Applicability of stable isotope analyses for ecological studies of abalone: estimation of fractionation values and natural diets.

ABSTRACT Controlled feeding experiments with juvenile abalone Haliotis discus hannai were performed to evaluate the suitability of stable isotope analyses for diet inference. Juvenile abalone were divided into five size groups with distinct isotopic ranges caused by different dietary regimes before the experiments. Three different foods were provided for 42 days: a benthic diatom Cylindrotheca closterium (BD), a macroalga Laminaria japonica (MA), and a formulated pellet (FP). For the FP +diet, the smallest juvenile group [8.3 mm in mean initial shell length (SL)] showed significant changes in carbon and nitrogen stable isotope ratios from the natural diet within approximately 1 wk. Using final tissue-diet isotopic differences in the two largest juvenile groups for the FP diet, the fractionation values were estimated as 1.6 [per thousand] and 2.2 [per thousand] for [delta] [sup.13]C and [delta] [sup.15]N, respectively. An exponential decay function of the change in [delta] [sup.13]C with time indicated that the shortest period to complete equilibrium fractionation was >120 days. Given the estimated fractionation values, the major diets of juvenile abalone (1.9-6.9 mm in SL) in the field were inferred to be benthic microalgae and several red macroalgae.

KEY WORDS: juvenile abalone, stable isotope ratios, fractionation values, feeding experiment, Haliotis discus hannai

INTRODUCTION

Stable isotope analyses have been increasingly used to identify the sources of dietary organic matter and to understand energy flow in food webs. Animals that show similar carbon and nitrogen stable isotope ratios are assumed to have similar food sources. Nitrogen isotope ratios become enriched at successive trophic levels, thereby allowing estimates of the consumer's trophic position (the fractionation value is 3.4 [per thousand]; Minagawa & Wada 1984). Carbon stable isotope ratios can indicate feeding and carbon flow pathways because there is little fractionation from prey to predator (the fractionation value is 1 [per thousand]; Peterson & Fry 1987). Thus, comparisons of [delta] [sup.13]C and [delta] [sup.15]N levels among coexisting plants and animals have enhanced the understanding of food webs and energy flows in aquatic ecosystems (Hecky & Hesslein 1995, Cabana & Rasmussen 1996, Vander Zanden & Rasmussen 1999, Post 2002).

Recently, stable isotope analyses have been used to elucidate ontogenetic changes such as diet shifts and habitat changes during the early developmental stages of several species, for example, energy sources of larval chironomids Chironomus acerbiphilus (Goedkoop et al. 2006, Doi et al. 2007), the dietary shift of larval red drum Sciaenops ocellatus (Herzka & Holt 2000), isotopic changes in early life stages of a juvenile crab Callinectes sapidus (Dittel et al. 2006), and ontogenetic shifts in the feeding of lake bass Micropterus salmoides (Post 2003). Coupled with these ontogenetic stable isotope studies, conventional fractionation values have often been re-evaluated and obtained for specific species (Rossi et al. 2004, Yokoyama et al. 2005) and developmental-stage- and diet-dependent diet-tissue fractionations (Post 2003, Barnes et al. 2007). Vander Zanden and Rasmussen (2001) reviewed studies of [sup.15]N (n = 35) and [sup.13]C (n = 42) fractionation and showed that the variability in fractionation values was larger in herbivores than in carnivores because of differences in the biochemical processes of metabolic and/or assimilative fractionation.

Abalone (Haliotis spp.) have been referred to as one of the major groups in subtidal rocky shore ecosystems (Ayling 1981, Bustamante & Branch 1996, Gordon & Cook 2001, Fowler-Walker & Connell 2002). Abalone depend on the yolk supply during the larval period and begin to feed after settlement (reviewed by Kawamura et al. 1998). They experience distinctive feeding transitions through successive developmental stages from microalgae to juvenile macroalgae and finally to adult macroalgae. The experimental evidence for these feeding transitions, however, has rarely been demonstrated in the natural habitat. The dramatic ontogenetic changes in the diet of abalone can provide a basis to test the applicability of stable isotope analyses because of the isotopic variation among primary producers such as microalgae and macroalgae (e.g., Peterson & Fry 1987). Stable isotope analyses could also be useful to identify foraging competitors and predators of abalone, as has been shown in food web studies (e.g., Yokoyama & Ishihi 2003, Moens et al. 2005, Behringer & Butler 2006).

