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Growth and gametogenesis in the lion-paw scallop Nodipecten (Lyropecten) subnodosus
Aquaculture 217 (2003) 335 – 349 www.elsevier.com/locate/aqua-online
Growth and gametogenesis in the lion-paw scallop Nodipecten (Lyropecten) subnodosus I.S. Racotta *, J.L. Ramirez, A.M. Ibarra, M.C. Rodrı´guez-Jaramillo, D. Carren˜o, E. Palacios Programa de Acuacultura, Centro de Investigaciones Biolo´gicas del Noroeste, Apdo. Postal 128, La Paz, Baja California Sur 23000, Mexico Received 10 December 2001; received in revised form 30 May 2002; accepted 16 July 2002
Abstract The lion-paw scallop (Nodipecten subnodosus) represents an important fishery resource along the Baja California Pacific coast in Mexico and has high potential for aquaculture. Although some previous studies have analyzed the growth rate of this species, knowledge of its reproduction is very limited, and no studies exist on the pattern of energy storage and utilization in relation to its reproductive cycle. Spat obtained in the laboratory from wild adults were cultured at Bahı´a Magdalena (Baja California Sur, Mexico). Growth and gametogenesis were analyzed over 18 months and biochemical composition of several tissues was analyzed during the last 6 months of culture. Growth rate, in terms of increase in shell height, averaged 0.22 mm/day. The weight of adductor muscle was 20 g after 12 months, and 55 g after 18 months of culture, respectively. Gonad maturity was observed mainly from July to September during the first year (1999) and started again in April in the second year (2000). Lipid and carbohydrate concentrations in female gonad increased with gonad development, but protein concentration remained unchanged. Adductor muscle index and carbohydrate concentration increased in parallel to gonad index during the second year (2000). However, a slight but significant decrease in adductor muscle proteins was observed as gonad developed. A continuous increase in digestive gland index concomitant to gonad development might indicate an increased availability of nutrients. From an aquaculture point of view, these results indicate that Bahı´a Magdalena is an adequate site for scallop growth and reproduction. From a physiological point of view, these results indicate that natural food availability could sustain the full cost of gametogenesis in this species in optimal environmental conditions, without or with minimal energy transfer from storage tissues. D 2003 Elsevier Science B.V. All rights reserved. Keywords: Reproduction; Pectinidae; Energy-allocation; Muscle; Gonad; Oocytes
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0044-8486/03/$ - see front matter D 2003 Elsevier Science B.V. All rights reserved. PII: S 0 0 4 4 - 8 4 8 6 ( 0 2 ) 0 0 3 6 6 - 6
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1. Introduction The lion-paw scallop (Nodipecten subnodosus) represents an important fishery resource along the Pacific coast of Baja California, Mexico. Its potential for aquaculture has been analyzed recently, although very few attempts at cultivation have been made in Mexico (Barrios et al., 1997; Felix-Pico et al., 1999) or for a related species (Nodipecten nodosus) in other countries (Lodeiros et al., 1998). Knowledge of the reproductive process in Nodipecten sp. is limited to description of conditioning and spawning (Rupp et al., 1997), or maturation and spawning in their natural habitat (Reinecke-Reyes, 1996). To our knowledge, only one study analyzes the cycle of energy storage and utilization in relation to growth and reproductive activity at an early age (6 months) in Nodipecten nodosus (Lodeiros et al., 2001). For other species of scallops, it is generally accepted that the energy assimilated from food is principally stored in the adductor muscle during non-reproductive periods. During gametogenesis, nutrients are transferred from the digestive gland (principally lipids) and the adductor muscle (carbohydrates and proteins) for yolk synthesis (Barber and Blake, 1981; Epp et al., 1988; Martinez, 1991; Pazos et al., 1997). The present study analyzed growth and timing of gametogenesis over an 18-month period in a population of lion-paw scallop introduced for aquaculture purposes to a site in the southern geographical range portion of the species. In the last 6 months, the specific pattern of nutrient accumulation and utilization in relation to gametogenesis was also analyzed.
2. Material and methods 2.1. Larval production and culture conditions Mature wild scallops were collected from Guerrero Negro, B.C.S., in October 1998, transported to the hatchery laboratory of the Centro de Investigaciones Biolo´gicas del Noroeste (CIBNOR), and mass spawning was induced by alternating cold (8 jC below normal temperature of 24 jC) and hot (6 jC above normal temperature of 24 jC) water shocks. After spawning, the scallops were removed from the tank, allowing for the fertilized eggs to develop into D-larvae. After 24 h, larvae were stocked at 10 larvae/ml in 500-l conical tanks, and reared to pediveliger larval stage. Water was changed every other day. When the pediveliger larval stage was reached, density was adjusted to 1 larva/ml, and plastic black mesh was introduced to the tank for settling. After settling, water was exchanged daily at a 100% rate. Feeding during larvae and settling stages consisted of a mixture of Monochrysis lutheri, Isochrysis galbana, and Chaetoceros calcitrans in approximately equal proportions, beginning with 30,000 cells/ml, and ending with 120,000 cells/ml. Water temperature was kept at 19 –20 jC, and salinity at 35 ppt during larval culture. In November 1998, the scallops spat (1– 2 mm) were placed in 0.7-mm mesh bags, and transported to the growout area in Bahı´a Magdalena, B.C.S., where they were grown suspended in Nestier trays (55 55 cm). After 3 weeks, scallops were transferred to 2-mm mesh bags, containing approximately
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1000 scallops per bag, and all subsequent growout was done in bottom structures to reduce fouling. One month later (January 1999), juveniles were transferred from the bags into three Nestier trays, and stocked at a density of 110 scallops per tray. These hatchery-produced scallops were grown and sampled during 18 months as indicated below. 2.2. Sampling procedure Ten to 15 scallops were collected monthly or bimonthly from the growout area in Bahı´a Magdalena from February 1999 to June 2000 and transferred to the laboratory at CIBNOR. For each individual, general morphometric traits, such as shell length (largest shell distance in an orientation ‘‘ear-to-ear’’), height (hinge line to the shell margin), and width (inflation or thickness), total weight, and total tissue (shell-free) wet weight (biomass), were recorded for all samples. Weight of adductor muscle and gonad was recorded from June and November 1999, respectively, to the end of the study. Starting in December 1999, mantle, digestive gland, adductor muscle, and gonad were sampled and stored at 80 jC for further biochemical analyses. Organ indices for the four tissues were calculated as the proportion of the organ weight to total tissue weight (Barber and Blake, 1991). In addition, a sample of gonad, taken from the mid-part of the gonad, was fixed in buffered formaldehyde for further histological analysis. For histological analysis, both female and male portions were fixed and processed. For biochemical analysis, dissection of enough sample of male gonad was not possible and only female gonad, taken form the most distal part, was analyzed. 2.3. Gonad histology The mid-part portion of gonad fixed in formaldehyde was embedded in a paraffin– paraplast mixture, sectioned (6 to 8 Am), and stained with Harris hematoxylin-eosin (Humason, 1972). Four stages (immature, maturing, ripe, and spawned) of female and male gonadal maturation were determined based on classifications proposed for this (Reinecke-Reyes, 1996) or other (Garcia-Dominguez et al., 1998) species of bivalve mollusks. It should be noted that spawned stage in the present study considers both partial spawning and spent stage (full spawned) described in previous works (Reinecke-Reyes, 1996; Garcia-Dominguez et al., 1998). The oocytes were classified according to their gametogenic developmental stage (oogonia, previtellogenic, mature, and reabsorption). The frequency of oocytes in each stage was estimated in three different regions of the ovary, counting the number of oocytes appearing in a predetermined test area of 0.3 mm2 (Briarty, 1975). The maximum and minimum diameters of oocytes, as well as the area occupied by each oocyte, were also determined in three different regions of the ovary, using an image analyzer. Only oocytes showing nuclei, sectioned approximately at the equatorial plane, were measured. Depending on the stage of maturation and individual variations, the number of oocytes that was measured ranged from 10 to 50 per scallop, considering 10 to 15 individuals per sampling.
