Effect of diet quality on nutrient allocation to the test and Aristotle's lantern in the sea urchin Lytechinus variegatus (Lamarck, 1816).
Article Type: Report
Subject: Sea urchins (Food and nutrition)
Authors: Heflin, Laura E.
Gibbs, Victoria K.
Powell, Mickie L.
Makowsky, Robert
Lawrence, Addison L.
Lawrence, John M.
Pub Date: 08/01/2012
Publication: Name: Journal of Shellfish Research Publisher: National Shellfisheries Association, Inc. Audience: Academic Format: Magazine/Journal Subject: Biological sciences; Zoology and wildlife conservation Copyright: COPYRIGHT 2012 National Shellfisheries Association, Inc. ISSN: 0730-8000
Issue: Date: August, 2012 Source Volume: 31 Source Issue: 3
Geographic: Geographic Scope: United States Geographic Code: 1USA United States
Accession Number: 303011414
Full Text: ABSTRACT Small, adult (19.50 [+ or -] 2.01g wet weight) Lytechinus variegatus (Lamarck, 1816) were fed 8 formulated diets with different protein (12-36% dry weight as fed) and carbohydrate (21-39% dry weight) levels. Each sea urchin (n = 8 per treatment) was fed a daily ration of 1.5% of the average body weight of all individuals for 9 wk. Akaike information criterion (AIC) scores were used to compare 6 different dietary composition hypotheses for 8 growth measurements. For each physical growth response, different mathematical models representing a priori hypotheses were compared using the AIC score. The AIC is one of many information theoretical approaches that allows for direct comparison of nonnested models with varying numbers of parameters. Dietary protein level and protein:energy ratio were the best models for prediction of test diameter increase. Dietary protein level was the best model of test with spines wet weight gain and test with spines dry matter production. When the Aristotle's lantern was corrected for size of the test, there was an inverse relationship with dietary protein level. Log-transformed lantern to test with spines index was also best associated with the dietary protein model. Dietary carbohydrate level was a poor predictor for growth parameters. However, the protein x carbohydrate interaction model was the best model of organic content (percent dry weight) of the test without spines. These data suggest that there is a differential allocation of resources when dietary protein is limiting and the test, but not the Aristotle's lantern, is affected by availability of dietary nutrients.

KEY WORDS: Aristotle's lantern, sea urchin, growth allometry, Lytechinus variegatus

INTRODUCTION

Natural environments are often highly variable. To maximize fitness, organisms need the ability to adjust not only from generation to generation, but also at the individual level (plasticity), with changes occurring within a lifetime (Newman 1992, Scheiner 1993, DeWitt 1998). The energetic cost associated with maintaining plasticity of traits is higher than that of maintaining a fixed trait (Newman 1992, Scheiner 1993, DeWitt 1998, Scheiner & Berrigan 1998, Lau et al. 2009). However, the additional energetic investment may be worthwhile if survival of the individual is increased. Phenotypic plasticity is important in plants (Schlichting 1986) and in animals that are either sessile or unable to travel quickly to a habitat with more suitable conditions (Ebert 1996, Russell 1998). Given the sedentary nature of most adult echinoderms, a high degree of plasticity would be predicted.

Phenotypic plasticity is an adaptive response to an environmental change or stress (Ebert 1980, Newman 1992, Russell 1998, Miner 2005). Plasticity may be interpreted as normal progression and variation in the pattern of iso- or allometric organ growth, defined herein as modeling. Alternatively, plasticity could be interpreted as a regression, including resorption (or shrinking) or shifting (movement), of organs in response to a biotic or abiotic stressor. This regression leading to an altered progression of growth would be defined as remodeling.

Low food quality and low food availability are both stressors that are suggested to lead to differential phenotypic expression in adult echinoderms (Ebert 1996, Fernandez & Boudouresque 1997, Guillou et al. 2000). Plasticity (relative increase or decrease in size) of the feeding apparatus (Aristotle's lantern) has been reported in several echinoderm species (Ebert 1968, Ebert 1980, Black et al. 1982, Fansler 1983, Black et al. 1984, Levitan 1991, Constable 1993, Fernandez & Boudouresque 1997, McShane & Anderson 1997, Guillou et al. 2000, Hagen 2008, Lau et al. 2009). In contrast, both Russell (1998) and Lawrence et al. (1996) observed no significant differences in relative lantern size among Strongylocentrotus droebachiensis (Muller, 1776) and Tetrapygus niger (Molina, 1782) fed varying feeds or varying feed rations.