Recently, stable isotope analyses have been used for ecological studies of abalone species. We have initiated a series of field studies using stable isotope ratios to elucidate the ecological niches of abalone species in Japanese coastal waters (Won 2007, Won et al. 2007). Guest et al. (2007) reported pioneering fieldwork on the natural diet of Australian abalone H. rubra, revealed using both fatty acid and stable isotope analyses. Pinnegar and Polunin (2000) reported stable isotope ratios of Mediterranean abalone Haliotis lamellose in association with rocky littoral fishes. The nitrogen stable isotope ratios reported from wild abalone in these studies are very low, indicating that the conventional nitrogen fractionation value (3.4[per thousand]) may not be applicable for interpreting the food sources of abalone. However, the ontogenetic dietary shifts of abalone species have never been investigated using stable isotope analyses, and the fractionation values of stable isotope ratios from diets to abalone remain undetermined.

Our aim was to evaluate the suitability of stable isotope ratios to detect the diet of the abalone Haliotis discus hannai. The tissue-diet fractionation values for five different size groups of the abalone were estimated as the first step in determining the applicability of stable isotope analyses in understanding abalone feeding habits in the natural habitat. The transitional pattern in abalone stable isotope ratios following a change in diet was observed to determine how fast the stable isotope ratio of a new diet would be incorporated into abalone tissues and to estimate the period required for abalone to attain the equilibrium stage of the diet-tissue fractionation. Finally, the estimated fractionation values were used to determine the potential food sources of juvenile H. discus hannai in the natural habitat.

MATERIALS AND METHODS

Experimental Design

Experiments were performed to examine the fractionation values of stable isotope ratios in juvenile abalone fed three different types of food: fresh fronds of an adult macroalga Laminaria japonica (MA), a commercial formulated pellet (FP), and a benthic diatom Cylindrotheca closterium (BD; Table 1). All food sources were given ad libitum and replaced every 3-4 days with fresh sources. EP is a commercial product from Nihon Nosan Industry Co. Ltd., Kanagawa (Awabi No. 3 as the trade name) and is used as an abalone feed in hatcheries. BD had been cultured on glass slides, which were placed at the bottom of test chambers.

The juvenile abalone used in the experiment had different growth histories and were divided into five different size groups: Group 1, 8.3 mm in mean initial shell length (SL); Group 2, 11.5 mm in SL; Group 3, 21.5 mm in SL; Group 4, 27.3 mm in SL; and Group 5, 34.9 mm in SL (Table 2). All individuals in Groups 1 and 2 originated from the same batch of postlarvae. All individuals were young-of-the-year and were fed benthic diatoms and/or juvenile macroalgae, which naturally occurred on the plates in abalone nursery tanks at Tohoku National Fisheries Research Institute (TNFRI), for >6 mo. However, Groups 1 and 2 were reared in different tanks. Group 1 was reared on plates covered with crustose coralline algae (CCA), whereas the rearing plates for Group 2 had few CCA. Because Groups 1 and 2 were not intentionally provided any food sources, they mainly fed on microalgae and juvenile macroalgae, which naturally occurred on the plates. Group 3 was composed of juveniles from several different batches that were reared together in a tank for approximately 1 y at TNFRI. Food sources for Group 3 included fresh fronds of the macroalga Undaria pinnatifida, which was intentionally provided, as well as naturally occurring microalgae and juvenile macroalgae. Groups 4 and 5 were fed FP, which was expected to be isotopically different from natural food sources. These individuals were born and reared for 15 mo at Miyagi Prefectural Center for Stock Enhancement, transferred to TNFRI, and then acclimated in the experimental aquarium for 1 wk before being used in the experiments.

The test chamber was constructed from a round PVC pipe (5 cm in diameter) that was 5 cm long (100 mL inner volume) to satisfy the individual rearing of abalone juveniles and to allow sufficient water flow. The pipe was wrapped with a 2-mm plastic net to prevent juvenile escape. The chambers were submerged in water in a plastic tray, with the top end of the mesh out of the water.

Two juveniles were placed in each chamber for Groups 1 and 2, three for Group 3, and one for Groups 4 and 5; individuals were reared for 42 days. Twenty-one chambers were prepared for each of Groups 1 and 2, but 15 chambers were created for Group 3 because of the small number of available juveniles. Twenty-three chambers were used for each of Groups 4 and 5. Juveniles were sampled from three chambers once a week during the first 3 wk; all survivors were collected at the end of the 42-day experiment.