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2.4. Biochemical analysis Samples of female gonad and digestive glands were mechanically homogenized in 1.5 ml of cold saline solution (NaCl, 35 ppt) to obtain a crude extract. From this crude extract, 100 Al was mixed with 1 ml sulfuric acid, heated to 90jC, and then lipids were analyzed by the sulpho-phosphovanillin method (Barnes and Blackstock, 1973) using commercial and standard solutions (Merck). Another part of the crude extract was centrifuged (3000 g, 5 jC, 10 min) and triglycerides were determined from the supernatant by a colorimetric-enzymatic kit (Merck). For carbohydrate determination, protein was first precipitated by mixing crude extract with 20% trichloroacetic acid (TCA) (1:2) and further centrifuged at 3000 g, 5 jC, for 10 min. The resulting supernatant was mixed with four parts of anthrone solution (0.1% dissolved in 76% sulfuric acid), incubated for 3 min at 90 jC, and cooled to 4 jC to stop further reaction (Van Handel, 1965). Total carbohydrates were quantified as glucosyl units, using a standard solution of glucose. The crude extract was also diluted 1:5 with 0.1N NaOH for determination of soluble protein (Bradford, 1976), using commercial chromogen reagent (Sigma) and bovine albumin serum (Sigma) as standard solution. For carbohydrate analyses in adductor muscle and mantle, samples of these tissues were homogenized directly in 5 ml of 10% TCA; the homogenate was centrifuged and processed as previously mentioned. Another sample of adductor muscle and mantle was digested for 24 h in 0.5N NaOH for protein analysis, as previously described. Lipids and triglycerides were not measured because the above methods lack sensitivity for these particular tissues. Another sample of all tissues was dried at 60 jC during 48 h for the estimation of water content. 2.5. Statistical analysis One-way analyses of variance followed by post hoc Tukey tests for unequal N (Statistica Version 5.0) were used to assess the significant differences between the months of sampling. The level of significance was set at P < 0.05. Organ indices were transformed to arcsine values for the analyses (Sokal and Rohlf, 1981).
3. Results 3.1. Scallop growth Scallop shell size and tissue weight increased continuously during the culture period (Fig. 1). Shell size increase corresponds to an average growth rate of 0.25 mm/day during Fig. 1. Growth of lion-paw scallops between February 1999 and June 2000: (A) shell length, height, and width; (B) total weight and wet tissue weight; and (C) adductor muscle and gonad weight. (D) Organ indices. AM: adductor muscle, M: mantle, DG: digestive gland, G: gonad. Data were analyzed by one-way ANOVA, followed by post hoc Tukey test for unequal N. Means not sharing the same superscript are significantly different; uppercase letters were used in the case of DG comparisons in order to avoid confusion with G comparisons.
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the first year and 0.22 mm/day for the overall duration of the study (Fig. 1A). Total weight reached 360 g in June 2000, of which 140 g (39%) was wet tissue weight (Fig. 1B). From June to December 1999 (1-year-old scallops), muscle weight increased to 17.2 g, which corresponds to a gain of 1.4 g/month (Fig. 1C). However, from January to June 2000, muscle growth was more pronounced, attaining a final weight of 55 g, which
Fig. 2. Proportion of organisms classified in each developmental stage of the female (A) or male (B) gonadal tissue of lion-paw cultured between February 1999 and June 2000.
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corresponds to a gain of 5.8 g/month. Gonad weight remained relatively constant from November 1999 to February 2000, and then increased until June 2000 (Fig. 1C). Adductor muscle index decreased significantly from June to November 1999, increased in December 1999, decreased again in January 2000 and then increased continuously until June 2000 (Fig. 1D). The mantle index was significantly lower in June 2000 than in previous months, whereas the digestive gland index showed an irregular pattern, with an increase from December 1999 to February 2000 followed by a decrease in April and an increase in June (Fig. 1D). The gonad index did not change significantly from November
Fig. 3. (A) Frequency of different oocytes, expressed as their absolute number per area unit (0.3 mm2) and (B) oocyte area and mean diameter. For oocyte area and diameter, data were analyzed by one-way ANOVA, followed by post hoc Tukey test for unequal N. Means not sharing the same superscript are significantly different.
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Table 1 Adductor muscle water content and biochemical composition (mg/g dry weight) of lion-paw scallop (Nodipecten subnodosus)
Water (%) Carbohydrate Protein
December 1999
February 2000
April 2000
June 2000
75.6 F 0.5b 32.0 F 7.3a 474.3 F 14.9b
73.7 F 0.8ab 116.7 F 4.5b 422.5 F 11.0a
71.8 F 0.8a 125.0 F 10b 430.9 F 11.4a
71.9 F 0.6a 200.1 F 16.7c 406.7 F 11.5a
All data are reported as mean F standard error. Data were analyzed by one-way ANOVA, followed by post hoc Tukey test for unequal N. Means not sharing the same superscript are significantly different ( P < 0.05).
1999 to February 2000 and then increased significantly between February and April 2000 (Fig. 1D). 3.2. Gonad histology Gametogenic activity (maturing individuals) in female and male portions of gonad was first observed in June 1999 (Fig. 2). In July, first maturity (individuals with ripe gonads) was attained principally for the female gonad (Fig. 2A), for which some spawning activity was recorded. In November 1999, scallops with maturing and ripe gonads were still present and spawning was recorded in some individuals in December. By January 2000, all organisms were in resting stage. By February, 50% of scallops had maturing or ripe female and male gonads. In April, most scallops had ripe gonads (67% for both sexes) or spawned gonads (27% for female gonad and 20% for male gonad, respectively). In June, all scallops had ripe female gonad and 70% had ripe male gonad. Previtellogenic oocytes were first observed in April 1999, increased dramatically in June and July, and then decreased by September (Fig. 3A). In parallel, mature oocytes were first observed in June and were more abundant in July and September. Both previtellogenic and mature oocyte progressively decreased from September 1999 to January 2000. In April and June 2000, mature oocytes increased dramatically again. Oocyte area and diameter followed a similar pattern with the highest values in September 1999 and April 2000 (Fig. 3B). However, between April and June 2000, a significant decrease in oocyte area and diameter was observed.