The functional significance of a comparatively large jaw apparatus is not entirely understood. It has been suggested that the increased size may, in part, be an effect of enlarging the muscles associated with the jaws as a result of an increased scraping effort (Ebert 1980). Another explanation is that a large lantern may help an individual obtain more food because it allows the urchin to graze upon a larger surface area (Ebert 1980, Ebert 1996, Black et al. 1984, Lawrence et al. 1995, Minor & Scheibling 1997, Lau et al. 2009).

The effect of specific nutrients on the relationship of the size of the test to the lantern has only been investigated in 1 study. Jones et al. (2010) suggested that dietary manipulation of the mineral selenium affected the ratio of dry lantern weight to the dry weight of the test with spines, indicating a specific nutrient can affect phenotypic variation in lantern and/or test size.

Comparatively, those nutrients that contribute to energy production or utilization may ultimately affect body organ allometry. In sea urchins, protein and carbohydrate are the 2 primary energy sources used for maintenance and growth (Marsh & Watts 2007). Protein, carbohydrate, and their value as anabolic precursors and energy sources affected total body weight gain in Lytechinus variegatus (Heflin 2010). Dietary changes in their profiles could potentially affect organ growth of the test, lantern, and spines. The purpose of this study is to examine the effect of variations in dietary protein and carbohydrate level, protein:energy ratio, protein:carbohydrate ratio, and total dietary energy level in relation to the size of the test relative to the lantern in growing L. variegatus. The results of this study will provide an understanding of allometric growth of calcified tissues in relation to nutrition, and has implications in optimizing strategies for mass culture of sea urchins.

MATERIALS AND METHODS

Collection and Initial Measurements

Small adult L. variegatus (ca. 19.50 [+ or -] 2.01 g initial wet weight) were collected from St. Joseph Bay (30[degrees] N, 85.5[degrees] W), FL, and transported to Texas AgriLIFE Mariculture Research Laboratory in Port Aransas, TX. Nineteen individuals were selected randomly for initial evaluation. Individuals were weighed (to the nearest milligram) and photographed for diameter measurements (to the nearest 0.01 mm) using ImageJ software. Urchins were dissected by making a circular incision around the peristomial membrane. The gut, gonads, and Aristotle's lantern (including muscles and pharynx) were removed. The interior surface of the test was scraped with a spatula to remove any soft tissue. The test and lantern were blotted with paper towels to remove excess moisture. The test with spines (including peristomial membrane) and Aristotle's lantern were weighed to the nearest milligram. The tests with spines and the lanterns were dried at 60[degrees]C for 48 h to a constant weight. Mean dry lantern and test with spines weights were calculated for the initial sample and used as an estimate for initial dry lantern and dry test with spines weights for urchins (n = 64) used in the study. Initial test diameters were measured for these urchins, and individuals were assigned randomly to 1 of 8 dietary treatments (n = 8 per diet). Initial test diameters did not differ significantly among dietary treatments.

Culture Conditions

Sea urchins were held in a semirecirculating system with both mechanical and biological filtration and UV sterilization of the seawater. The culture system (2,400 L) consisted of 16 interconnected 20-L fiberglass tanks containing water distributed from a central sump. Each tank held 4 cylindrical plastic mesh cages (diameter, 12 cm; height, 30 cm; open mesh, 4 mm). Each plastic cage was inserted into a PVC coupling (internal diameter, 11.5 cm) and elevated with PVC spacers to allow unimpeded seawater circulation throughout the cage. Each cage housed one individual. Cages were identical to eliminate the potential effects of vessel shape on test phenotype (Hernandez & Russell 2010).

Water volume in each tank was maintained by a central standpipe, and natural seawater was supplied to each mesh enclosure at an approximate rate of 25 L/hr (water exchange rate, 3,000% per day). Fresh seawater was passed through a sand filter and a stratified Diamond water filter (Diamond Water Conditioning, Horton, WI). Seawater in the entire culture system was exchanged at a rate of 10% per day. Water quality parameters measured included total ammonia nitrogen, nitrite nitrogen, nitrate nitrogen, pH, temperature, and salinity based on methods modified from those of Mullen and Riley (1955), Solarzano (1969), Strickland and Parsons (1972), and Spotte (1979).

