Effect of food concentration on protein and carbohydrate production during larval development of the sea urchin Lytechinus variegatus.
KEY WORDS: Lytechinus, larvae, development, proximate composition, proteins, carbohydrates, Florida, sea urchin
Studies on aquaculture of sea urchins have focused on postmetamorphic individuals (Robinson 2004). Little attention has been given to developmental stages although production of seed for aquaculture is a major undertaking (Sakai et al. 2004). Although fundamental to understanding nutrition of larvae, few studies have measured production of proximate constituents during development, probably because of the number of larvae required for analysis. Most studies have focused primarily on embryos for which large numbers are easily obtained (Cognetti 1982, Guidice 1973, Isono & Isono 1975, Yanigasawa 1975a, Yanigasawa 1975b). The embryonic stage involves cell division during which protein synthesis involves protein turnover (Guidice 1973, Leong & Manahan 1997). Net production begins with the feeding larva. Protein content of larval Lytechinus pictus increases quickly after feeding begins (Pace & Manahan 2006). Carbohydrate and protein increase in amount during larval development of Paracentrotus lividus (Fenaux et al. 1985, Fenaux et al. 1992b, George et al. 1990).
Although several studies have compared fed and starved echinoid larvae (Fenaux et al. 1980, George et al. 1997, Marsh et al. 1999, Sewell 2005, Pace & Manahan 2006, Pace & Manahan 2007), few have addressed the question of the effect of food concentration on larval production. Fenaux et al. (1988) reported food concentration affects growth (dimensional) in larval Paracentrotus lividus. Reitzel et al. (2005) fed larvae of two closely related sand dollar species (Mellita tenuis, Leodia sexiesperforata) limiting and nonlimiting food concentrations during the facultative period (when food is not required) or throughout development or during the obligate feeding period (when food is required) only. Large differences in newly meta morphosed individuals were found only for protein with L. sexiesperforata and carbohydrate for M. tenuis. Feeding during the facultative period increased the amount of carbohydrates and lipids at metamorphosis only for M. tenuis in the nonlimiting food concentration. The facultative period is the early feeding stage when feeding structures are least developed.
These observations suggest production of carbohydrate and protein are an integral part of sea urchin larval development and that capacity for their production progresses with development. Varying availability of food can provide insight into the capacity for production. In this paper we report proximate content of proteins and carbohydrates during development of larvae of the sea urchin Lytechinus variegatus, a candidate species for aquaculture (Watts et al. 1998), raised at two concentrations of food.
Sea urchins, Lytechinus variegatus (50-80 rum diameter) were collected at Lido Beach, west Florida in October and December 2004. They were maintained in aquaria at 21[degrees]C and 30 [per thousand] salinity with recirculating filtered sea water. They were fed three times a week with an artificial feed of encapsulated pellets obtained from A. L. Lawrence. Feces and uneaten food were removed, and about 20% of the water was replaced prior to feeding.
Sea urchins were spawned 3 mo after collection by injection of 1-2 mL of 0.5 M KCl. Eggs from several females were pooled and fertilized with a few drops of sperm from several males (Fenaux et al. 1992a, George et al. 1997). Fertilized eggs were placed into 1-L beakers with 1-[micro]m millipore-filtered and UV sterilized seawater (1 [micro]m). Fertilization success was >95%. A day after fertilization, the total number of embryos was estimated by counting five 1-mL samples from the beakers. Between 1,050-1,200 embryos were placed into each Imhoff cone, for a total of 10 cones (5 for each diet treatment) containing 1,000 mL of 1 [micro]m millipore-filtered, UV sterilized sea water with a continuous supply of about 50 small bubbles of air per minute (George et al. 2004). Feeding began 24 h after fertilization. Larvae were fed a mixed diet of Dunaliella tertolecta and Isochrysis galbana. The high concentration diet consisted of 1,500 and 4,500 cells [ml.sup.-1] [day.sup.-1] of each respectively, and the low concentration diet consisted of 150 and 450 cells [ml.sup.-1] [day.sup.-1] respectively (Schiopu & George 2004). Between 800-900 ml of the water was replaced three times a week (Mondays, Wednesdays and Fridays). The water in the Imhoff cone was poured into a 1-L beaker and the water was siphoned gently through a flat funnel measuring 5 cm in diameter with a mesh of 50-[micro]m. The Imhoff cone was washed with warm tap water and then rinsed with deionized water before filling it with filtered seawater and returning the larvae.