Table 2 Mantle water content and biochemical composition (mg/g dry weight) of lion-paw scallop (Nodipecten subnodosus)
Water (%) Carbohydrate Protein
December 1999
February 2000
April 2000
June 2000
81.9 F 0.2a 16.9 F 1.3a 312.6 F 18.4a
84.7 F 0.7b 30.2 F 0.9b 515.2 F 38.6b
83.6 F 0.2b 32.3 F 2.2b 496.9 F 34.1b
81.3 F 0.3a 62.7 F 4.7c 511.1 F 25.5b
See Table 1 for data analysis.
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Table 3 Digestive gland water content and biochemical composition (mg/g dry weight) of lion-paw scallop (Nodipecten subnodosus)
Water (%) Carbohydrate Protein Total lipids Triglycerides
December 1999
February 2000
April 2000
June 2000
62.6 F 2.2bc 124.3 F 3.4a 164.1 F 4.4a 132.4 F 16.6a 67.2 F 7.2a
64.7 F 1.4c 115.9 F 3.5a 180.4 F 5.8a 141.4 F 12.9ab 100 F 8.4a
57.6 F 1.1ab 112.5 F 8.3a 172.0 F 7.0a 192.4 F 14.7bc 80.1 F 9.6a
56.3 F 1.1a 125.3 F 9.8a 169.7 F 7.8a 242.7 F 17.9c 97.8 F 20.0a
See Table 1 for data analysis.
3.3. Biochemical composition In adductor muscle, carbohydrate concentration progressively increased from December 1999 to June 2000, whereas protein concentration was significantly higher in December 1999 than in the following months (Table 1). Water content in adductor muscle was significantly lower in April and June 2000 than in December 1999 (Table 1). Mantle carbohydrate concentration progressively increased from December 1999 to June 2000, and protein concentration was significantly lower in December 1999 than in the following months (Table 2). Water content in mantle was significantly higher in February and April 2000 compared to December 1999 and June 2000 (Table 2). In digestive gland, carbohydrate, protein, and triglycerides concentration remained constant through time, whereas total lipids progressively increased from December 1999 to June 2000 (Table 3). Higher water content in digestive gland was observed in December 1999 and February 2000 than in April and June 2000 (Table 3). In female gonad, carbohydrates, total lipids, and triglycerides were significantly higher in February, April and June 2000 compared to December 1999 (Table 4). In addition, triglycerides in June were also significantly higher than in February and April. Protein concentration in gonad did not change significantly between December 1999 and June 2000. Water content was significantly lower in June 2000 than in December 1999 and February 2000 (Table 4).
Table 4 Gonad water content and biochemical composition (mg/g dry weight) of lion-paw scallop (Nodipecten subnodosus)
Water (%) Carbohydrate Protein Total lipids Triglycerides
December 1999
February 2000
April 2000
June 2000
86.7 F 1.0bc 27.0 F 3.7a 234.0 F 25.6a 25.5 F 2.5a 8.6 F 1.5a
88.1 F 0.9c 41.4 F 3.5b 288.9 F 33.1a 43.1 F 4.3b 25.3 F 4.3b
83.3 F 0.5ab 41.9 F 3.2b 252.4 F 17.3a 43.2 F 3.7b 22.4 F 1.8b
80.8 F 0.6a 50.4 F 1.8b 254.1 F 21.4a 45.0 F 3.1b 33.5 F 3.1c
See Table 1 for data analysis.
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4. Discussion 4.1. Growth The daily growth rate of Nodipecten subnudosus in the present study was 0.25 mm/day, if considering only the first year (1999), which is the maximum period considered in previous studies done in different regions of the Peninsula of Baja California. One-year studies report values of 0.14 mm/day for Bahı´a de la Paz (Barrios et al., 1997), 0.21 mm/ day for the area near Guerrero Negro (Garcia-Dominguez et al., 1992), and 0.24 mm/day for Bahı´a de Loreto (Felix-Pico et al., 1999). On a shorter term basis some studies report daily growth of 0.33 mm/day during 107 days in Bahı´a de La Paz or 0.26 mm/day during 286 days in Bahı´a Magdalena (Morales-Hernandez and Caceres-Martinez, 1996). From the present study and the data reported by Morales-Hernandez and Caceres-Martinez (1996), Bahı´a Magdalena appears as an adequate site for the culture of this species, even if it is located at the southern limit of its natural geographical range. During the second year (2000), the rate of increase in shell height was lower, although total weight and adductor muscle weight increased at higher rates in this year. From the aquaculture point of view, the present results show that the expected value on final product yield is a function of the duration of culture. The advantage of a longer culture period at this site is reinforced by the simultaneous somatic growth and gonad development occurring during the second year. The relationship between growth and reproduction is fully discussed below, but it is worth noting that harvesting scallops during the second year of culture will have the advantage of a higher muscle weight, which does not appear to be affected by gonad development. In addition, the presence of a more developed gonad could be of commercial importance, depending on market preferences for roe-on product. 4.2. Reproduction Maturation and spawning were observed from July to September during 1999 and started again in April during 2000, in which unfortunately it was not possible to further analyze the reproductive pattern because all animals were already sampled by June. In the natural population from the area of Guerrero Negro, maturity was observed in June, although the major spawning activity was recorded in August – September (ReineckeReyes, 1996). In culture conditions at Bahı´a de La Paz, the highest gonad index values were observed during July, with a decrease in September and another increase in November (Barrios et al., 1997). In the present study, in addition to the first maturity attained in July 1999, a second partial maturity and spawning seemed to be occurring in December 1999. Reproductive quiescence was then evident in January 2000, and reproductive activity started again in February, attaining its peak in April. In June 2000, a second maturation was probably occurring, at least in some organisms, an assumption supported by the occurrence of spawned scallops in April, in contrast to 100% ripe female gonads in June. In addition, lower values of oocyte size were recorded in June, indicating that they were probably still growing towards a second maturity. In a general way, biochemical composition of gonad in the present study reinforces the histological data. Total lipids and triglycerides were increased in gonad by February,