Diet and Diet Preparation

Eight semipurified diets were formulated and produced using both purified and practical ingredients. Levels of dietary protein and carbohydrate (Tables 1 and 2) ranged from 12-36% protein (using purified plant and animal sources) and 21-39% carbohydrate (using a purified starch source). Levels of protein and carbohydrate were adjusted with acid-washed diatomaceous earth, which has no effect on sea urchins at these levels (unpubl. data). All other nutrients were constant among treatments. The proximate components are shown in Table 2.

Dry ingredients were mixed with a PK twin shell blender for 10 min. Dry ingredients were then transferred to a Hobart stand mixer and blended for 40 min. Liquid ingredients were added, and the mixture was blended for an additional 10 min to a mashlike consistency. The diets were extruded using a meat chopper attachment fitted with a 4.8-mm die. Feed strands were separated and dried on wire trays in a forced air oven (35[degrees]C) for 48 h. Final moisture content of all feed treatments was 8-10%. Feed was stored in air-tight storage bags at 4[degrees]C until used.

Feeding Rate

Each sea urchin was proffered a limiting daily ration (subsatiation) equal to 1.5% of the initial average wet body weight of all individuals. Feeding at subsatiation ensured that urchins consumed all food proffered within a 24-h period and allowed for direct measure of feed intake. A subsatiation feeding regime also prevented individuals from compensating for a dietary deficiency by increasing consumption (Taylor 2006). Individuals were photographed for diameter measurements every 3 wk, and feed rations were adjusted to be equivalent to 1.5% of the average body weight at that time. Consumption of all food proffered was confirmed by direct observation. Feces were removed by siphon immediately prior to feeding each day.

Daily feeding rate was calculated as

Average wet weight of individuals in the study (g) x 0.015 (1)

Protein:energy ratio of each feed was calculated as

Protein (mg)/Energy content (Kcal) (2)

Protein:carbohydrate ratio of each feed was calculated as

Protein (mg)/Carbohydrate (mg) (3)

Total energy content of each feed was calculated with the caloric equivalents of Phillips (1972)

%Protein/100 x 5,650 (cal/g)+ %Carbohydrate/100 x 4,000 (cal/g) + %Lipid/100 x 9,450 (cal/g) (4)

After 9 wk, urchins were dissected as previously described.

Growth

To measure test diameters, urchins were photographed from above and diameters were measured (0.01 mm) using ImageJ software. For each individual, 2 diameters were recorded across the ambitus (each perpendicular to the other) and averaged. Test diameter was measured every 3 wk. Diameter increase was calculated as

Final diameter (mm) - Initial diameter (mm) (5)

Estimated Aristotle's lantern and test with spines dry matter production were calculated for each individual as

Final dry weight of lantern (or test with spines and peristomial membrane (g)) - Initial average dry weight of lantern (or test with spines and peristomial membrane (g)) (6)

Final dry Aristotle's lantern or test with spines index of an individual was calculated as

Dry weight of Aristotle's lantern (or test with spines and peristomial membrane (g)) - Dry weight of individual (g) X 100 (7)

Final dry Aristotle's lantern to final dry test with spines index was calculated as

Dry weight of Aristotle's lantern (g)/Dry weight of test with spines and peristomial membrane (g) x 100 (8)

Percent Organic Matter of Test and Spines

For each individual, a quarter section of the dry test was analyzed, with oral and aboral plates and spines removed from a lateral quadrant (not including the peristomial membrane). Test plates and spines were placed separately in preweighed crucibles. The combined crucible and tissue weight was recorded, and tissues were "ashed" in a muffle furnace at 500[degrees]C for 4 h. After cooling, the combined weight of the crucible and tissue was recorded, and the percent organic matter was calculated as

(Dry tissue weight - Weight of inorganic matter (g))/ Dry weight of tissue (g) X 100 (9)

Statistics

To determine the relationship between carbohydrate and protein levels on lantern and test growth measurements, multiple linear regressions were conducted in R 2.11.1 (www.r-project. org). We opted to model the independent variables on a linear scale (deemed appropriate after examining the residuals) as opposed to a categorical scale (i.e., ANOVA-type analysis) to have maximal statistical power. For each physical growth response, different mathematical models representing a priori hypotheses were compared using the Akaike information criterion (AIC) score (Burnham & Anderson 2002). The AIC is one of many information theoretical approaches that allows for direct comparison of nonnested models with varying numbers of parameters. The a priori hypotheses compared were that lantern and test size (see Table 3 for the complete list of response variables examined) are dependent on: (1) protein level, carbohydrate level, and their interaction; (2) total energy; (3) the protein:energy ratio, and (4) the protein:carbohydrate ratio (descriptions of equations used to derive values are provided earlier). Because initial analyses showed that, at the dietary levels used, the interaction between protein and carbohydrate levels as well as carbohydrate levels themselves were often statistically unimportant, 2 parameter-reduced models were considered. These included models with protein and carbohydrate level, and only the protein level. Model fit was checked by examining the residuals for normality and homoscedasticity visually. For the lantern/total size index, lantern was included as the response and total size was a covariate. One response variable, the lantern/test index, was log transformed to satisfy linearity more completely.

