ABSTRACT Triploid organisms have a limited capacity to develop gonads, and thus are considered sterile or partially sterile. The objective of this study is to compare triploid and diploid organisms of the same age, and grown under similar conditions during a 1-year period, to determine to what extent sterility affects size and number of adductor muscle cells, and accumulation of biochemical reserves at first maturation. Adult catarina scallops (Argopecten ventricosus) were matured, and eggs were treated with cytochalasin-B to induce triploidy. Untreated diploid controls and triploids were grown in Bahia Magdalena, Mexico in Nestier trays at 3 m depth from April 2000 to March 2001. Scallops were sampled monthly from June 2000. The gonadosomatic index (GSI) was significantly larger in diploid organisms, whereas the muscle index was larger in triploid organisms. A small proportion (40%) of diploid scallops exhibited reproductive activity during the first year. In spite of high GSI, the gonads of triploid scallops were mostly immature, except for some individuals that formed mature oocytes by March 2001. Triploids had 123% higher adductor muscle weight than diploid scallops at the end of the sampling period, and most of this increased gain was a result of adductor muscle cell size (hypertrophy), and not cell number (hyperplasia). Total lipids and proteins concentrations were significantly larger in the gonads of diploid scallops. Muscle carbohydrates were significantly larger in triploid scallops, which suggests decreased transference of carbohydrates form muscle to gonad in triploid sterile organisms.
KEY WORDS: energy-of-allocation, gonad, maturation, muscle, oocytes. Pectinidae, reproduction, Argopecten
During the reproductive period of pectinids, there is an important accumulation of lipids and proteins in the gonads (Barber & Blake 1981, Barber & Blake 1985, Martinez 1991, Pazos et al. 1996), mostly provided by food or mobilization of endogenous reserves when food is not readily available (Barber et al. 1991). The adductor muscle of pectinids is the site of one of the largest reserves of glycogen and protein mobilized during maturation (Barber & Blake 1981, Barber & Blake 1985, Epp et al. 1988). When reproductive activity begins, there is a large transfer of biochemical components to the gonad, and the weight of the muscle decreases. Also, water content in the adductor muscle is modified, and this can affect its taste and texture and thus its market price (Allen & Downing 1991). Triploid organisms have limited capacity to mature, and in most species where triploidy is attained, individuals do not produce gametes or viable larvae, and are considered sterile. An advantage of sterile organisms is that they transfer less biochemical energy to the gonad, so other tissues can gain more weight over time. For this reason, triploidy has been induced in pectinids (Tabarini 1984, Beaumont 1986, Komaru & Wada 1989, Ruiz-Verdugo et al. 2000, Yang et al. 2000, Maldonado-Amparo et al. 2004) and other mollusks of commercial interest (for review, see Beaumont & Fairbrothers 1991). The adductor muscle weight of triploid catarina scallops can be 182% that of diploids after 280 days of grow-out (Ruiz-Verdugo et al. 2000). In catarina scallop, reproduction has a significant impact on meat (muscle), because commercial scallops are harvested at 1 year (6-cm length), and first maturation age can be as early as 4 months or 2-cm length (Cruz et al. 2000). Although triploid muscle weight gain is assumed to result from less transfer of biochemical energy from the muscle to the gonads (Ruiz-Verdugo et al. 2001b), no studies have analyzed the effect of increased muscle energy reserves on muscle fiber growth (hypertrophy) or number (hyperplasia). An increase in muscle fiber numbers or size can have different effects on the texture of the muscle. The objective of this study is to compare triploid and diploid organisms of the same age and grown under the same conditions during a 1-year period, to determine to what extent sterility affects the biochemical reserves accumulated in muscle, and the size of adductor muscle cells.