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which corresponds to the onset of gametogenesis during 2000. In accordance, lipid accumulation in gonad during gametogenesis is an important and well-documented process occurring during maturation in scallops (Vassallo, 1973; Barber and Blake, 1991; Couturier and Newkirk, 1991; Martinez, 1991; Pazos et al., 1997). Total lipid levels were not significantly different from February to June. However, a further increase in triglycerides was observed from April to June. As supported by histological data, in April, several organisms had spawned and probably had lower levels of triglycerides than mature animals. In June, all organisms were maturing and average triglycerides were higher than in April. This is supported by the particular accumulation of this lipid class in ripe ovaries, which has been documented in pectinids (Pollero et al., 1979; Couturier and Newkirk, 1991; Soudant et al., 1996; Pazos et al., 1996; Racotta et al., 1998). In the present study, the levels of protein in gonadal tissue did not show significant differences during the gametogenic process of Nodipecten subnodosus. Although some studies have shown an important accumulation of proteins in gonad during maturation (Barber and Blake, 1981; Epp et al., 1988), other studies have not shown an increase in protein concentration in ripe female gonads of several pectinids (Thompson, 1977; Couturier and Newkirk, 1991; Pazos et al., 1996; Racotta et al., 1998; Ruiz-Verdugo et al., 2001). In contrast, carbohydrate levels in gonad increased with gametogenic activity in the present study. Previous studies have not shown changes in total carbohydrates (Barber and Blake, 1981; Couturier and Newkirk, 1991; Martinez, 1991; Pazos et al., 1997) or free glucose (Pazos et al., 1996, 1997) in gonad in relation to the reproductive cycle. However, during conditioning, higher total carbohydrates (Martinez et al., 1992) or free glucose (Racotta et al., 1998) were associated with gonad development. In addition, higher levels of carbohydrates were observed in gonads of diploid scallops than triploid ones, which were associated with their higher gonad development (Ruiz-Verdugo et al., 2001). As previously suggested (Racotta et al., 1998; Ruiz-Verdugo et al., 2001), it seems likely that accumulation of carbohydrates in developing gonad can be used as immediate available energy for synthesis of lipids or for spawning, and also as precursors for lipogenesis from carbohydrates, which is known to occur in mollusks (Gabbott, 1975; Barber and Blake, 1985a). 4.3. Energy allocation for growth and reproduction The relative importance of reserves vs. recently ingested food to sustain gametogenesis in pectinids has been clearly demonstrated by several studies, and it depends on several factors (for review, see Barber and Blake, 1991). When mobilization of previously stored reserves is occurring, the adductor muscle has been considered the most important site of energy storage in scallops, which is later used during gametogenesis. This energy storage and utilization pattern between adductor muscle and gonad is supported by a decrease in muscle index (Barber and Blake, 1981, 1983; VillalejoFuerte and Ceballos-Vazquez, 1996; Barrios et al., 1997), a decrease in carbohydrate muscle concentration (Barber and Blake, 1981; Martinez, 1991; Pazos et al., 1997; Martinez and Mettifogo, 1998; Strohmeier, 1999; Brokordt et al., 2000; Lodeiros et al., 2001), and a decrease in the protein muscle concentration (Epp et al., 1988; Couturier and Newkirk, 1991; Pazos et al., 1997; Strohmeier, 1999; Brokordt et al., 2000) associated
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with the reproductive cycle. In the present study, muscle index decreased from June to November during 1999 in parallel to gonad development and spawning during this period. In contrast, during 2000 muscle index and carbohydrate concentration increased from April to June, despite the important development of gonads during this period. However, utilization of muscle protein is suggested by the slight, but significant decrease in proteins from December 1999 compared to further months (February to June 2000). Muscle protein could be used during the vitellogenic process itself, as shown by a higher relative mobilization of protein than carbohydrates (Epp et al., 1988; Couturier and Newkirk, 1991; Faveris and Lubet, 1991; Pazos et al., 1997; Racotta et al., 1998). Alternatively, muscle protein may be used at the end of the gametogenic cycle to meet the maintenance demands for final maturation and spawning (Barber and Blake, 1981, 1985b; Brokordt et al., 2000). The digestive gland has also been suggested to supply nutrients during the gametogenic process, although its role in the energy storage and utilization pattern is considered to be on a short-term basis (Barber and Blake, 1983). Lipids are the principal components mobilized from the digestive gland during gametogenesis (Vassallo, 1973; Barber and Blake, 1981; Pazos et al., 1997; Lodeiros et al., 2001). A more generalized use of other components was also suggested by a decrease in protein concentration (Lodeiros et al., 2001), a decrease in digestive gland index (Sastry, 1968), and 14C loss from lipid, carbohydrate, and protein (Barber and Blake, 1985a). In contrast, in the present study, the continuous increase in digestive gland index might indicate an increased availability of nutrients, without a mobilization of lipids, proteins and carbohydrates to gonads. Mobilization of nutrients stored in tissues other than muscle and digestive gland, such as mantle, has been poorly analyzed for scallops (Martinez, 1991; Couturier and Newkirk, 1991; Lodeiros et al., 2001), although it is well known for other mollusks, such as mussels (Gabbott, 1975; Bayne et al., 1982; Mathieu and Lubet, 1993). For Argopecten purpuratus, it was suggested that mantle biochemical composition was more related to growth than to reproduction (Martinez, 1991). However, a decrease in mantle proteins was observed in parallel to gametogenesis in Nodipecten nodosus (Lodeiros et al., 2001). In the present study, protein concentration in mantle increase concomitant to gametogenic activity and muscle growth, and thus there was no evidence of reserve mobilization from the mantle. The decrease in mantle index observed from April to June may be explained by a higher proportional increase in the weight of other tissues such as adductor muscle, digestive gland and gonad. These results indicate that gametogenesis in this population of lion-paw scallop in Bahı´a Magdalena, and during the time-period considered, is not importantly supported by mobilization of reserves. Several authors have suggested that the degree of utilization of reserves in muscle or other tissues depends on an adequate food supply (Sastry, 1968; Barber and Blake, 1983; Pazos et al., 1997; Racotta et al., 1998; Martinez et al., 2000; Lodeiros et al., 2001). Unfortunately, food availability through primary productivity was not measured in this study, but it seems likely that it was enough to sustain the overall gametogenic process. Similarly, Pazos et al. (1997) reported that gametogenic activity in Pecten maximus does not depend on stored reserves during spring when food availability is maximal.
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5. Conclusions From the aquaculture point of view, these results indicate that Bahı´a Magdalena is an adequate site for scallop growth and reproduction. From the physiological point of view, these results indicate that the typical energy storage and utilization patterns observed for other pectinids are not always evident, and that food could sustain the full cost of gametogenesis in optimal environmental conditions.
Acknowledgements We acknowledge the collaboration of Miguel Robles for donation of larvae, Roberto Herna´ndez-Herrera for technical support in the biochemical analyses, Teresa Arteche and Adriana Green for histological processing of gonads, Ira Fogel for editing the Englishlanguage text, and anonymous reviewers for their useful comments. This research was supported by SIMAC project BCS7001 and institutional projects CIBNOR-PAC14, PAC15 and PAC31.