[FIGURE 1 OMITTED]

RESULTS

Water Quality

Water conditions were maintained as follows: salinity, 32 [+ or -] 0.5 ppt; DO, 22 [+ or -] 2[degrees]C; ammonia, 7 [+ or -] 2 ppm; nitrite, <0.1 ppm; nitrate, <0.1 ppm; and pH, 8.2. A 12-h:12-h light:dark photoperiod was maintained.

[FIGURE 2 OMITTED]

Test and Spines Analyses

The best models of test diameter increase included dietary protein level and protein:energy ratio (Table 3). Parameter estimates indicated test diameter of individuals increased by 0.19 mm for every 1%increase in dietary protein level (Table 4, Fig. 1), and by 0.09 mm for every 1 mg protein/kcal increase in protein:energy ratio (Table 4).

The dietary protein level model was also the best predictor of test with spines wet weight gain and dry matter production (Table 3). Test with spines wet weight gain and dry matter production were directly proportional to protein consumption (Table 5, Figs. 2 and 3). Parameter estimates showed that test with spines wet weight of individuals increased by 0.201 g, and dry matter production increased by 0.085 g for every 1%increase in dietary protein level (Table 4). Dietary carbohydrate level was a poor model of test with spines growth in terms of diameter increase, wet weight gain, and dry matter production.

[FIGURE 3 OMITTED]

[FIGURE 4 OMITTED]

Organic content (measured as a percentage) of the test (without spines) varied among dietary treatments (Table 5). The protein:carbohydrate interaction effect model was best associated with variations in organic content (Table 3, Fig. 4). When the carbohydrate level was held constant, organic content of the test varied directly with dietary protein at levels less than 19%. However, at dietary protein levels greater than 19%, there was an inverse correlation between dietary protein level and test organic content. Test organic content correlated inversely with the dietary carbohydrate levels used in this study.

Lantern Analysis

Lantern wet weight gain (corrected for size of the individual) and dry matter production were found to be associated minimally with the predictor variables explored (Table 6, all [R.sup.2] < 0.01). As a result, we excluded these response variables from the tables and figures.

Lantern to Test with Spines Index

The dietary protein level model was the best predictor of the log-transformed dry lantern to dry test with spines index (Table 3, Fig. 5). Parameter estimates showed that for every 1% increase in dietary protein, the log of the lantern to test with spines index decreased by 0.011 (P < 0.001, Table 4).

DISCUSSION

Water-quality parameters maintained in this study were within acceptable ranges for sea urchins (Basuyaux & Mathieu 1999). This is supported further by the high survivorship and high growth rates exhibited by all treatments.

Among all factors examined, growth of individuals varied within dietary treatments. This variation can be attributed to intrinsic, most likely genetic, differences in growth rates among individuals (Pawson & Miller 1982, Grosjean 2001, Vadas et al. 2002; unpubl, data).

Aside from intrinsic differences in growth rates, the growth rate of sea urchins, like most organisms, is influenced greatly by the quality and quantity of food available (Lawrence & Lane 1982). Urchins increase growth rate with an increase in intake of nutrients. Because feed rations in this study were below satiation, any variations in the relationship of the size of the test with spines to the size of the Aristotle's lantern can be attributed directly to variations in nutrition quality instead of nutrient quantity. In addition, feeding at defined levels below satiation removed the potential effect of compensatory feed intake.

During severe food limitation, sea urchins may exhibit plasticity of the test, remodeling the body wall to use stored nutrients for metabolism, which results in a decrease in test size (Ebert 1968, Ebert 1980, Levitan 1991, Guillou et al. 2000). This process may be slow to occur (Lares & Pomory 1998) and, although it may increase fitness of an individual (because a smaller body size requires fewer resources to maintain (Ebert 1996)), test remodeling may be costly energetically and probably occurs only under conditions of extreme food restriction. Lares and Pomory (1998) starved L. variegatus for 2.5 mo and did not observe changes in test diameter; however, nutrient stores in the test of starved individuals decreased, suggesting that the test diameter of L. variegatus may shrink under extended conditions of extreme food restriction. Individuals in the current study were not starved or subjected to significant food restriction, and all tests increased in diameter and weight. These data suggest that the nutrient limitations in this study were not restrictive enough to induce an adaptive plastic response in test remodeling.