MATERIAL AND METHODS
Adult catarina scallops (Argopecten ventricosus) were matured under laboratory conditions at CIBNOR, as described by Ramirez et al. (1999). For triploid induction, eggs were treated with 0.5 mg/L cytochalasin-B (CB) during 15 min, after 50% of eggs were in the first polar division, as described by Ruiz-Verdugo et al. (2000). Untreated diploid controls and treated organisms (containing triploids and diploids) were transported to Laguna Rancho Bueno (Bahia Magdalena, B.C.S., Mexico) where they were grown separately in Nestier trays at 3 m depth (Ruiz-Verdugo et al. 2000).
A total of 30 scallops (from both CB-untreated and treated groups) were collected at approximately monthly intervals from the grow-out area at Laguna Rancho Bueno from June 2000 to March 2001 and transferred to the laboratory at CIBNOR. Upon arrival, total live animal weights, and shell-free tissue weights (biomass) of all samples were recorded. Adductor muscle and gonad weight were recorded from July 2000 to the end of the study in March 2001. Organ indices for the two tissues were calculated as the proportion of organ wet weight to total tissue weight minus gonad weight, to eliminate any effect of on-going maturation.
GSI (%) = (gonad wet weight (g) x100)/(total wet biomass (g) - gonad wet weight (g))
MSI (%) - (muscle wet weight (g) x100)/(total wet biomass [g] - gonad wet weight [g])
Triploidy condition was verified individually in samples of mantle tissue using flow cytometry (Maldonado-Amparo et al. 2004). Untreated control organisms and CB-treated diploid scallops were grouped together for statistical analyses. True triploids ranged from 60% to 93.3% in the CB-treated group along each sampling period (Fig. 1), which is similar to the induced triploids of catarina scallop reported by Ruiz-Verdugo et al. (2001a).
[FIGURE 1 OMITTED]
Gonad development was evaluated macroscopically during sampling based on stages described by Sastry (1968)--immaturity, partial maturity, maturity, and spent--but including also a "non-active" stage. In addition, a portion of the gonad was fixed in formaldehyde, embedded in paraffin-paraplast mixture, sectioned (6-8 [micro]m), and stained with Harris hematoxylin-eosin (Humanson 1972). Maturation stages of 10 to 15 scallops per sampling were assessed using the classification for triploid and diploid organisms of this species proposed by Ruiz-Verdugo et al. (2000a), and Maldonado-Amparo and Ibarra (2002a).
A portion of the adductor muscle was fixed in 10% formaldehyde, embedded in paraffin-paraplast mixture, sectioned transversely (4-6 [micro]m), and stained with Harris hematoxylin-eosin (Humanson 1972). The number of fibers in a fixed area of striated muscle in diploid and triploid scallops was counted. The diameter of individual striated muscle fibers (n = 30 fibers) was assessed using an image analyzer (Image-Pro), and photographed using a microscope (x40). It was observed that the surrounding connective tissue allowed for an increase in muscle fiber size and it was assumed that this increase was not affecting the number of cells/ area.
Gonad and muscle samples were homogenized with a mechanical homogenizer in 1.5 mL of cold, saline solution (NaCl, 35 g/L) to obtain a crude extract, from which analyses for total protein, carbohydrates, and lipids, were done as described in previous works (Ruiz-Verdugo et al. 2001b, Racotta et al. 2003). Lipids were analyzed by the sulpho-phosphovanillin method after mixing 100 [micro]L of the crude extract with 1-mL sulfuric acid and heating the mixture to 90 [degrees]C (Barnes & Blackstock 1973). The amounts of gonad in July and August 2000 samples were not sufficient for analysis. Carbohydrates were analyzed after precipitating proteins from the crude extract with 20% trichloroacetic acid (1:2) and centrifuging at 3000 x g, 5 [degrees]C for 10 min and mixing the supernatant with four parts of anthrone solution (0.1% dissolved in 76% sulfuric acid), incubated 3 min at 90 [degrees]C and cooled to 4 [degrees]C to stop further reaction (Van Handel 1965). Soluble proteins were determined in diluted crude extract (1:5 with 0.1N NaOH) (Bradford 1976). The energy conversion factors used were 17.2 kJ/g for carbohydrates, 17.9 kJ/g for proteins, and 33.0 kJ/g for lipids, as described in Heras et al. (1998).