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Growth and gametogenesis in the lion-paw scallop Nodipecten (Lyropecten) subnodosus I.S. Racotta *, J.L. Ramirez, A.M. Ibarra, M.C. Rodrı´guez-Jaramillo, D. Carren˜o, E. Palacios Programa de Acuacultura, Centro de Investigaciones Biolo´gicas del Noroeste, Apdo. Postal 128, La Paz, Baja California Sur 23000, Mexico Received 10 December 2001; received in revised form 30 May 2002; accepted 16 July 2002
Abstract The lion-paw scallop (Nodipecten subnodosus) represents an important fishery resource along the Baja California Pacific coast in Mexico and has high potential for aquaculture. Although some previous studies have analyzed the growth rate of this species, knowledge of its reproduction is very limited, and no studies exist on the pattern of energy storage and utilization in relation to its reproductive cycle. Spat obtained in the laboratory from wild adults were cultured at Bahı´a Magdalena (Baja California Sur, Mexico). Growth and gametogenesis were analyzed over 18 months and biochemical composition of several tissues was analyzed during the last 6 months of culture. Growth rate, in terms of increase in shell height, averaged 0.22 mm/day. The weight of adductor muscle was 20 g after 12 months, and 55 g after 18 months of culture, respectively. Gonad maturity was observed mainly from July to September during the first year (1999) and started again in April in the second year (2000). Lipid and carbohydrate concentrations in female gonad increased with gonad development, but protein concentration remained unchanged. Adductor muscle index and carbohydrate concentration increased in parallel to gonad index during the second year (2000). However, a slight but significant decrease in adductor muscle proteins was observed as gonad developed. A continuous increase in digestive gland index concomitant to gonad development might indicate an increased availability of nutrients. From an aquaculture point of view, these results indicate that Bahı´a Magdalena is an adequate site for scallop growth and reproduction. From a physiological point of view, these results indicate that natural food availability could sustain the full cost of gametogenesis in this species in optimal environmental conditions, without or with minimal energy transfer from storage tissues. D 2003 Elsevier Science B.V. All rights reserved. Keywords: Reproduction; Pectinidae; Energy-allocation; Muscle; Gonad; Oocytes
*
Corresponding author. Tel.: +52-612-125-36-33x3414; fax: +52-612-125-36-25. E-mail address: [email protected] (I.S. Racotta).
0044-8486/03/$ - see front matter D 2003 Elsevier Science B.V. All rights reserved. PII: S 0 0 4 4 - 8 4 8 6 ( 0 2 ) 0 0 3 6 6 - 6
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1. Introduction The lion-paw scallop (Nodipecten subnodosus) represents an important fishery resource along the Pacific coast of Baja California, Mexico. Its potential for aquaculture has been analyzed recently, although very few attempts at cultivation have been made in Mexico (Barrios et al., 1997; Felix-Pico et al., 1999) or for a related species (Nodipecten nodosus) in other countries (Lodeiros et al., 1998). Knowledge of the reproductive process in Nodipecten sp. is limited to description of conditioning and spawning (Rupp et al., 1997), or maturation and spawning in their natural habitat (Reinecke-Reyes, 1996). To our knowledge, only one study analyzes the cycle of energy storage and utilization in relation to growth and reproductive activity at an early age (6 months) in Nodipecten nodosus (Lodeiros et al., 2001). For other species of scallops, it is generally accepted that the energy assimilated from food is principally stored in the adductor muscle during non-reproductive periods. During gametogenesis, nutrients are transferred from the digestive gland (principally lipids) and the adductor muscle (carbohydrates and proteins) for yolk synthesis (Barber and Blake, 1981; Epp et al., 1988; Martinez, 1991; Pazos et al., 1997). The present study analyzed growth and timing of gametogenesis over an 18-month period in a population of lion-paw scallop introduced for aquaculture purposes to a site in the southern geographical range portion of the species. In the last 6 months, the specific pattern of nutrient accumulation and utilization in relation to gametogenesis was also analyzed.
2. Material and methods 2.1. Larval production and culture conditions Mature wild scallops were collected from Guerrero Negro, B.C.S., in October 1998, transported to the hatchery laboratory of the Centro de Investigaciones Biolo´gicas del Noroeste (CIBNOR), and mass spawning was induced by alternating cold (8 jC below normal temperature of 24 jC) and hot (6 jC above normal temperature of 24 jC) water shocks. After spawning, the scallops were removed from the tank, allowing for the fertilized eggs to develop into D-larvae. After 24 h, larvae were stocked at 10 larvae/ml in 500-l conical tanks, and reared to pediveliger larval stage. Water was changed every other day. When the pediveliger larval stage was reached, density was adjusted to 1 larva/ml, and plastic black mesh was introduced to the tank for settling. After settling, water was exchanged daily at a 100% rate. Feeding during larvae and settling stages consisted of a mixture of Monochrysis lutheri, Isochrysis galbana, and Chaetoceros calcitrans in approximately equal proportions, beginning with 30,000 cells/ml, and ending with 120,000 cells/ml. Water temperature was kept at 19 –20 jC, and salinity at 35 ppt during larval culture. In November 1998, the scallops spat (1– 2 mm) were placed in 0.7-mm mesh bags, and transported to the growout area in Bahı´a Magdalena, B.C.S., where they were grown suspended in Nestier trays (55 55 cm). After 3 weeks, scallops were transferred to 2-mm mesh bags, containing approximately
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1000 scallops per bag, and all subsequent growout was done in bottom structures to reduce fouling. One month later (January 1999), juveniles were transferred from the bags into three Nestier trays, and stocked at a density of 110 scallops per tray. These hatchery-produced scallops were grown and sampled during 18 months as indicated below. 2.2. Sampling procedure Ten to 15 scallops were collected monthly or bimonthly from the growout area in Bahı´a Magdalena from February 1999 to June 2000 and transferred to the laboratory at CIBNOR. For each individual, general morphometric traits, such as shell length (largest shell distance in an orientation ‘‘ear-to-ear’’), height (hinge line to the shell margin), and width (inflation or thickness), total weight, and total tissue (shell-free) wet weight (biomass), were recorded for all samples. Weight of adductor muscle and gonad was recorded from June and November 1999, respectively, to the end of the study. Starting in December 1999, mantle, digestive gland, adductor muscle, and gonad were sampled and stored at 80 jC for further biochemical analyses. Organ indices for the four tissues were calculated as the proportion of the organ weight to total tissue weight (Barber and Blake, 1991). In addition, a sample of gonad, taken from the mid-part of the gonad, was fixed in buffered formaldehyde for further histological analysis. For histological analysis, both female and male portions were fixed and processed. For biochemical analysis, dissection of enough sample of male gonad was not possible and only female gonad, taken form the most distal part, was analyzed. 2.3. Gonad histology The mid-part portion of gonad fixed in formaldehyde was embedded in a paraffin– paraplast mixture, sectioned (6 to 8 Am), and stained with Harris hematoxylin-eosin (Humason, 1972). Four stages (immature, maturing, ripe, and spawned) of female and male gonadal maturation were determined based on classifications proposed for this (Reinecke-Reyes, 1996) or other (Garcia-Dominguez et al., 1998) species of bivalve mollusks. It should be noted that spawned stage in the present study considers both partial spawning and spent stage (full spawned) described in previous works (Reinecke-Reyes, 1996; Garcia-Dominguez et al., 1998). The oocytes were classified according to their gametogenic developmental stage (oogonia, previtellogenic, mature, and reabsorption). The frequency of oocytes in each stage was estimated in three different regions of the ovary, counting the number of oocytes appearing in a predetermined test area of 0.3 mm2 (Briarty, 1975). The maximum and minimum diameters of oocytes, as well as the area occupied by each oocyte, were also determined in three different regions of the ovary, using an image analyzer. Only oocytes showing nuclei, sectioned approximately at the equatorial plane, were measured. Depending on the stage of maturation and individual variations, the number of oocytes that was measured ranged from 10 to 50 per scallop, considering 10 to 15 individuals per sampling.