[FIGURE 5 OMITTED]

Dietary protein levels influence test growth directly in sea urchins (reviewed by McBride et al. (1998)). Hammer et al. (2006) reported larger diameters and higher wet and dry test weights in L. variegatus fed 20% protein compared with individuals fed 9% protein. McBride et al. (1998) found no differences in test diameter among Strongylocentrotus franciscanus (Agassiz, 1863) fed prepared diets with protein levels ranging from 30-50%. Although diets used in these studies were not isocaloric, the observed growth differences in the current study were not the result of differences in dietary energy, but it seems they were affected primarily by dietary protein content. The 12% protein diets, although adequate for maintenance and survival, do not provide enough protein for maximal test with spines growth.

Dietary protein and carbohydrate levels both correlated with nutrient storage in the body wall. The concentration (percent dry weight) of organic matter in the tests of urchins fed diets with 12% protein was lower than that of urchins fed diets with 19% protein. However, at the 19% and 12% levels of dietary protein, we observed an inverse relationship between dietary carbohydrate level and the concentration of organic matter in the test, with the largest effect observed at the 19% protein level. We hypothesize that, under the conditions of this study, high levels of dietary carbohydrate in some way inhibited the processing and/or assimilation of protein. Because protein is the major component of the sea urchin test (Lawrence & Lane 1982), we believe that the difference in organic matter concentration in the test indicates most likely that minimal protein was allocated to storage in the test of the individuals fed 12% protein, suggesting this level is not adequate for optimal test growth and nutrient storage. Greater than 19%, the decrease in percent organic matter suggests that, in addition to dietary protein and carbohydrate levels, another factor also influenced the storage of organic matter in the tests of these individuals. We hypothesize that the high metabolic cost of absorbing and assimilating high levels of dietary protein may decrease the deposition of organic matter to the test. A similar trend was observed by Hammer et al. (2006) in L. variegatus fed at 9%, 20%, and 31% protein (decrease observed at 31% protein).

Final wet weight gain and dry matter production of the Aristotle's lantern did not vary among treatments, indicating little or no variation in the growth rate of the lantern in response to large differences in dietary protein and carbohydrate content during the 9-wk study period. Although the size of the lantern did not vary among treatments, the relative size of the lantern (indexed to the size of the test with spines) did vary significantly with diet, primarily as a result of differences in test growth. These data indicate that growth rates of the test with spines increased directly with dietary protein, but the growth rates of the lantern did not, under the conditions of this study. Consequently, resource allocation to the test with spines varied with the quality of the diet (particularly protein), but resource allocation to the Aristotle's lantern remained constant, regardless of dietary quality.

In this study, specific nutrients affected resource allocation to the test and lantern in L. variegatus, inducing allometry in calcified tissues. Thus, allometry of calcified tissues in sea urchins appears to be influenced not only by the quantity of food available, but also by availability of specific nutrients. Future studies can use varying levels of specific nutrients proffered at varying rations to evaluate the effects of both food quality and quantity (including starvation) on modeling and remodeling of the calcareous tissues (test and lantern).

ACKNOWLEDGMENTS

We thank Jeff Barry, Patty Waits Beasley, and the rest of the staff at the Texas AgriLIFE Research Mariculture Laboratory for providing technical support and facilities for this study. We thank Dorothy Moseley and Warren Jones for technical assistance. This report was prepared by S. A. W. under award NA07OAR4170449 from the University of Alabama at Birmingham, U.S. Department of Commerce. R. M. was funded through postdoctoral training grant T32 HL072757. The statements, findings, conclusions, and recommendations are those of the authors and do not necessarily reflect the views of NOAA or the U.S. Department of Commerce.