Data are reported as mean [+ or -] standard error (S). Two-way analyses of variance (ANOVA) followed by a Tukey test for unequal n post-hoc mean comparisons (Statistica Version 5.0) were used to assess significant differences among months of sampling and ploidy groups. The level of significance was set at P < 0.05. Organ indices were arcsine transformed for the analyses (Sokal & Rohlf 1981), but untransformed data are presented. When there was a significant interaction, a Tukey post hoe to evaluate for differences between diploid and triploid means was performed.
Scallop growth in terms of live weight and tissue biomass are shown in Figure 1. Total or live weight increased continuously from 0.6 g in June 2000, to 33.3 g for diploids and 62.5 g for triploids in March 2001, but significant differences were seen from November 2000 on. Biomass represented 36% of total weight in diploids and 45% in triploids by the end of the sampling period. In March 2001, the biomass of triploids was 88% higher than that of diploids, but biomass differences between diploids and triploids were significant by November 2000.
Gonad weights began to increase earlier in diploid than in triploid scallops, but triploid gonads reached larger weights by March 2001, 2.1 g or 61% higher than the 1.3 g of diploids at that same time (Table 1). Triploid scallops had generally lower gonadosomatic indices (GSI), and higher gonad water content than diploids (P < 0.01). Gonad carbohydrates decreased in the triploid group throughout the sampling period, and although the two were not significantly different, the interaction was. In general, there was a greater variation in gonad carbohydrate for triploids than for diploids, with the highest and lowest values both for triploid scallops in October 2000 and February 2001, respectively, which were significantly different to those in the diploid group. Protein and lipid concentrations in the gonads of diploid and triploid scallops were similar from September 2000 to January 2001, but by February 2001 proteins and lipids in diploid gonads increased significantly, decreasing again in March. In the gonads of triploid scallops no increase in protein and lipid was seen in the same period. Energy reserves were similar in gonads of diploid and triploid scallops from September 2000 to January 2001, but increased significantly in gonads of diploid scallops by February 2001, when energy reserves were the lowest ones for triploid scallops, and remained still higher than in triploids in March 2001.
Adductor muscle weight of triploids was 123% greater than that of diploids by the end of the sampling period (Table 2). The muscle index was larger in triploid organisms, except at the beginning of the sampling period, when it was similar to that of diploid scallops. Water content in diploid and triploid muscle was not significantly different, but showed differences related to sampling time, with the lowest values in January 2001 for both groups. Muscle carbohydrates and lipids varied seasonally in both ploidy groups, but were generally higher in triploid scallops. In contrast, no differences between diploid and triploid scallops in muscle protein and energy reserves were observed, although seasonal variations were observed in both, with the highest values in October 2000.
Gonad development of diploid scallops varied seasonally, with reproductive activity in July to August and in October 2000, although the distribution of gonads into different macroscopic stages showed mature and spent gonads throughout the sampling period (Fig. 2A). In contrast, triploid scallops were mostly inactive or immature, with partially mature gonads in October 2000, and January and March 2001, and mature gonads only in March 2001. However, no spent gonads were found in triploids during the sampling period. Gametogenesis evaluated in diploid and triploid gonads of catarina scallops during 382 days did not reveal maturation in gonads of triploid scallops (Ruiz-Verdugo et al. 2000). The distribution of the macroscopic stages was in accordance with the maturation stages evaluated by histology (Maldonado-Amparo & Ibarra 2002a).
[FIGURE 2 OMITTED]
The adductor muscle cells of triploids were significantly larger than those of diploids on all sampling dates (Fig. 3A). In January 2001, triploid muscle cells were 53% larger than muscle cells of diploid scallops. The largest difference in cell size was observed in February 2001, when the triploid muscle cell size was three times larger than that in diploids. In March 2001, the cells of triploid scallops increased 31% compared with February, and were 79% larger than those of diploid scallops. In contrast, the number of muscle fibers per area (fiber density) was not significantly different between ploidy groups or as a result of sampling time (see Fig. 3B).