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2.4. Biochemical analysis Samples of female gonad and digestive glands were mechanically homogenized in 1.5 ml of cold saline solution (NaCl, 35 ppt) to obtain a crude extract. From this crude extract, 100 Al was mixed with 1 ml sulfuric acid, heated to 90jC, and then lipids were analyzed by the sulpho-phosphovanillin method (Barnes and Blackstock, 1973) using commercial and standard solutions (Merck). Another part of the crude extract was centrifuged (3000 g, 5 jC, 10 min) and triglycerides were determined from the supernatant by a colorimetric-enzymatic kit (Merck). For carbohydrate determination, protein was first precipitated by mixing crude extract with 20% trichloroacetic acid (TCA) (1:2) and further centrifuged at 3000 g, 5 jC, for 10 min. The resulting supernatant was mixed with four parts of anthrone solution (0.1% dissolved in 76% sulfuric acid), incubated for 3 min at 90 jC, and cooled to 4 jC to stop further reaction (Van Handel, 1965). Total carbohydrates were quantified as glucosyl units, using a standard solution of glucose. The crude extract was also diluted 1:5 with 0.1N NaOH for determination of soluble protein (Bradford, 1976), using commercial chromogen reagent (Sigma) and bovine albumin serum (Sigma) as standard solution. For carbohydrate analyses in adductor muscle and mantle, samples of these tissues were homogenized directly in 5 ml of 10% TCA; the homogenate was centrifuged and processed as previously mentioned. Another sample of adductor muscle and mantle was digested for 24 h in 0.5N NaOH for protein analysis, as previously described. Lipids and triglycerides were not measured because the above methods lack sensitivity for these particular tissues. Another sample of all tissues was dried at 60 jC during 48 h for the estimation of water content. 2.5. Statistical analysis One-way analyses of variance followed by post hoc Tukey tests for unequal N (Statistica Version 5.0) were used to assess the significant differences between the months of sampling. The level of significance was set at P < 0.05. Organ indices were transformed to arcsine values for the analyses (Sokal and Rohlf, 1981).
3. Results 3.1. Scallop growth Scallop shell size and tissue weight increased continuously during the culture period (Fig. 1). Shell size increase corresponds to an average growth rate of 0.25 mm/day during Fig. 1. Growth of lion-paw scallops between February 1999 and June 2000: (A) shell length, height, and width; (B) total weight and wet tissue weight; and (C) adductor muscle and gonad weight. (D) Organ indices. AM: adductor muscle, M: mantle, DG: digestive gland, G: gonad. Data were analyzed by one-way ANOVA, followed by post hoc Tukey test for unequal N. Means not sharing the same superscript are significantly different; uppercase letters were used in the case of DG comparisons in order to avoid confusion with G comparisons.
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the first year and 0.22 mm/day for the overall duration of the study (Fig. 1A). Total weight reached 360 g in June 2000, of which 140 g (39%) was wet tissue weight (Fig. 1B). From June to December 1999 (1-year-old scallops), muscle weight increased to 17.2 g, which corresponds to a gain of 1.4 g/month (Fig. 1C). However, from January to June 2000, muscle growth was more pronounced, attaining a final weight of 55 g, which
Fig. 2. Proportion of organisms classified in each developmental stage of the female (A) or male (B) gonadal tissue of lion-paw cultured between February 1999 and June 2000.
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corresponds to a gain of 5.8 g/month. Gonad weight remained relatively constant from November 1999 to February 2000, and then increased until June 2000 (Fig. 1C). Adductor muscle index decreased significantly from June to November 1999, increased in December 1999, decreased again in January 2000 and then increased continuously until June 2000 (Fig. 1D). The mantle index was significantly lower in June 2000 than in previous months, whereas the digestive gland index showed an irregular pattern, with an increase from December 1999 to February 2000 followed by a decrease in April and an increase in June (Fig. 1D). The gonad index did not change significantly from November
Fig. 3. (A) Frequency of different oocytes, expressed as their absolute number per area unit (0.3 mm2) and (B) oocyte area and mean diameter. For oocyte area and diameter, data were analyzed by one-way ANOVA, followed by post hoc Tukey test for unequal N. Means not sharing the same superscript are significantly different.
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Table 1 Adductor muscle water content and biochemical composition (mg/g dry weight) of lion-paw scallop (Nodipecten subnodosus)
Water (%) Carbohydrate Protein
December 1999
February 2000
April 2000
June 2000
75.6 F 0.5b 32.0 F 7.3a 474.3 F 14.9b
73.7 F 0.8ab 116.7 F 4.5b 422.5 F 11.0a
71.8 F 0.8a 125.0 F 10b 430.9 F 11.4a
71.9 F 0.6a 200.1 F 16.7c 406.7 F 11.5a
All data are reported as mean F standard error. Data were analyzed by one-way ANOVA, followed by post hoc Tukey test for unequal N. Means not sharing the same superscript are significantly different ( P < 0.05).
1999 to February 2000 and then increased significantly between February and April 2000 (Fig. 1D). 3.2. Gonad histology Gametogenic activity (maturing individuals) in female and male portions of gonad was first observed in June 1999 (Fig. 2). In July, first maturity (individuals with ripe gonads) was attained principally for the female gonad (Fig. 2A), for which some spawning activity was recorded. In November 1999, scallops with maturing and ripe gonads were still present and spawning was recorded in some individuals in December. By January 2000, all organisms were in resting stage. By February, 50% of scallops had maturing or ripe female and male gonads. In April, most scallops had ripe gonads (67% for both sexes) or spawned gonads (27% for female gonad and 20% for male gonad, respectively). In June, all scallops had ripe female gonad and 70% had ripe male gonad. Previtellogenic oocytes were first observed in April 1999, increased dramatically in June and July, and then decreased by September (Fig. 3A). In parallel, mature oocytes were first observed in June and were more abundant in July and September. Both previtellogenic and mature oocyte progressively decreased from September 1999 to January 2000. In April and June 2000, mature oocytes increased dramatically again. Oocyte area and diameter followed a similar pattern with the highest values in September 1999 and April 2000 (Fig. 3B). However, between April and June 2000, a significant decrease in oocyte area and diameter was observed.