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LAURA E. HEFLIN, (1) * VICTORIA K. GIBBS, (2) MICKIE L. POWELL, (1) ROBERT MAKOWSKY, (3) ADDISON L. LAWRENCE (4) AND JOHN M. LAWRENCE (5)

(1) University of Alabama at Birmingham, Department of Biology, 1720 2nd Avenue South, CH 374, Birmingham, A L 35294; (2) Villanova University, 800 Lancaster A venue, Mendel Hall Rm G63C, Biology, Villanova, PA 19085; (3) FDA-Center for Biologics Evaluation and Research, 1401 Rockville Pike, HFM 210, Rockville, MD 20852; (4) Texas A & M University, AgriLife Research Mariculture Laboratory, 1300 Port Street, Port Aransas, TX 78343; (5) University of South Florida, Department of Integrative Biology, 4202 East Fowler Avenue, SCA 132, Tampa, FL 33620

* Corresponding author. E-mail: leheflin@uab.edu

DOI: 10.2983/035.031.0335
TABLE 1.
Calculated protein (P) and carbohydrate (C) levels, total
energy, protein:energy ratios, and protein:carbohydrate ratios
in each of the 8 diets.

Protein   Carbohydrate    Total     Protein:       Protein:
(% dry       (% dry      Energy      Energy      Carbohydrate
weight)     weight)      (cal/g)   (mg P/kcal)   (mg P:mg C)

36             21         3,749        95            1.7
28             30         3,299        76            0.93
19             21         2,783        68            0.90
19             30         3,130        60            0.63
19             39         3,478        54            0.49
12             21         2,380        50            0.57
12             30         2,727        44            0.40
12             39         3,075        39            0.31

TABLE 2.
Proximate composition of the formulations used to produce diets
varying in protein and carbohydrate levels.

                      12%       12%       12%       19%
                    P:21% C   P:30% C   P:39% C   P:21% C

Crude protein (%)   12        12        12        19
Carbohydrate (%)    21        30        39        21
Fiber (%)            4.5       4.5       4.5       4.5
DE (%)              27        18         9        19
Non-DE ash (%)      24        24        24        24
Crude fat (%)        7         7         7         7

                      19%       19%       28%       36%
                    P:30% C   P:39% C   P:30% C   P:21% C

Crude protein (%)   19        19        28        36
Carbohydrate (%)    30        39        30        21
Fiber (%)            4.5       4.5       4.6       4.6
DE (%)              10         1         0         0
Non-DE ash (%)      24        24        24        25
Crude fat (%)        7         7         7         7

All values are calculated, and on an as-fed basis. All diets
contain up to 28% marine ingredients, 28.7% plant ingredients,
1.1 % carotenoids, 0.7% vitamin premix, 24% mineral mix, 7.2%
binder, and antifungal-antioxidant agents. C, carbohydrate; DE,
diatomaceous earth; P, protein.

TABLE 3.
Akaike information criterion scores for each growth model.

Response                  P + C +     Protein +        Protein
Variable                  (P X C)    Carbohydrate   (% dry weight)

Test with spines wet      243.02     241.09         239.14
  weight gain (g)
Test with spines dry      150.94     148.94         146.97
  matter production (g)
Log (lantern/test with    -70.58     -70.93         -72.91
  spines index)
Test diameter             298.36     287.40         285.40
  increase (mm)
Test percent              162.8      167.5          165.5
  organic matter *

                          Total Energy
Response                   (cal/g dry        P:E           P:C
Variable                    weight)      (mg P/kcal)   (mg P/mg C)

Test with spines wet      250.10         241.35        245.26
  weight gain (g)
Test with spines dry      157.18         149.44        151.99
  matter production (g)
Log (lantern/test with    -64.25         -69.92        -67.52
  spines index)
Test diameter             290.99         285.74        288.69
  increase (mm)
Test percent              177.2          173.65        182.14
  organic matter *

* For this response, all models included a quadratic term
(protein in the multiple regression).

Rows are the response variable; columns are the variables in the
model. Scores are only comparable for models with the same
response variable. All models within one information unit of the
best model are in bold type, whereas the best model is in bold
type and underlined.

P+ C + (P X C) = Protein + Carbohydrate + (Protein X
Carbohydrate).

P:C, protein: carbohydrate ratio; P:E, protein:energy ratio; TE,
total dietary energy.

TABLE 4.
Parameter estimates and tests of significance for various measures
of Lytechinus variegatus growth models.