Field Sampling

Sampling was conducted using SCUBA at the Tomarihama coast, Oshika Peninsula, Miyagi Prefecture, Japan, in October and November 2006 (38[degrees] 20.8'N, 141[degree] 31.6'E; Fig 1). Juvenile abalone were collected from rocky reefs that were approximately 4-7 m deep. The numerous boulders and cobblestones were covered with crustose coralline algae (CCA), and a few macroalgae grew on the bedrock. CCA is recognized as a major habitat of abalone species during the early life stages (e.g., Sasaki & Shepherd 2001). Several cobblestones that were 15-20 cm in the longest dimension, which were densely covered with CCA, were brought to the laboratory so that benthic microalgae could be collected from their surfaces. Common macroalgae found in the same area were also collected. All samples were immediately transferred to TNFRI and frozen at -80[degrees]C after processing for stable isotope analyses.

[FIGURE 1 OMITTED]

Sample Preparation and Measurement of Stable Isotope Ratios

Muscle tissue of abalone was used for stable isotope analyses after lipid extraction (Folch et al. 1957).

Benthic microalgae were collected from the surfaces of several cobblestones by brushing and carefully examined under a dissecting microscope to exclude any macroalgal material. Microalgal suspensions were filtered through 0.1-mm mesh net and concentrated by centrifugation (X3,000g). The pellets were decalcified using 0.1 N HCl and freeze-dried. All of the procedures for benthic microalgae were completed on the day of field sampling. Macroalgae were identified to species and picked free of all visible meiofauna and detrital fragments. When contamination such as epiphytic calcareous algae was likely, samples were rinsed with 0.1 N HCl. Samples were freeze-dried after a final wash with distilled water.

The stable isotope ratios of the MA and FP used in the feeding experiments were analyzed after samples were washed with distilled water to remove surface contaminants, including carbonates. BD were scratched off of the glass slides using a cooking spatula and were concentrated by centrifugation (X3000g) after being washed with distilled water.

All samples were freeze-dried, homogenized to a fine powder, and kept at -80[degrees]C until analysis. One day before the analyses, each sample was placed in a tin container that was combusted for the stable isotope analyses. All prepared samples were combusted in a Flash Elemental Analyzer (Flash-EA 1112; Thermo Electron, Milano, Italy) that was interfaced to an Isotope Ratio Mass Spectrometer (IRMS; [DELTA.sup.plus] XP; Thermo Finnigan, Bremen, Germany) via a continuous flow device (Conflo-III; Thermo Finnigan). Stable isotope ratios are given in the conventional delta notation ([delta] [sup.l3]C; [delta] [sup.15]N) per mil ([per thousand]) according to the formula

[delta]X = [([R.sub.sample]/[R.sub.standard]) - 1] x [10.sup.3]

where X = [sup.13]C or [sup.15]N and R = [sup.13]C/[sup.12]C or [sup.15]N/[sup.14]N. Standards for C and N were Pee Dee Belemnite and atmospheric nitrogen, respectively.

Statistical Analyses

The differences in stable isotope ratios among samples were tested using ANOVA with Tukey-Kramer multiple comparisons. Dunnett test was also used to find the difference between a control and each developmental stage. All statistical analyses were performed using JMP version 5.0 (SAS Institute Inc., Cary, NC. USA).

RESULTS

Initial Stable Isotope Ratios of Abalone and Foods

Carbon and nitrogen stable isotope ratios were significantly different among the three food types (Tukey-Kramer multiple comparison, P < 0.05): fresh L. japonica (-12.8 [+ or -] 2.0 [per thousand], 4.9 [+ or -] 1.2 [per thousand]), C. closterium (-14.2 [+ or -] 2.7 [per thousand] 0.8 [+ or -] 1.4 [per thousand]), and formulated pellet (-21.8 [+ or -] 0.2 [per thousand], 5.0 [+ or -] 0.3 [per thousand]; Fig. 2, Table 2).

The initial stable isotope ratios of juvenile abalone were significantly different between Groups 1-3 and Groups 4 and 5 for both [delta] [sup.13]C and [delta] [sup.15]N (Tukey-Kramer multiple comparison, P < 0.05; Fig. 2, Table 2).

Changes in Stable Isotope Ratios of Juvenile Abalone During Experiments

Groups that were fed FP showed a remarkable carbon isotopic change, whereas the other groups did not show any significant isotopic shift, except for some juveniles that were fed the macroalga L. japonica (Table 3). For the groups that were fed FP, significant isotopic changes were observed first in Group 1, then in Group 2, and finally in Group 3. At the end of the experiment, the tissue-diet differences of stable isotope ratios between juvenile abalone and food sources (FP) were 1.4 [per thousand] to 6.1 [per thousand] for [delta] [sup.13]C and 1.8 [per thousand] to 4.0 [per thousand] for [delta] [sup.15]N (Table 4).