Larvae were collected from the cones twice a week. An equal number of larvae was taken from each cone to maintain the proportion of developing larvae among the cones. The larvae were placed into 1.5 mL Eppendorf tubes, centrifuged at x 30,000 rpm for 1 min and the water removed by siphon. The larvae were frozen at -4[degrees]C (Miloslavich & Dufresne 1994). Because echinoid larvae do not develop synchronously, especially in later larval stages (McEdward & Herrera 1999), stages were determined at each time point when at least 75% of the individuals were at the same morphological stage. Between 50 and 80 larvae were used for estimation of protein and between 100 and 120 larvae were used for estimation of carbohydrate. Because of the extraction of larvae during the experiment, larval density in the cones decreased over time and therefore, the concentration of algal cells available per larvae increased from the first to the last feeding day of the experiment (from 6-60 cells/larva/day for the high diet treatment and from 0.64 cells/ larva/day for the low diet treatment). This increase over time in food concentration per larva, incremented even more the difference between the two treatments (Fig. 1).
To determine the protein and carbohydrate content of the juveniles after metamorphosis, four weeks after fertilization, (eight days after rudiment development and four days after pedicellaria had started to develop in well fed larvae), all the larvae that remained from the high concentration diet were pooled together and placed into 1-L beakers with water from the aquaria in which the adults were kept (about 30 larvae/ beaker). To obtain metamorphosis more rapidly, the larvae were fed with a diet of 18,000 cells [ml.sup.-1] [day.sup.-1] of the combined diet (13,500 cells [ml.sup.-1] [day.sup.-1] of D. tertolecta and 4,500 cells [ml.sup.-1] [day.sup.-1] of I. galbana) When metamorphosis began, the juveniles were placed into 10 Eppendorf tubes with 12-27 individuals in each for protein and carbohydrate determinations (5 replicates each).
Measurement of Proximate Constituents
NaOH-soluble protein was measured by the Bradford method (Bio-Rad microassay procedure). Larvae in the Eppendorf tubes were homogenized in 200 [micro]L of 0.5 N NaOH with an Eppendorf pestle attached to a Grobet shaft machine, held overnight at 2[degrees]C, vortexed and absorbance measured at 595 nm. Protein concentrations of the standard curve varied between 1-10 [micro]g x mL. Bovine serum albumin was the standard.
[FIGURE 1 OMITTED]
Total carbohydrate was measured by a modification of the Herbert et al. (1971) phenol method as described in Penchaszadeh and Miloslavich (2001). Samples were homogenized as mentioned earlier in 1 mL of citrate buffer (0.1 M, pH 5.0) and held overnight at 2[degrees]C. Fifty [micro]L of 80% phenol were added, mixed with a vortex and held at 21[degrees]C for 40 min. then 5 mL of concentrated sulfuric acid was added and vortexed. Absorbance was measured at 480 nm after the samples cooled to room temperature. Carbohydrate concentrations of the standard curve were between 1 and 50 [micro]g [ml.sup.-1]. Sacharose was the standard.
Differences between the two diets and changes in the amounts of protein and carbohydrate throughout development were analyzed with a 2-way ANOVA (factors: high or low concentration diet, Time) using Statistica 6.0 with untransformed data. Differences in larval survival with the two diet treatments throughout the experiment was performed with an independent samples t-test, by using the difference between the initial and final counts of individuals per cone and subtracting the number of larvae collected for proximate analysis.
Percent survival with the high concentration diet (77 [+ or -] 18%, n = 5) and low concentration diet (66 [+ or -] 24%, n = 5) did not differ significantly (P = 0.470). Between 52% to 100% (mean: 77 [+ or -] 18, n = 5) of the larvae survived up to day 24 with the high concentration treatment, and between 28% to 86% (mean: 66 [+ or -] 24, n = 5) survived with the low concentration diet; the difference is not significant (t-test, P = 0.470). Larvae with the high concentration diet developed fully and metamorphosed after 30 days. Larvae with the low concentration diet developed more asynchronously. In one of the low treatment cones, all the larvae remained in the 4-arm stage, whereas in the other four cones, about 50% of the larvae reached the 8-arm stage but developed no further. Despite the morphological differences of the larvae between the two treatments, the 2-way ANOVA analyzing the interaction between food concentration and time showed no significant difference in the protein and carbohydrate content of poorly and well fed larvae (P = 0.3799 for proteins, and P = 0.0873 for carbohydrates). However, protein and carbohydrate larval content increased significantly in time (Table 1, Fig. 2 and Fig. 3). This increase only happened after day 20 for both constituents (ANOVA P = 0.0003 for proteins, and P < 0.0001 for carbohydrates), meaning that with both diets, protein and carbohydrate content remained relatively constant through day 17 after fertilization, significantly increased by day 20, and remained high until day 24. In larvae, day 20 coincides with the first appearance of the rudiment stage and day 24 coincides with the first appearance of pedicellariae. From this stage to metamorphosis, the increase in protein content was 3 fold, and the increase in carbohydrates was 5 fold (Table 2).