                                    Separate Effects

                                  Protein          Carbohydrate
                                  (% dry              (% dry
Response Variable                 weight)             weight)

Test with spines wet           0.201 ***                --
  weight gain (g)
Test with spines dry           0.085 ***                --
  matter production (g)
Log (lantern/test with        -0.011 ***                --
  spines index) ([dagger])
Lantern/total index           -0.005 **                 --
Diameter increase (mm)         0.19 ***                 --
Test percent organic           0.989 ***          0.162 *
  matter (quadratic term)    (-0.015 ***)
Spines percent                      --                  --
  organic matter

                                     Combined Effects

                                                   Total energy
                                 Protein X          (cal/g dry
Response Variable              Carbohydrate           weight)

Test with spines wet                --             0.0031 ***
  weight gain (g)
Test with spines dry                --             0.0013 ***
  matter production (g)
Log (lantern/test with              --            -0.00017 ***
  spines index) ([dagger])
Lantern/total index                 --            -0.00007 *
Diameter increase (mm)              --                   --
Test percent organic         -0.01 *               0.013 **
  matter (quadratic term)                        (-0.000002 *)
Spines percent                      --                   --
  organic matter

                                     Combined Effects

                                 Protein:            Protein:
                                  Energy           Carbohydrate
Response Variable               (mg P/kcal)         (mg P/mg C)

Test with spines wet           0.089 ***           4.498 ***
  weight gain (g)
Test with spines dry           0.037 ***           1.912 ***
  matter production (g)
Log (lantern/test with        -0.0049 ***         -0.248 ***
  spines index) ([dagger])
Lantern/total index           -0.0019 *           -0.092 *
Diameter increase (mm)         0.09 ***            3.29 ***
Test percent organic           0.207 ***           4.08 *
  matter (quadratic term)    (-0.0014 **)        (-1.73 *)
Spines percent                 0.015 *                   --
  organic matter

([dagger]) Parameters shown for this response are given in terms
of the log scale of the lantern to test with spines index but can
be converted for any dietary protein level (as a percentage) by
using [e.sup.-0.011] x protein level.

Only statistically significant terms (P < 0.05) are included (if
an interaction was found to be significant, main effects were
included regardless of associated P values). Associated P values
for parameter estimates being significantly different than 0 are
included as * P < 0.05, ** P < 0.01, and P < 0.001.

C, carbohydrate; P, protein.

TABLE 5.
Mean values ([+ or -]SEM) at the end of the experiment for test
with spines wet weight, test with spines wet weight gain, dry
test with spines index, test with spines production test diameter
increase, and test and spine organic matter of Lytechinus
variegatus fed diets with varying protein and carbohydrate
levels, protein:energy ratios (P:E), total energy (TE), and
protein:carbohydrate ratios (P:C).

                           P:E                   P:C
Protein   Carbohydrate    (mg P/       TE        (mg/
(%)           (%)         kcal)     (cal/g)      mg)

12                  21       50.8      2,380       0.57
12                  30       44.3      2,728       0.40
12                  39       39.3      3,075       0.31
19                  21       68.6      2,783       0.90
19                  30       60.9      3,131       0.63
19                  39       54.9      3,478       0.49
28                  30       76.9      3,647       0.93
36                  21       95.6      3,749       1.71

               Final Wet
               Test with               Wet Test
                 Spines              with Spines
Protein          Weight                 Weight
(%)               (g)                  Gain (g)

12        13.72 [+ or -] 0.69     6.87 [+ or -] 0.69
12        14.65 [+ or -] 0.57     7.80 [+ or -] 0.57
12        14.57 [+ or -] 0.65     7.72 [+ or -] 0.65
19        16.60 [+ or -] 0.68     9.75 [+ or -] 0.68
19        16.39 [+ or -] 0.90     9.54 [+ or -] 0.90
19        15.31 [+ or -] 0.41     8.46 [+ or -] 0.41
28        18.22 [+ or -] 0.97    11.37 [+ or -] 0.97
36        18.84 [+ or -] 0.57    11.99 [+ or -] 0.57

                Dry Test              Final Dry
              with Spines             Test with
Protein        Production               Spines
(%)               (g)                 Weight (g)

12         3.50 [+ or -] 0.33     7.19 [+ or -] 0.33
12         3.77 [+ or -] 0.24     7.46 [+ or -] 0.24
12         3.89 [+ or -] 0.33     7.58 [+ or -] 0.33
19         4.56 [+ or -] 0.29     8.26 [+ or -] 0.29
19         4.44 [+ or -] 0.36     8.14 [+ or -] 0.36
19         3.91 [+ or -] 0.25     7.60 [+ or -] 0.25
28         5.39 [+ or -] 0.42     9.08 [+ or -] 0.42
36         5.61 [+ or -] 0.25     9.31 [+ or -] 0.25

                 Final                   Test
                  Test                 Diameter
Protein         Diameter               Increase
(%)               (mm)                   (mm)

