formula tissue fresh weight/shell dry weight. GOD-PAP/Trinder e Spinreact). Each specimen was examined histologically to determine All determinations were done in triplicate, applying the sex and the gametogenic stage according to the scale average value to the graphic representations. 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 Environmental parameters spawning, (IIIC) restoration, and (IV) exhaustion. The Centro de Control do Medio Marinõ (CCMM) recorded temperature and chlorophyll a concentration of the water column at a point located in the Rıá de Vigo Biochemical analyses (42(13#75$N8(52#20$W) on a weekly basis. Values e e e For each sample, we pooled tissues of specimens of the referred to depth ranges of 0 5, 5 10, and 10 15 m. same sex and gametogenic stage. The use of pooled tissue Temperature was measured using a CTD temperature of many individuals to determine the average biochemical sensor and chlorophyll by spectrofluorometry. composition was recommended by Giese (1966) and Giese et al. (1967). Statistical analysis e ( Each pool obtained was homogenized, at 0 4 C, in Statistical analyses were performed using the statistical e a buffer (40 mM Tris HCl buffer, pH 7, 0.12 M KCl) with package SPSS for Windows, version 9.0.1, applying the a 1:2 ratio (weight:volume) for the gonad and digestive ManneWhitney U-test. 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 Results homogenized at 2000e2500 rpm with a Tri-R homogenizer (Tri-R Stir-R model 43), and hard tissues (anterior adductor Environmental parameters muscle and foot) were homogenized at 15000 rpm with Figure 2a shows temperature data in three depth ranges a Tempest IQ squared homogenizer (Virtix). The homo- during the sampling period. From January to May 1998 genate obtained was frozen at ÿ80(C in 1 ml aliquots for temperature was similar at all depths, ranging between 888 S. Darriba et al. 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Figure 1. Location of the natural bed of Ensis arcuatus. The left arrow signifies the Cıés Islands at the mouth of the Rıá de Vigo.

14(C and 15(C, which would indicate a vertical mixing of onwards, vertical mixing re-occurred and the temperature the water column. In May the water was colder in the of the water column decreased progressively, reaching 10e15 m range than at the surface. This situation re- values ! 13(C at the end of the year. During the ﬁrst 4 occurred in July and August after a warming of the water months of 1999 the vertical mixing of water continued, and column in the ﬁrst half of June. The surface temperature from May onwards, the temperature was higher on the was O 15(C after the middle of May and the highest surface, as in the previous year. temperatures were recorded at the end of June and Figure 2b shows the concentration of chlorophyll a in the beginning of September. From the middle of October water column during the same period. From January to the Energy storage and utilization in Ensis arcuatus 889

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). Downloaded from https://academic.oup.com/icesjms/article/62/5/886/856445 by guest on 29 September 2021 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 (Julye 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 Figure 2. Environmental parameters. Variation of temperature (a) to analysis. Total proteins were the most important and chlorophyll a (b) from January 1998 to September 1999 near component in quantitative terms (Figure 4c), varying the natural bed of Ensis arcuatus in three depth ranges (0e5, 5e10, between 75 and 150 mg gÿ1, with no statistically significant and 10e15 m). differences between sexes (p O 0.05, ManneWhitney U-test). Lipid contents were significantly higher in females, beginning of May 1998, concentrations of chlorophyll a in both total lipids and triacylglycerols (Figure 4b) Z e were very low, except in February. From May to the (p 0.000, Mann Whitney U-test). beginning of November, the highest values of the year Figure 5 shows the biochemical contents of the digestive alternated with low, values. From November 1998 to May gland during the sampling period and in relation to the 1999 values were very low, and from May 1999 onwards gametogenic stages. Lipids were the most important the highest values were obtained (maximum values for the component in quantitative terms (Figure 5a). During the first 2 years), and these alternated with low values. half of 1998 and 1999, the total lipid concentration was less than 250 mg gÿ1, but it was higher from July 1998 to December 1998. Triacylglycerols (Figure 5b) represented Condition indices 73.7% of total lipids in males and 69.7% in females. Thus, Figure 3 shows the seasonal evolution of the condition they were the most important lipids in the digestive gland in indices (left) and the average of the condition indices per terms of quantity, showing statistical significance between gametogenic stage (right). A similar pattern can be the sexes (p ! 0.005, ManneWhitney U-test). Total proteins observed between sexes. (Figure 5c) were the second most important biochemical The gonadal condition index (GCI; Figure 3a) showed component in terms of quantity, showing no seasonal pattern. minimum values, close to zero, from July to November Glycogen and glucose were minor components. As regards 1998 (stages 0 and I), increasing to maximum values glycogen, there were two annual minima (February and May (stages IIIA and IIIB) from February to June 1999. The first 1998, and February 1999) and a period of maximum half of the 2 years studied showed consecutive maxima concentration in summer. In relation to the gametogenic interspersed with lower values because of drops in the stages, the decrease in lipids and glycogen from the early index (stage IIIC). stages to ripeness (IIIA) was noteworthy (Figure 5a, b, d). In 1998 the digestive gland condition index (DCI; The anterior adductor muscle and the foot were Figure 3b) had two maxima e not very high e in March composed mainly of proteins (Figures 6c, 7c). Lipids were and April. From May to July the variation in the DCI was found in very low concentrations, less than in the other not statistically significant (p O 0.05; ManneWhitney U), tissues (Figures 6a, 7a), principally the triacylglycerols but it reached its highest values in summer. During (Figures 6b, 7b). Glycogen concentrations were higher than autumn the DCI declined gradually to a minimum level in the other tissues and the seasonal pattern was similar to reached at the beginning of 1999. The first months of 1999 that of the digestive gland, with two annual minima and showed a similar pattern as in 1998. The highest DCI a peak in summer (Figures 6d, 7d). As far as the 890 S. Darriba et al. Downloaded from https://academic.oup.com/icesjms/article/62/5/886/856445 by guest on 29 September 2021