Development of Lytechinus variegatus in this work was much slower than reported by McEdward and Herrera (1999), who obtained full development of the rudiment and pedicellariae by days 8-9 at a higher temperature of 26[degrees]C and a richer diet either of 8,000 cells [ml.sup.-1] of Rhodomonas (8 days) or 8,000 cells [ml.sup.-1] Dunaliella (9 days). These authors also showed evidence that the larvae can complete development to metamorphosis very rapidly when culture conditions are suitable in terms of food. Mazur and Miller (1971) obtained metamorphosis of L. variegatus larvae in 33-37 days with a low food quality and high larval densities. In this work, we increased by 3-fold the amount of food given to the larvae to induce metamorphosis because larvae had reached 28 days and still showed no signs of metamorphic competence. This delay in metamorphosis in our experiment could be because of, in the first place, too low temperature (21[degrees]C), in comparison with that reported by McEdward and Herrera (1999) who obtained complete metamorphosis in 9-11 days at a temperature of 26[degree]C. Delay in metamorphosis could also be because of the fact that larvae were raised in cones, with the air bubbles coming from the narrow bottom, making settlement very difficult. Fenaux and Pedroti (1988) reported that echinoid larvae may still metamorphose in the water column, however, metamorphosis will be delayed for about 1-2 wk and survival may be lower. McEdward and Herrera (1999) also reported that growth (measured as arm length) of starved larvae of L. variegatus only reached the four-arm stage (postoral and anterolateral pairs). Growth of the starved larvae was equivalent to well fed larvae until day 4 after which they failed to grow any further and deteriorated. Difference in egg quality may have contributed to this difference. George et al. (1990) reported that different populations of Arbacia punctulata produced eggs with different protein and lipid content that affected rate of development.
[FIGURE 2 OMITTED]
Regarding the survival of the larvae during the experiment, and that no significant differences were observed between diets, the analysis performed (independent samples t-test) does not adequately represent the variation in the survival of the larvae during the time of the experiment because the remaining larvae in each cone were not counted at each collecting date. To correctly evaluate this factor, a formal survival analysis should be applied to the individual counts of each cone at each sampling time. The estimated survival curves could show evidences of different survival rates during intermediate times and present more solid evidences of the effect of the diet in growth, at the different stages of larval development. However, this additional manipulation of the larvae could have as a consequence an increase in mortality.
Measurement of changes in proximate content of sea urchin larvae during development has been probably hampered by the large number of individuals necessary for analysis. Table 3 summarizes some of the previous studies aimed to measure major biochemical components such as proteins, carbohydrates, and lipids in various premetamorphic stages of echinoid species, specifying the number of eggs, embryos or larvae necessary to carry out the analysis. The present work shows that by using the Imhoff cones, it is possible to carry out protein and carbohydrate analyses with the appropriate number of replicas without having to false huge amounts of larvae. This represents a useful advance in methodology to perform studies of larval bioenergetics.
[FIGURE 3 OMITTED]
Our analysis indicates that larvae raised at a low food diet, also increase significantly in energy despite they are only reaching the 4- or 8-arm stage, and their energy content is not significantly different from that of the larvae raised at a high food diet. A possible explanation for this is that such larvae have expressed phenotypic plasticity, a well-studied phenomenon in echinoderm larvae (Hart & Strathmann 1994, George 1999, Miner et al. 2005, Sewell et al. 2004). These studies indicate that larval response to different food availability is a species-specific trait, which depends in part on the level of dependence of the larvae to exogenous food (Sewell et al. 2004). Larvae respond to food scarcity by changing their morphology, however, such changes could be insufficient to ensure continued larval development under food-limiting conditions; suggesting that changes in internal morphology such as increase in the size of the digestive tract may also be necessary (George 1999). Hart and Strathmann (1994) reported that starved larvae develop a longer ciliated band than well-fed larvae, and demonstrated that such phenotypic plasticity is functionally significant, because longer ciliated bands allow higher feeding rates when food is scarce. Despite this morphological compensation, longer ciliated bands do not completely counterbalance for reduced food supply, because starved larvae needed more time to complete larval development and produced smaller juveniles in comparison with well fed larvae.