12        49.94 [+ or -] 0.97    11.19 [+ or -] 0.91
12        49.96 [+ or -] 0.97    11.74 [+ or -] 0.82
12        50.38 [+ or -] 0.78    12.23 [+ or -] 0.69
19        52.47 [+ or -] 0.84    14.39 [+ or -] 0.76
19        50.89 [+ or -] 0.67    12.80 [+ or -] 0.88
19        51.02 [+ or -] 1.78    12.63 [+ or -] 1.43
28        53.47 [+ or -] 0.68    15.47 [+ or -] 1.17
36        55.37 [+ or -] 0.82    16.30 [+ or -] 1.22

                  Test
                Organic
                 Matter
Protein          (% dry
(%)             weight)

12        12.36 [+ or -] 0.34
12        12.78 [+ or -] 0.19
12        12.16 [+ or -] 0.28
19        14.26 [+ or -] 2.03
19        14.54 [+ or -] 0.42
19        14.01 [+ or -] 0.22
28        13.23 [+ or -] 0.24
36        13.51 [+ or -] 0.16

P:E represents milligram of protein per kilocalorie. TE
represents total dietary energy in calories per gram. P:C
represents protein: carbohydrate ratio in milligrams per
milligram.

Initial average wet test with spines weight was 6.85 [+ or -]
0.17 g. Initial average dry test with spines weight was 3.65 [+
or -] 0.33 g.

TABLE 6.
Mean ([+ or -] SEM) final lantern wet weight, lantern wet weight
gain, lantern production, dry lantern/dry test with spines index
of Lytechinus variegatus fed diets with varying protein and
carbohydrate levels, protein:energy ratios (P:E), total dietary
energy (TE), and protein:carbohydrate ratios (P:C).

Protein   Carbohydrate       P:E          TE        P:C
(%)           (%)        (mg P/kcal)   (cal/gl)   (mg/mg)

12                  21          50.8      2,380      0.57
12                  30          44.3      2,728      0.40
12                  39          39.3      3,075      0.31
19                  21          68.6      2,783      0.90
19                  30          60.9      3,131      0.63
19                  39          54.9      3,478      0.49
28                  30          76.9      3,647      0.93
36                  21          95.6      3,749      1.71

               Final Wet            Wet Lantern
Protein         Lantern               Weight
(%)           Weight (g)             Gain (g)

12        1.07 [+ or -] 0.09    0.34 [+ or -] 0.09
12        1.19 [+ or -] 0.05    0.45 [+ or -] 0.05
12        1.17 [+ or -] 0.05    0.43 [+ or -] 0.05
19        1.17 [+ or -] 0.05    0.43 [+ or -] 0.05
19        1.19 [+ or -] 0.07    0.45 [+ or -] 0.07
19        1.04 [+ or -] 0.08    0.30 [+ or -] 0.07
28        1.09 [+ or -] 0.05    0.35 [+ or -] 0.05
36        1.16 [+ or -] 0.05    0.43 [+ or -] 0.05

               Final Dry            Lantern Dry
Protein         Lantern               Matter
(%)           Weight (g)          Production (g)

12        0.64 [+ or -] 0.05    0.22 [+ or -] 0.05
12        0.67 [+ or -] 0.01    0.25 [+ or -] 0.01
12        0.69 [+ or -] 0.02    0.27 [+ or -] 0.02
19        0.69 [+ or -] 0.02    0.27 [+ or -] 0.02
19        0.71 [+ or -] 0.03    0.29 [+ or -] 0.03
19        0.63 [+ or -] 0.05    0.21 [+ or -] 0.05
28        0.64 [+ or -] 0.04    0.22 [+ or -] 0.04
36        0.66 [+ or -] 0.32    0.24 [+ or -] 0.32

Protein         Lantern
(%)            Index (%)

12        8.93 [+ or -] 0/60
12        9.04 [+ or -] 0.22
12        9.20 [+ or -] 0.30
19        8.43 [+ or -] 0.47
19        8.72 [+ or -] 0.27
19        8.33 [+ or -] 0.67
28        7.08 [+ or -] 0.40
36        7.12 [+ or -] 0.31

P:E represents milligram of protein per kilocalorie. TE
represents total dietary energy in calories per gram. P:C
represents protein:carbohydrate ratio in milligrams per
milligram.

Initial average lantern wet weight was 0.74 [+ or -] 0.04 g.
Initial average lantern dry weight was 0.64 [+ or -] 0.05 g.
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