[FIGURE 2 OMITTED]

Stable Isotope Ratios of Abalone and Wild Micro- and Macroalgae

Twelve juvenile abalone (1.9-6.9 mm in SL) were analyzed and showed [delta] [sup.13]C and [delta] [sup.15]N of-17.0 [+ or -] 0.8 [per thousand] and 8.0 [+ or -] 0.9 [per thousand], respectively. Seven macroalgae from three taxa (Chlorophyceae, Phaeophyceae, and Rodophyceae) were identified, and their [delta] [sup.13]C and [delta] [sup.15]N values ranged from -20.0 [per thousand] to -16.4 [per thousand] and from 3.1 [per thousand] to 5.7 [per thousand], respectively (Table 5). Benthic microalgae showed [delta] [sup.13]C and [delta] [sup.15]N of-18.8 [+ or -] 0.8 [per thousand] and 5.8 [+ or -] 0.2 [per thousand], respectively. When the fractionation values of 1.6 [per thousand] for [delta] [sup.13]C and 2.2 [per thousand] for [delta] [sup.15]N were applied, two species of red algae (i.e., Gelidium elegans and Pterocladiella tenuis) and benthic microalgae were estimated as the major food sources of juvenile abalone (Fig. 3).

DISCUSSION

Suitability of Stable Isotope Ratios for the Detection of Abalone Diets

Clear isotopic shifts were shown in the groups that were fed FP, whereas those that were fed other foods showed only weakly significant (MA) or nonsignificant (BD) changes (Table 3). Because the diet-tissue fractionation is a growth-rate-dependent process that results from tissue turnover, fast growth and isotopically distinct food sources are two critical factors that are required to ensure the success of stable isotope experiments (Herzka & Holt 2000, Witting et al. 2004, Watanabe et al. 2005, Yokoyama et al. 2005, Goedkoop et al. 2006). The isotopically depleted formulated pellets were beneficial to the stable isotope study (Fig. 2). The significant isotopic changes were detected earlier in the size-dependent order from the smallest individuals, i.e., Group 1, to the largest individuals, i.e., Group 5. Groups 4 and 5 were fed FP and did not show any carbon changes, with the exception that the 17-day-old Group 5 individuals showed a weak, but significant change (Table 2). These two groups can be used to calculate the fractionation values of juvenile abalone. Large juvenile (>10 mm in SL) and adult H. discus hannai prefer adult brown macroalgae and grow quickly on this diet (Kikuchi et al. 1967, Uki 1981, Uki et al. 1986). Juvenile H. discus hannai show distinct feeding on juvenile macroalgae when they attain 5 mm in SL, but they are able to feed on juvenile macroalgal diets at approximately 2 mm in SL when they develop functional radula and digestive enzymes for macroalgal polysaccharides (Kawamura et al. 1998, Takami et al. 1998, Kawamura et al. 2001, Takami et al. 2003). Group 1 individuals were large enough (8.3 mm in SL) to feed on juvenile macroalgae. Because FP is a commercial abalone feed that satisfies juvenile and adult nutritional requirements, it can be considered a substitute diet for juvenile growth. Group 1 was the first to show significant isotopic changes, manifesting in the first 7 days (Table 3).

The maximum growth increment was 0.71 mm in SL in 7 days (0.1 mm/day), although the mean growth rate for all treatments was 4 [micro]m/day. The previously reported growth rates for juvenile H. discus hannai (>3 mm in SL) varied depending on the food source (reviewed by Kawamura et al. 1998), with rates > 0.1 mm/day in juveniles of approximately 4 mm in SL (Maesako et al. 1984) and 30 mm in SL (Uki 1981). The growth rates in our experiment varied among individuals, and length changes were sometimes too small to detect, but the maximum growth rate was comparable to the fastest rates reported in previous studies.

Although previous studies have reported diet-tissue equilibriums from long-term feeding experiments with more than 4-fold biomass increases, the first isotopic shifts were observed within the 2-fold biomass increase after the change in diet (Herzka & Holt 2000, Herzka et al. 2001, Witting et al. 2004, Yokoyama et al. 2005). When the carbon contribution of FP was estimated using the mixing equation (e.g., Fredriksen 2003) with the expected [delta] [sup.13]C of the initial food sources (-13.4[per thousand], -15.4 [per thousand], and -14.4 [per thousand] of [delta] [sup.13]C for Group 1,2, and 3, respectively) and FP (-21.8 [per thousand] of [delta] [sup.13]C), the carbon distribution of FP during the first 7-day period of growth was 22.2 [+ or -] 8.7% for Group 1 (Table 5).