Table 2 Mantle water content and biochemical composition (mg/g dry weight) of lion-paw scallop (Nodipecten subnodosus)
Water (%) Carbohydrate Protein
December 1999
February 2000
April 2000
June 2000
81.9 F 0.2a 16.9 F 1.3a 312.6 F 18.4a
84.7 F 0.7b 30.2 F 0.9b 515.2 F 38.6b
83.6 F 0.2b 32.3 F 2.2b 496.9 F 34.1b
81.3 F 0.3a 62.7 F 4.7c 511.1 F 25.5b
See Table 1 for data analysis.
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Table 3 Digestive gland water content and biochemical composition (mg/g dry weight) of lion-paw scallop (Nodipecten subnodosus)
Water (%) Carbohydrate Protein Total lipids Triglycerides
December 1999
February 2000
April 2000
June 2000
62.6 F 2.2bc 124.3 F 3.4a 164.1 F 4.4a 132.4 F 16.6a 67.2 F 7.2a
64.7 F 1.4c 115.9 F 3.5a 180.4 F 5.8a 141.4 F 12.9ab 100 F 8.4a
57.6 F 1.1ab 112.5 F 8.3a 172.0 F 7.0a 192.4 F 14.7bc 80.1 F 9.6a
56.3 F 1.1a 125.3 F 9.8a 169.7 F 7.8a 242.7 F 17.9c 97.8 F 20.0a
See Table 1 for data analysis.
3.3. Biochemical composition In adductor muscle, carbohydrate concentration progressively increased from December 1999 to June 2000, whereas protein concentration was significantly higher in December 1999 than in the following months (Table 1). Water content in adductor muscle was significantly lower in April and June 2000 than in December 1999 (Table 1). Mantle carbohydrate concentration progressively increased from December 1999 to June 2000, and protein concentration was significantly lower in December 1999 than in the following months (Table 2). Water content in mantle was significantly higher in February and April 2000 compared to December 1999 and June 2000 (Table 2). In digestive gland, carbohydrate, protein, and triglycerides concentration remained constant through time, whereas total lipids progressively increased from December 1999 to June 2000 (Table 3). Higher water content in digestive gland was observed in December 1999 and February 2000 than in April and June 2000 (Table 3). In female gonad, carbohydrates, total lipids, and triglycerides were significantly higher in February, April and June 2000 compared to December 1999 (Table 4). In addition, triglycerides in June were also significantly higher than in February and April. Protein concentration in gonad did not change significantly between December 1999 and June 2000. Water content was significantly lower in June 2000 than in December 1999 and February 2000 (Table 4).
Table 4 Gonad water content and biochemical composition (mg/g dry weight) of lion-paw scallop (Nodipecten subnodosus)
Water (%) Carbohydrate Protein Total lipids Triglycerides
December 1999
February 2000
April 2000
June 2000
86.7 F 1.0bc 27.0 F 3.7a 234.0 F 25.6a 25.5 F 2.5a 8.6 F 1.5a
88.1 F 0.9c 41.4 F 3.5b 288.9 F 33.1a 43.1 F 4.3b 25.3 F 4.3b
83.3 F 0.5ab 41.9 F 3.2b 252.4 F 17.3a 43.2 F 3.7b 22.4 F 1.8b
80.8 F 0.6a 50.4 F 1.8b 254.1 F 21.4a 45.0 F 3.1b 33.5 F 3.1c
See Table 1 for data analysis.
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4. Discussion 4.1. Growth The daily growth rate of Nodipecten subnudosus in the present study was 0.25 mm/day, if considering only the first year (1999), which is the maximum period considered in previous studies done in different regions of the Peninsula of Baja California. One-year studies report values of 0.14 mm/day for Bahı´a de la Paz (Barrios et al., 1997), 0.21 mm/ day for the area near Guerrero Negro (Garcia-Dominguez et al., 1992), and 0.24 mm/day for Bahı´a de Loreto (Felix-Pico et al., 1999). On a shorter term basis some studies report daily growth of 0.33 mm/day during 107 days in Bahı´a de La Paz or 0.26 mm/day during 286 days in Bahı´a Magdalena (Morales-Hernandez and Caceres-Martinez, 1996). From the present study and the data reported by Morales-Hernandez and Caceres-Martinez (1996), Bahı´a Magdalena appears as an adequate site for the culture of this species, even if it is located at the southern limit of its natural geographical range. During the second year (2000), the rate of increase in shell height was lower, although total weight and adductor muscle weight increased at higher rates in this year. From the aquaculture point of view, the present results show that the expected value on final product yield is a function of the duration of culture. The advantage of a longer culture period at this site is reinforced by the simultaneous somatic growth and gonad development occurring during the second year. The relationship between growth and reproduction is fully discussed below, but it is worth noting that harvesting scallops during the second year of culture will have the advantage of a higher muscle weight, which does not appear to be affected by gonad development. In addition, the presence of a more developed gonad could be of commercial importance, depending on market preferences for roe-on product. 4.2. Reproduction Maturation and spawning were observed from July to September during 1999 and started again in April during 2000, in which unfortunately it was not possible to further analyze the reproductive pattern because all animals were already sampled by June. In the natural population from the area of Guerrero Negro, maturity was observed in June, although the major spawning activity was recorded in August – September (ReineckeReyes, 1996). In culture conditions at Bahı´a de La Paz, the highest gonad index values were observed during July, with a decrease in September and another increase in November (Barrios et al., 1997). In the present study, in addition to the first maturity attained in July 1999, a second partial maturity and spawning seemed to be occurring in December 1999. Reproductive quiescence was then evident in January 2000, and reproductive activity started again in February, attaining its peak in April. In June 2000, a second maturation was probably occurring, at least in some organisms, an assumption supported by the occurrence of spawned scallops in April, in contrast to 100% ripe female gonads in June. In addition, lower values of oocyte size were recorded in June, indicating that they were probably still growing towards a second maturity. In a general way, biochemical composition of gonad in the present study reinforces the histological data. Total lipids and triglycerides were increased in gonad by February,