[FIGURE 3 OMITTED]
One advantage of producing triploid mollusks is that individuals grow more (Allen & Downing 1986, Child & Watkins 1994, Hawkins et al. 1994, Hand et al. 1998, 1999, Kesarcodi-Watson et al. 2001). Several hypothesis have been proposed to explain the higher growth rate of triploids over diploids (Garnier-Gere et al. 2002). A genetic hypothesis is based on the expected higher heterozygosity of triploids (Guo & Allen 1994, Hawkins et al. 2000). Triploids may perform better because of the potential for faster transcription due to the three copies of the same gone (Magoulas et al. 2000). A physiologic hypothesis is based on the sterility of triploids, which would divert more metabolic flux to growth (Allen & Downing 1986, Hand et al. 1999). Guo and Allen (1994) proposed a third hypothesis (i.e., that greater growth in triploid mollusks results from larger size polyploid cells) based on the assumption that a larger nucleus requires a larger cytoplasm so that nutrients and organelles are adequately proportioned during cell division and growth.
Guo and Allen (1994) hypothesized that this resulted in individuals with polyploid gigantism, caused by increased cell volume and a lack of cell number compensation. They stated that the polyploidy gigantism hypothesis needs to be tested directly by studies on cell size, cell number, and organ size in diploids and triploids. Differences in cell size between triploid and diploid mollusks have been reported for eggs (Guo & Allen 1994, Eversole et al. 1996, Ruiz-Verdugo et al. 2001a), sperm (Maldonado-Amparo & Ibarra, 2002b), adductor muscle diameter (Gardner et al. 1996), gill tissue cells and hemolymph cell nuclei (Child & Watkins 1994). However, polyploid gigantism might be more apparent in some tissues than in others. For example, in bivalves, the adductor muscle seems particularly susceptible to polyploid gigantism (Guo & Allen 1994). In the present study, we observed that triploid catarina scallops had significantly larger muscle cells diameter (hypertrophy) than their diploid counterparts grown in similar conditions. However, the number of fibers (hyperplasia) in the adductor muscle of triploids was not significantly different from that of diploids, at least during the period tested. Thus, if the number of muscle cells did not decrease to compensate for hypertrophy, a lack of regulation in number of muscle cells is consistent with the polyploid gigantism hypothesis proposed by Guo and Allen (1994).
However, it is difficult to separate hypertrophy caused by polyplodism, and that caused by an increase in energy accumulated in storage tissues per se. This is because, although in most tissues bigger cells might compensate for fewer cells, storage tissues can follow a different pattern. As an example, the adipose tissue in mammals presents hypertrophy as a result of acylglycerides storage (Palacios et al. 1996). This usually happens in adult animals, and it is not accompanied by a decrease in cell number, although there can be an increase in the number of fat cells (hyperplasia). In this work, muscle carbohydrates in triploids increased 5-fold from January to February, and 2-fold from February to March 2001 (Table 2), corresponding to similar increases in muscle cell size during the same times (see Fig. 3A). In pectinids, where muscle cells are implicated in the storage and mobilization of nutrients to meet reproductive requirements (Mathieu & Lubet 1993), the observed glycogen storage in triploid adductor muscle could be a normal mechanism to accumulate excess energy during periods of high productivity. Thus, we should expect muscle hypertrophy when the conditions are adequate even in diploids, as was the case for diploids sampled at the end of the experiment and compared with those sampled in January 2001 (see Fig. 3A). Therefore, an increase in cell size can be compatible with either larger polyploid cell volume or changes in allocation of energy reserves from muscle to gonad.