Therefore, changes in stable isotope ratios could be detected for Group 1 (<10 mm in SL), even during the short growth period, with approximately 22% tissue turnover in 7 days, when a favorable food was provided and its isotopic value was distinctly different from the previous food.

[FIGURE 3 OMITTED]

Estimation of Fractionation Values

Groups 1-3, which were reared on natural food sources before the experiment, showed significant changes in carbon stable isotope ratios when fed FP. Groups 4 and 5, which were reared on FP before the experiment, retained their stable isotope values and showed no changes after the 6-wk experimental period. These results indicate that Groups 4 and 5, which were fed FP, were already at the equilibrium state of diet-tissue fractionation before the start of the experiment. Therefore, the mean fractionation values for juvenile abalone were estimated from the values of Groups 4 and 5 as 1.6 [per thousand] and 2.2 [per thousand] for [delta] [sup.13]C, and [delta] [sup.15]N respectively.

Suzuki and Suzuki (2000) reported fractionation values of adult Haliotis discus hannai (82-95 mm in SL) of 1.9 [per thousand] and 3.2 [per thousand] for [delta] [sup.13]C and [delta] [sup.15]N, respectively, which were inferred from stable isotope ratios of individuals that were fed mainly the natural green alga Ulva pertusa. The value for [delta] [sup.13]C was similar to that of the juvenile H. discus hannai fed FP measured here, but we estimated a smaller fractionation value for [delta] [sup.15]N. Pinnegar and Polunin (2000) pointed out the unusually low value of [delta] [sup.15]N (2.36 [+ or -] 0.35 [per thousand]) of Mediterranean abalone Haliotis lamellose, which possessed the lowest value among the herbivorous molluscs that they measured. Won et al. (2007) showed that Japanese abalone H. diversicolor had the lowest value of [delta] [sup.15]N (9.3 [+ or -] 0.5 [per thousand]) among those of the herbivorous gastropods collected in the same habitat and indicated that the conventional fractionation value of [delta] [sup.15]N (3.4 [per thousand]) is not suitable for inferring food sources such as brown macroalgae. Guest et al. (2007) also reported that the [delta] [sup.15]N of Australian abalone H. rubra is low and cannot be distinguished from that of their major food, brown macroalgae.

During the experimental period (42 days), none of the juvenile groups reached equilibrated values of diet-tissue fractionation following the change in diet. Longer feeding experiments are required to achieve fractionation values at the equilibrium state. A remarkable change in [delta] [sup.13]C, however, was measured in juveniles that were fed FP. According to Tieszen et al. (1983), the change in [delta] [sup.13]C versus the rearing time can be fitted to an exponential decay function. The initial changes in [delta] [sup.13]C after the change to a diet of FP can be shown for Group 1, and the equilibrium state can be obtained from the mean final value for Groups 4 and 5. When the initial changes in Group 1 and the mean final value of Groups 4 and 5 were used to model the whole changes in [delta] [sup.13]C after introducing a FP diet, a desirable experimental period to satisfy the equilibrium state was indicated as at least 120 days (Fig. 4). Because the growth rate is temperature dependent, the turnover time should be carefully considered under various temperature regimes, for example, different seasons.

[FIGURE 4 OMITTED]

Several isotopic discrimination processes that occur during feeding activities, which are more prevalent in herbivores than carnivores, can influence the fractionation values (Vander Zanden & Rasmussen 2001, Mill et al. 2007). Food qualities such as the biochemical composition may affect the assimilation efficiency and result in biased fractionation values (Mill et al. 2007). Further experimental studies using natural food sources are needed to examine fractionation values for different diets in abalone.