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which corresponds to the onset of gametogenesis during 2000. In accordance, lipid accumulation in gonad during gametogenesis is an important and well-documented process occurring during maturation in scallops (Vassallo, 1973; Barber and Blake, 1991; Couturier and Newkirk, 1991; Martinez, 1991; Pazos et al., 1997). Total lipid levels were not significantly different from February to June. However, a further increase in triglycerides was observed from April to June. As supported by histological data, in April, several organisms had spawned and probably had lower levels of triglycerides than mature animals. In June, all organisms were maturing and average triglycerides were higher than in April. This is supported by the particular accumulation of this lipid class in ripe ovaries, which has been documented in pectinids (Pollero et al., 1979; Couturier and Newkirk, 1991; Soudant et al., 1996; Pazos et al., 1996; Racotta et al., 1998). In the present study, the levels of protein in gonadal tissue did not show significant differences during the gametogenic process of Nodipecten subnodosus. Although some studies have shown an important accumulation of proteins in gonad during maturation (Barber and Blake, 1981; Epp et al., 1988), other studies have not shown an increase in protein concentration in ripe female gonads of several pectinids (Thompson, 1977; Couturier and Newkirk, 1991; Pazos et al., 1996; Racotta et al., 1998; Ruiz-Verdugo et al., 2001). In contrast, carbohydrate levels in gonad increased with gametogenic activity in the present study. Previous studies have not shown changes in total carbohydrates (Barber and Blake, 1981; Couturier and Newkirk, 1991; Martinez, 1991; Pazos et al., 1997) or free glucose (Pazos et al., 1996, 1997) in gonad in relation to the reproductive cycle. However, during conditioning, higher total carbohydrates (Martinez et al., 1992) or free glucose (Racotta et al., 1998) were associated with gonad development. In addition, higher levels of carbohydrates were observed in gonads of diploid scallops than triploid ones, which were associated with their higher gonad development (Ruiz-Verdugo et al., 2001). As previously suggested (Racotta et al., 1998; Ruiz-Verdugo et al., 2001), it seems likely that accumulation of carbohydrates in developing gonad can be used as immediate available energy for synthesis of lipids or for spawning, and also as precursors for lipogenesis from carbohydrates, which is known to occur in mollusks (Gabbott, 1975; Barber and Blake, 1985a). 4.3. Energy allocation for growth and reproduction The relative importance of reserves vs. recently ingested food to sustain gametogenesis in pectinids has been clearly demonstrated by several studies, and it depends on several factors (for review, see Barber and Blake, 1991). When mobilization of previously stored reserves is occurring, the adductor muscle has been considered the most important site of energy storage in scallops, which is later used during gametogenesis. This energy storage and utilization pattern between adductor muscle and gonad is supported by a decrease in muscle index (Barber and Blake, 1981, 1983; VillalejoFuerte and Ceballos-Vazquez, 1996; Barrios et al., 1997), a decrease in carbohydrate muscle concentration (Barber and Blake, 1981; Martinez, 1991; Pazos et al., 1997; Martinez and Mettifogo, 1998; Strohmeier, 1999; Brokordt et al., 2000; Lodeiros et al., 2001), and a decrease in the protein muscle concentration (Epp et al., 1988; Couturier and Newkirk, 1991; Pazos et al., 1997; Strohmeier, 1999; Brokordt et al., 2000) associated
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with the reproductive cycle. In the present study, muscle index decreased from June to November during 1999 in parallel to gonad development and spawning during this period. In contrast, during 2000 muscle index and carbohydrate concentration increased from April to June, despite the important development of gonads during this period. However, utilization of muscle protein is suggested by the slight, but significant decrease in proteins from December 1999 compared to further months (February to June 2000). Muscle protein could be used during the vitellogenic process itself, as shown by a higher relative mobilization of protein than carbohydrates (Epp et al., 1988; Couturier and Newkirk, 1991; Faveris and Lubet, 1991; Pazos et al., 1997; Racotta et al., 1998). Alternatively, muscle protein may be used at the end of the gametogenic cycle to meet the maintenance demands for final maturation and spawning (Barber and Blake, 1981, 1985b; Brokordt et al., 2000). The digestive gland has also been suggested to supply nutrients during the gametogenic process, although its role in the energy storage and utilization pattern is considered to be on a short-term basis (Barber and Blake, 1983). Lipids are the principal components mobilized from the digestive gland during gametogenesis (Vassallo, 1973; Barber and Blake, 1981; Pazos et al., 1997; Lodeiros et al., 2001). A more generalized use of other components was also suggested by a decrease in protein concentration (Lodeiros et al., 2001), a decrease in digestive gland index (Sastry, 1968), and 14C loss from lipid, carbohydrate, and protein (Barber and Blake, 1985a). In contrast, in the present study, the continuous increase in digestive gland index might indicate an increased availability of nutrients, without a mobilization of lipids, proteins and carbohydrates to gonads. Mobilization of nutrients stored in tissues other than muscle and digestive gland, such as mantle, has been poorly analyzed for scallops (Martinez, 1991; Couturier and Newkirk, 1991; Lodeiros et al., 2001), although it is well known for other mollusks, such as mussels (Gabbott, 1975; Bayne et al., 1982; Mathieu and Lubet, 1993). For Argopecten purpuratus, it was suggested that mantle biochemical composition was more related to growth than to reproduction (Martinez, 1991). However, a decrease in mantle proteins was observed in parallel to gametogenesis in Nodipecten nodosus (Lodeiros et al., 2001). In the present study, protein concentration in mantle increase concomitant to gametogenic activity and muscle growth, and thus there was no evidence of reserve mobilization from the mantle. The decrease in mantle index observed from April to June may be explained by a higher proportional increase in the weight of other tissues such as adductor muscle, digestive gland and gonad. These results indicate that gametogenesis in this population of lion-paw scallop in Bahı´a Magdalena, and during the time-period considered, is not importantly supported by mobilization of reserves. Several authors have suggested that the degree of utilization of reserves in muscle or other tissues depends on an adequate food supply (Sastry, 1968; Barber and Blake, 1983; Pazos et al., 1997; Racotta et al., 1998; Martinez et al., 2000; Lodeiros et al., 2001). Unfortunately, food availability through primary productivity was not measured in this study, but it seems likely that it was enough to sustain the overall gametogenic process. Similarly, Pazos et al. (1997) reported that gametogenic activity in Pecten maximus does not depend on stored reserves during spring when food availability is maximal.
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5. Conclusions From the aquaculture point of view, these results indicate that Bahı´a Magdalena is an adequate site for scallop growth and reproduction. From the physiological point of view, these results indicate that the typical energy storage and utilization patterns observed for other pectinids are not always evident, and that food could sustain the full cost of gametogenesis in optimal environmental conditions.
Acknowledgements We acknowledge the collaboration of Miguel Robles for donation of larvae, Roberto Herna´ndez-Herrera for technical support in the biochemical analyses, Teresa Arteche and Adriana Green for histological processing of gonads, Ira Fogel for editing the Englishlanguage text, and anonymous reviewers for their useful comments. This research was supported by SIMAC project BCS7001 and institutional projects CIBNOR-PAC14, PAC15 and PAC31.
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