Figure 3. Condition indices. Left: seasonal evolution of the condition indices (GCI: gonadal condition index; DCI: digestive gland condition index; MCI: anterior adductor muscle condition index; FCI: foot condition index) from January 1998 to June 1999. Right: Average variation of the condition indices per gametogenic stage. gametogenic stages were concerned, there was a major chlorophyll a was high, with high surface temperatures decrease from the sexual rest (stage 0) to the mature stage and lower temperatures at depth (Figure 2). (stage IIIA). Compared with the environmental parameters analysed, we observed that, in the period of high lipid concentrations, Discussion particularly triacylglycerols in the digestive gland (Figure 5a, b) and glycogen in the anterior adductor muscle (Figure By combining the results of the reproductive cycle study of 6d) and the foot (Figure 7d), the concentration of Ensis arcuatus, the biomass and the biochemical contents Energy storage and utilization in Ensis arcuatus 891

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 Downloaded from https://academic.oup.com/icesjms/article/62/5/886/856445 by guest on 29 September 2021 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 Figure 4. Gonadal biochemical contents. Evolution of total lipids, between the size of the digestive gland and the development triacylglycerols, total proteins, glycogen, and glucose in males and females in the period sampled. of the gonad was found in some species, suggesting the transfer of nutrients from the digestive gland to the gonad 892 S. Darriba et al. Downloaded from https://academic.oup.com/icesjms/article/62/5/886/856445 by guest on 29 September 2021

Figure 5. Digestive gland biochemical contents. Left: Evolution of total lipids, triacylglycerols, total proteins, glycogen, and glucose in males and females in the period sampled. Right: Average variations of the biochemical components per gametogenic stage. Energy storage and utilization in Ensis arcuatus 893 Downloaded from https://academic.oup.com/icesjms/article/62/5/886/856445 by guest on 29 September 2021

Figure 6. Anterior adductor muscle biochemical contents. Left: Evolution of total lipids, triacylglycerols, total proteins, glycogen, and glucose in males and females in the period sampled. Right: Average variations of the biochemical components per gametogenic stage. 894 S. Darriba et al. Downloaded from https://academic.oup.com/icesjms/article/62/5/886/856445 by guest on 29 September 2021

Figure 7. Foot biochemical contents. Left: Evolution of total lipids, triacylglycerols, total proteins, glycogen, and glucose in males and females in the period sampled. Right: Average variations of the biochemical components per gametogenic stage. Energy storage and utilization in Ensis arcuatus 895

(Giese, 1969). This was conﬁrmed using 14C as a marker References (Sastry and Blake, 1971; Voogt, 1972; Vassallo, 1973). In E. arcuatus, the digestive gland appears to act as an Adachi, K. 1979. Seasonal changes of the protein level in the important reserve storage site for lipids, which were adductor muscle of clam, Tapes philippinarum (Adams and Reeve) with reference to the reproductive seasons. Comparative transferred to the gonad during gamete development, as Biochemistry and Physiology, 64: 85e89. the triacylglycerol concentration decreased while gonadal Ansell, A. D. 1974. Seasonal changes in biochemical composition biomass increased before spawning. Furthermore, the of the bivalve Chlamys septemradiata from the Clyde Sea area. relationship between digestive gland biochemical content Marine Biology, 25: 85e99. and the gametogenic stages clearly showed a decrease in Barber, B. J., and Blake, N. L. 1981. Energy storage and utilization in relation to gametogenesis in Argopecten irradians concen- lipids from the sexual rest to the ripe stage.

tricus (Say). Journal of Experimental Marine Biology and Downloaded from https://academic.oup.com/icesjms/article/62/5/886/856445 by guest on 29 September 2021 A similar situation was observed with glycogen in the Ecology, 52: 121e134. analysed muscle tissues (anterior adductor muscle and foot), Barber, B. J., and Blake, N. L. 1983. Growth and reproduction of as well as in the digestive gland. When the availability of food the bay scallop, Argopecten irradians (Lamarck) at its southern was high in summer, glycogen was stored, but it was distributional limit. Journal of Experimental Marine Biology and Ecology, 66: 247e256. mobilized when gamete development started in autumn. Barber, B. J., and Blake, N. L. 1991. Reproductive physiology These glycogen reserves may allow the synthesis of lipids in In Scallops: Biology, Ecology and Aquaculture, pp. 377e409. the gametes, as reported by several authors (Voogt, 1972; Ed. by S. E. Shumway. Elsevier, Amsterdam. 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Seasonal variations in condition, reproductive activity and gross biochemical compo- genesis, has been described for several different scallops sition of two species of adult clam reared in a common habitat: (Taylor and Venn, 1979; Barber and Blake, 1981; Lubet Tapes decussatus L. (Jeffreys) and Tapes philippinarum (Adams et al., 1987b; Romań et al., 1996, Pazos et al., 1997). & Reeve). Journal of Experimental Marine Biology and However, in E. arcuatus the foot also appears to act as Ecology, 79: 19e37. a storage site for glycogen, with a high quantity of reserves, Crespo, C. A. 1989. Histofisiologıá de las reservas bioenerge´ticas del manto de Mytilus galloprovincialis Lmk. PhD thesis, because of its size. Universidad de Santiago de Compostela. 215 pp. Gametogenesis is a process that requires energy. In Darriba, S., San Juan, F., and Guerra, A. 2004. 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