Estimation of Diets in the Natural Habitat

The feeding transitions of abalone have been well explained by many experiments (reviewed by Kawamura et al. 1998). Until recently, however, few studies have reported isotopic evidence for abalone diets in natural habitats, although stable isotope ratios are widely used to explore feeding relations in aquatic ecosystems (Guest et al. 2007, Won et al. 2007). The major dietary components can gradually change from benthic microalgae to macroalgae in the juvenile stage, which is enabled by the development of the digestive organ and an increase in digestive enzyme activities for macroalgae (Takami et al. 1998, Kawamura et al. 2001). With our estimated fractionation values, benthic microalgae and red macroalgae were likely the major food sources of juvenile abalone (Table 6, Fig. 3). This result can be considered as direct evidence indicating that benthic microalgae are important food sources for abalone in early life stages in natural habitats. Red macroalgal species have scarcely been considered as major diets for Haliotis discus hannai, whereas brown macroalgae are regarded as favorite diets for adults (e.g., Uki et al. 1986). Wild juvenile abalone usually have a rosy or brownish shell color, whereas artificially cultured juveniles that are fed adult brown macroalgae such as Laminaria spp. and/or formulated diets have green shells. Sakai (1962) reported that artificially cultured H. discus hannai that are fed adult brown and green algae usually have bluish-green shells, but they began to form brown shells if they were provided with red algae (Polyopes affinis and Mazzaella japonica), which was the normal shell color of wild juveniles. These results indicate that juvenile abalone do not exclusively use adult brown algae and that red macroalgae can be important diets and affect the shell color of juvenile abalone, although the shell color of juvenile abalone feeding on juvenile brown algae has never been reported. Because juvenile and adult Laminaria spp. are likely to have different isotopic values (e.g., Fredriksen 2003, Won et al. 2007) and may produce brown-colored shells, the dietary effects of juvenile brown macroalgae on juvenile abalone remains to be examined.

Although further analyses of field samples of abalone with a wider range of shell lengths from different fields and seasons are needed to understand the ontogenetic transitions in the diet of H. discus hannai in the natural habitat, our results indicate that stable isotope analysis is a reliable tool for examining abalone feeding ecology.

ACKNOWLEDGMENTS

The authors thank T. Miyajima at the University of Tokyo for IRMS assistance and helpful discussions. This research was supported by a grant-in-aid Development of Seed Production and Releasing Techniques for Stock Enhancement of Marine Resources Considering the Conservation of Ecosystems, from the Fisheries Agency, Japan.

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NAM-IL WON, (1) * TOMOHIKO KAWAMURA, (1) HIDEKI TAKAMI (2) AND YOSHIRO WATANABE (1)

(1) Ocean Research Institute, the University of Tokyo, Nakano, Tokyo 164-8639, Japan; (2) Tohoku National Fisheries Research Institute, Shiogama, Miyagi 985-0001, Japan

* Corresponding author. E-mail: [e-mail]

TABLE 1.
Design of feeding experiments.

               Initial
Juvenile        Mean                                Temperature
  Group        SL (mm)            Food Type         ([degrees]C)

Group 1     8.3 [+ or -]    No food                      15
                 1.8        Macroalga                    15
                            (Laminaria japonica)
                            Formulated pellets           15
                            Benthic diatom               15
                            (Cylindrotheca
                            closterium)
Group 2     11.5 [+ or -]   No food                      15
                 1.7        Macroalga                    15
                            (Laminaria japonica)
                            Formulated pellets           15
                            Benthic diatom               15
                            (Cylindrotheca
                            closterium)
Group 3     21.5 [+ or -]   No food                      15
                 3.7        Macroalga                    15
                            (Laminaria japonica)
                            Formulated pellets           15
Group 4     27.3 [+ or -]   No food                      15
                 2.1        Macroalga                    15
                            (Laminaria japonica)
                            Formulated pellets           15
Group 5     34.9 [+ or -]   No food                      15
                 1.6        Macroalga                    15
                            (Laminaria japonica)
                            Formulated pellets           15
                            Benthic diatom               15
                            (Cylindrotheca
                            closterium)

                                                       Number of
                          Duration     Replication       Abalone
Food Type                  (days)          (n)        (ind/chamber)

No food                      42             21              2
Macroalga                    42             21              2
(Laminaria japonica)
Formulated pellets           42             21              2
Benthic diatom               42             21              2
(Cylindrotheca
closterium)
No food                      42             21              2
Macroalga                    42             21              2
(Laminaria japonica)
Formulated pellets           42             21              2
Benthic diatom               42             21              2
(Cylindrotheca
closterium)
No food                      42             15              3
Macroalga                    42             15              3
(Laminaria japonica)
Formulated pellets           42             15              3
No food                      42             24              1
Macroalga                    42             24              1
(Laminaria japonica)
Formulated pellets           42             24              1
No food                      42             24              1
Macroalga                    42             24              1
(Laminaria japonica)
Formulated pellets           42             24              1
Benthic diatom               42             24              1
(Cylindrotheca
closterium)

TABLE 2.
Initial stable isotope ratios of diets and juvenile abalone.