Leong and Manahan (1997) reported the rate of protein synthesis was low and protein content constant in Lytechinus pictus embryos. These increased when larvae began to feed, continuing to increase with time. Fenaux et al. (1992b) reported that the increase in protein and lipid in Arbacia punctulata larvae was greatest during rudiment development. McEdward (1984) similarly reported production in protein is greater during the period of rudiment development of Dendraster excentricus. We found significant increases in proximate content of protein and carbohydrate with larval development of Lytechinus variegatus, however, this increase happens after the feeding structures, the arms, have fully developed. The amount of production in this work is similar to that reported in the studies cited earlier. As reported in these studies, the major increase in protein and carbohydrate production occurs in the last part of development during rudiment formation. Allen et al. (2006) have discussed the implications for the evolution of large egg size and its importance for postlarval stages that result from the benefit of an increased energy supply to echinoderm larvae. Protein content changed in parallel with metabolic activity during larval development of Dendraster excentricus, both increasing greater in the late stages (McEdward 1984). McEdward (1984) pointed out that most of the growth throughout larval development occurs as an increase in arm length. This growth apparently requires little production but is more a change in dimension and larval form that increases feeding capacity. As mentioned earlier, phenotypic plasticity of feeding capacity determined by food availability occurs in sea urchin larvae. The length of the ciliated arm band relative to larval size of Lytechinus variegatus increases by an increase in arm length under conditions of low food supply (Boidron-Metairon 1988). Our results showed morphological differences between the larvae fed with different diets, however, these differences were not reflected in the protein and carbohydrate content of the larvae, indicating that such plasticity might not involve a cost at least for these two components. However, because plasticity in any form usually involves a cost of some type, the energetic expenditure to produce the plastic phenotype could come from another source such as lipids. Sewell (2005) studied lipid utilization during early development of Evechinus chloroticus and found that lipid stored during the planktonic larval phase, in combination with other proximate constituents of the larval body, provided the energy for the metamorphic and perimetamorphic periods during development.
Fenaux et al. (1985) reported that protein content of the pluteus larva of Paracentrotus lividus almost doubles from day 8 to day 10, approaching rudiment formation, whereas the carbohydrate content increases slowly but constantly from day 1 to day 16. This is the period during which larval P. lividus are most sensitive to food concentration (Fenaux et al. 1988). Fenaux et al. (1988) reported the increase in ingestion rate of with development of larval Paracentrotus lividus is similar to that of a logarithmic growth curve. It seems a threshold exists for a high capacity for production necessary to support rudiment formation. Rates of larval development of Sterechinus neumayeri were not affected by food availability from the four-arm to the six-arm stage (Marsh et al. 1999). During early larval development, respiration was accounted for by cell-specific metabolic rate, not cell number. During the advanced larval stage, size-specific metabolic rate was determined at a level of physiological regulation independent of cell numbers of feeding history.
Rates of development of larval Sterechinus neumayeri are not affected by food availability from the four-arm to the six-arm stage (Marsh et al. 1999). This observation can be related to the results of Reitzel et al. (2005). Reitzel et al. (2005) fed larvae of two closely related sand dollar species (Mellita tenuis, Leodia sexiesperforata) fed limiting and nonlimiting food concentrations during the facultative period (when food is not required) or throughout development or during the obligate feeding period (when food is required) only. Large differences in newly metamorphosed individuals were found only for protein with L. sexiesperforata and carbohydrate for M. tenuis. Feeding during the facultative period increased the amount of carbohydrates and lipids at metamorphosis only for M. tenuis in the nonlimiting food concentration. The facultative period is the early feeding stage when feeding structures are least developed. Our results indicate that even if fed, minimal requirements are necessary. The amount of production by larval Lytechinus variegatus fed the low concentration of food was insufficient for development to continue.