The energy allocation hypothesis is based on the sterility or partial sterility of triploid mollusks, and is characterized by triploids growing larger because of energy diverted from reproduction to growth (Stanley et al. 1984, Allen & Downing 1986, Akashige 1990, Barber & Mann 1991, Shpigel et al. 1992, Hawkins et al. 1994, Hand et al. 1998). Differences in growth arc not usually detected until the organisms reach first sexual maturity, or until after first spawn (Stanley et al. 1984, Tabarini 1984, Barber & Mane, 1991, Beaumont & Fairbrothers 1991, Hand et al. 1998, Ruiz-Verdugo et al. 2001b). The losses attributable to spawning in a 40-mm shell length hard clam had been calculated by Ansell and Lander (1967) at 20% to 25% of the total energy used for growth. Eversole et al. (1996) concluded that this amount of energy diverted into growth rather than reproduction may account for the difference in size between diploids and triploids. In accordance, we observed higher energy values in the gonads of diploid scallops by the end of the sampling period (see Table 1) than can be interpreted as energy loss for growth. Guo and Allen (1994) concluded that unless diploids spawned thus decreasing their total weight, and triploids did not, the energy reallocation hypothesis could not explain the larger total weight in triploids before sexual maturation. However, in pectinids the adductor muscle is the most important storage tissue with the digestive gland secondary, shown by the decrease in weights during the reproductive period (Barber & Blake 1981, 1985, Epp et al. 1988, Couturier & Newkirk 1991, Martinez 1991, Pazos et al. 1997), and the effects of maturation on weight and condition index of the adductor muscle are significant. For example, Barber and Blake (1981) have shown that in A. irradians adductor muscle dry weight decreased by two-thirds during maturation. Muscle weight decline has been associated mostly with decreases in muscle glycogen, and on increase in gonad lipids (Comely 1974, Taylor & Venn 1979). A diploid scallop transforming muscle glycogen to lipids theoretically could lose wet weight during maturation without releasing eggs. This is because glycogen can be converted to lipids through lipogenesis in mollusks (Gabbot 1975), but glycogen is stored in a highly hydrated form; for each gram of stored carbohydrates, there are 4 to 5 g of stored water (Randall et al. 1998). In contrast, lipids are accumulated in tissues with very little water. Thus, to produce 1 g of lipid, an organism must use a much larger weight of glycogen. We observed that carbohydrate concentrations were lower in the muscle of diploid organisms in October 2000 and March 2001, whereas lipid concentrations in the gonads increased by the end of the sampling period, as expected for organisms going through gametogenesis-vitellogenesis and previously observed for the same species (Ruiz-Verdugo et al. 2001b). With no difference in carbohydrates and less total proteins and lipids in the gonads of triploid organisms, we might have expected a smaller gonad, but water content in triploid gonads was higher than in diploids, thus contributing to higher total gonad weight. In muscle of triploid scallops, we expect greater muscle weight, with no differences in water and protein and with more carbohydrates and lipids. Thus, greater total and muscle weights in triploid scallops theoretically could be attained without spawning.
In conclusion, triploid sterility increases adductor muscle weight in catarina scallops, through an increase in carbohydrate storage that affects the size of adductor muscle fibers (hypertrophy), which is a result of a lower energy allocation to gonads. Thus, the increase in muscle size in the gigantism hypothesis may be a direct consequence of the energy allocation hypothesis.
The authors thank Rosalio Maldonado, Juan H. Macliz, and Gabriel Gonzalez for support in the sample processing; Carmen Rodriguez-Jaramillo and Teresa Arteche for histologic processing of gonads and muscles; and English editing staff at CIBNOR. The authors also thank two anonymous reviewers for critical comments. This research was supported by SIMAC project BCS7001 and CONACyT project 28256B.
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E. PALACIOS, * I. S. RACOTTA, A. M. IBARRA, J. L. RAMIREZ, A. MILLAN AND S. AVILA
Programa de Acuacultura, Centro de Investigaciones Biologicas del Noroeste (CIBNOR), Mar Bermejo 195, Col. Playa Palo de Santa Rita, La Paz, B.C.S. 23090, Mexico
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