                                               [delta][sup.13]C
                                               ([per thousand])

                                                            P < 0.05
  Sample Type                        n     Mean      SD        (a)

Diets
  Formulated pellet (FP)            12     -21.8     0.2       B
  Laminaria japonica (MA)           12     -12.8     2.0       A
  Cylindrotheca closterium (BD)     12     -14.2     2.7       A
Juvenile abalone (initial SL)
  Group 1 (8.3 [+ or -] 1.8 mm)     10     -11.8     1.0       A
  Group 2 (11.5 [+ or -] 1.7 mm)     9     -13.8     0.3       B
  Group 3 (21.5 [+ or -] 3.7 mm)     4     -12.2     0.2       A
  Group 4 (27.3 [+ or -] 2.1 mm)     5     -20.1     0.2       C
  Group 5 (34.9 [+ or -] 1.6 mm)     5     -19.9     0.1       C

                                               [delta][sup.15]C
                                               ([per thousand])

  Sample Type                              Mean      SD     p < 0.05

Diets
  Formulated pellet (FP)                    5.0      0.3       A
  Laminaria japonica (MA)                   4.9      1.2       A
  Cylindrotheca closterium (BD)             0.8      1.4       B
Juvenile abalone (initial SL)
  Group 1 (8.3 [+ or -] 1.8 mm)             9.2      0.3       A
  Group 2 (11.5 [+ or -] 1.7 mm)            9.5      0.2       A
  Group 3 (21.5 [+ or -] 3.7 mm)            9.4      0.1       A
  Group 4 (27.3 [+ or -] 2.1 mm)            5.9      0.4       B
  Group 5 (34.9 [+ or -] 1.6 mm)            6.3      0.3       B

(a) Difference in stable isotope ratio among diets and abalone groups
was tested by Turkey Kramer multiple comparisons. Data not connected
by the same letter are significantly different.

TABLE 3.
Summary of carbon stable isotope ratios of juveniles in each diet
treatment.

                                    No food (Control)

            Rearing           Mean
Juvenile      Time            ([per            Dunnett's   P < 0.05
Group        (day)     n    thousand])   SD      Value       (a)

Group 1         0      10     -11.8      1.0     -1.0      control
                7       4     -11.9      0.8     -1.2
               14       4     -11.6      0.5     -1.2
               26
               42       2     -11.2      0.3     -1.1
Group 2         0       9     -13.8      0.3     -0.7      control
                7       7     -13.5      0.9     -0.4
               14       3     -13.1      0.4     -0.3
               26
               42
Group 3         0      10     -13.2      1.1     -1.1      control
                7       6     -14.1      0.3     -0.4
               17       9     -13.5      1.0     -0.9
               42      10     -12.7      1.2     -0.7
Group 4         0       5     -20.1      0.2     -0.4      control
                7       3     -19.9      0.2     -0.3
               17       3     -20.0      0.1     -0.4
               26
               42       3     -19.7      0.3      0.0
Group 5         0       5     -19.9      0.1     -0.6      control
                7       3     -20.0      0.4     -0.6
               17       3     -20.0      0.4     -0.5
               26
               42       3     -19.3      0.4     -0.1

                           Macroalga (Laminarla japonica)

            Rearing           Mean
Juvenile      Time            ([per            Dunnett's   P < 0.05
Group        (day)     n    thousand])   SD      Value       (a)

Group 1         0      10     -11.8      1.0     -1.0      control
                7       5     -11.0      0.6     -0.4
               14       4     -11.6      0.8     -1.1
               26
               42       3     -11.8      0.1     -1.4
Group 2         0       9     -13.8      0.3     -0.4      control
                7       7     -13.6      0.3     -0.2
               14       6     -13.3      0.5      0.0         *
               26
               42       3     -13.1      0.1      0.2         *
Group 3         0      11     -12.8      1.7     -1.2      control
                7       6     -14.1      0.2     -0.2
               17       9     -13.3      1.1     -0.8
               42       9     -13.0      0.8     -1.1
Group 4         0       5     -20.1      0.2     -0.8      control
                7       3     -19.2      0.6     -0.1
               17       3     -19.3      0.6     -0.2
               26
               42       3     -18.5      0.5      0.6         *
Group 5         0       5     -19.9      0.1     -0.5
                7       3     -19.4      0.2     -0.1
               17       3     -19.1      0.5      0.3         *
               26
               42       3     -18.7      0.3      0.6         *

                              Formulated Pellets

            Rearing           Mean
Juvenile      Time            ([per            Dunnett's   P < 0.05
Group        (day)     n    thousand])   SD      Value       (a)