The larval stage of sea urchin development itself thus can be divided into two stages. The first involves growth of the larva that results in an increase in feeding capacity. After a threshold, sufficient feeding occurs for production in the last part of the second stage for formation of the rudiment. This involves production of protein and carbohydrate necessary for the newly metamorphosed individual. Because artificial food can support complete sea urchin larval development (George et al. 2004), it will be possible to manipulate quantity and quality of larval food to meet larval nutritional requirements.
The authors thank Elizabeth Huck, Universidad Simon Bolivar for her assistance in the laboratory. Comments by ah anonymous referee greatly improved the manuscript. This work was supported by the University of South Florida and by the Decanato de Investigacion y Desarrollo, Universidad Simon Bolivar, Caracas, Venezuela.
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PATRICIA MILOSLAVICH, (1,2) * JOHN M. LAWRENCE, (1) DANIELA SCHIOPU (1) AND EDUARDO KLEIN (2)
(1) Department of Biology, University of South Florida, Tampa, Florida 33620; (2) Departamento de Estudios Ambientales, Universidad Simon Bolivar, Caracas, Venezuela
* Corresponding author. E-mail: email@example.com.
TABLE 1. Regression results of protein and carbohydrate content in time with the two diet treatments. Low diet treatment Proteins [R.sup.2] = 0.127, P = 0.039 Carbohydrates [R.sup.2] = 0.623, P < 0.0001 High diet treatment Proteins [R.sup.2] = 0.193, P = 0.005 Carbohydrates [R.sup.2] = 0.559, P < 0.0001 TABLE 2. Chronology of development and proximate content of protein and carbohydrate ([micro]g/larva) during larval development of Lytechinus variegatus with a high concentration diet. Food concentration was increased 3-fold after 24 days to induce metamorphosis. (Numbers represent mean [+ or -] SD, n = 5 determinations, except for day 1: n = 10 determinations). For larvae, protein determinations had 50-80 larvae/replica and carbohydrate determinations had 100-120 larvae/replica. Juveniles had 12-27 juveniles/replica for both determinations. Age Stage Protein (Days) 1 24 h Fertilized egg 0.056 [+ or -] 0.021 6 4-arm pluteus 0.030 10 8-arm pluteus 0.047 [+ or -] 0.018 13 8-arm pluteus 0.076 [+ or -] 0.026 17 8-arm pluteus 0.064 [+ or -] 0.053 20 8-arm pluteus 0.115 [+ or -] 0.075 and rudiment 24 8-arm pluteus 0.121 [+ or -] 0.081 and rudiment. First appearance of pedicellariae >30 Juvenile at 0.371 [+ or -] 0.135 metamorphosis Age Carbohydrate (Days) 1 0.022 [+ or -] 0.013 6 0.017 [+ or -] 0.011 10 0.041 [+ or -] 0.015 13 0.049 [+ or -] 0.011 17 0.077 [+ or -] 0.039 20 0.078 [+ or -] 0.027 24 0.148 [+ or -] 0.070 >30 0.764 [+ or -] 0.201 TABLE 3. Summary of measurement of changes in proximate content of sea urchin larvae during development and number of larvae needed. Species Number of individuals Dendraster excentricus 5-50 larvae (according to stage) Arbacia lixula 2,000 eggs and 70-80 larvae and postlarvae Encope michelini 50-1,000 embryos and larvae (according to stage) Arbacia lixula Not stated iu original paper * 70-1,500 larvae Mellita isometra 250-500 larvae Mellita tenuis and Leodia 30-40 eggs and 2-8 juveniles sexiesperforata Lytechinus variegatus 50-80 larvae (protein) and 100-120 (carbohydrate) Species Analysis Dendraster excentricus Protein Arbacia lixula Protein and lipid Encope michelini Protein and lipid Arbacia lixula Protein, carbohydrate and lipid Mellita isometra Protein Mellita tenuis and Leodia Protein, carbohydrate sexiesperforata and lipid Lytechinus variegatus Protein and carbohydrate Species Reference Dendraster excentricus McEdward (1984) Arbacia lixula George et al. (1990) Encope michelini George et al. (1997) Arbacia lixula Fenaux et al. (1985, 1988) * George et al. (1997) Mellita isometra Schiopu and George (2004) Mellita tenuis and Leodia Reitzel et al. (2005) sexiesperforata Lytechinus variegatus Present work
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|Author:||Miloslavich, Patricia; Lawrence, John M.; Schiopu, Daniela; Klein, Eduardo|
|Publication:||Journal of Shellfish Research|
|Date:||Dec 1, 2007|
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