Group 1         0      10     -11.8      1.0     -1.3      control
                7       5     -13.7      0.7      0.3         *
               14       3     -15.3      0.1      1.6         *
               26       4     -15.0      1.2      1.5
               42       5     -17.4      1.7      4.0         *
Group 2         0       9     -13.8      0.3     -0.8      control
                7       5     -14.1      0.4     -0.7
               14       4     -16.0      0.6      1.1         *
               26       4     -15.7      1.1      0.8
               42       4     -17.7      0.8      2.8
Group 3         0      10     -13.2      1.1     -1.4      control
                7       6     -14.3      0.4     -0.5
               17       9     -14.6      1.4     -0.1
               42       9     -15.7      1.7      1.1
Group 4         0       5     -20.1      0.2     -0.7      control
                7       4     -20.3      0.4     -0.5
               17       3     -20.2      0.7     -0.7
               26       3     -19.8      0.3     -0.6
               42       3     -20.4      0.4     -0.5
Group 5         0       5     -19.9      0.1     -0.2      control
                7       3     -19.8      0.1     -0.2
               17       3     -20.2      0.2      0.1         *
               26       3     -20.1      0.2      0.0
               42       3     -20.0      0.3     -0.3

                                  Benthic Diatom
                            (Cylindrotheca closterium)

            Rearing           Mean
Juvenile      Time            ([per            Dunnett's   P < 0.05
Group        (day)     n    thousand])   SD      Value       (a)

Group 1         0      10     -11.8      1.0     -0.9      control
                7       5     -11.8      0.4     -1.1
               14       4     -11.9      0.4     -1.1
               26
               42       2     -11.1      1.0     -0.9
Group 2         0       9     -13.8      0.3     -0.4      control
                7       7     -13.7      0.4     -0.3
               14       3     -13.8      0.4     -0.6
               26
               42
Group 3         0
                7
               17
               42
Group 4         0
                7
               17
               26
               42
Group 5         0       5     -19.9      0.1     -0.4      control
                7       3     -19.8      0.3     -0.4
               17       3     -19.9      0.3     -0.5
               26
               42       3     -19.4      0.3     -0.02

(a) Initial stable isotope ratio of each juvenile group was used as
the control of Dunnette's test.

* Difference in stable isotope ratio between control and each
treatment was tested by Dunnette's test.

TABLE 4.
Tissue-diet differences in stable isotope ratios of abalone fed
fornulated pellets.

                       [delta] [sup.13]C       [delta] [sup.15]C

  Item           n      Mean         SD       Mean     SD

Food            12      -21.8        0.2       5.0    0.3
Abalone
  Group 1        5      -17.4        1.7       7.8    0.3
  Group 2        4      -17.7        0.8       8.4    0.6
  Group 3        9      -15.7        1.7       9.0    0.7
  Group 4        3      -20.4        0.4       6.8    0.8
  Group 5        3      -20.0        0.3       7.5    0.3
Fractionation
values (a)

                      Difference ([per thousand])

  Item                 [delta]     [delta]
                      [sup.13]C   [sup.15]C
Food
Abalone
  Group 1                4.4         2.8
  Group 2                4.1         3.4
  Group 3                6.1         4.0
  Group 4                1.4         1.8
  Group 5                1.8         2.5
Fractionation            1.6         2.2
values (a)

(a) The fractionation values were calculated by the values of
Groups 4 and 5.

TABLE 5.
[delta][sup.13]C and [delta][sup.15]N values of micro and macroalgae
and juvenile abalone.

                                [delta][sup.13]C    [delta][sup.13]N
                                ([per thousand)     ([per thousand)

            Species             Mean       SD       Mean       SD

Potential food source
  Chlorophyceae
    Cladophorajaponica          -16.4      --        4.7       --
  Phaeophyceae
    Eisenia bicyclis            -16.9      1.7       4.6       2.2
    Pachydictyon coriaceum      -20.0       -        4.4       --
    Undaria pinnatida           -15.9      1.5       3.1       1.4
  Rodophyceae
    Gelidium elegans            -18.5      1.3       5.1       0.9
    Grateloupia lanceolata      -17.7      --        5.1       --
    Pterocladiella tenuis       -19.0      --        5.7       --
    Benthic microalgae          -18.8      0.8       5.8       0.2
Abalone
  Haliotis discus
    hannai (l.9-6.9 mm SL)      -17.0      0.8       8.0       0.9

                                          Group
            Species               n        No.

Potential food source
  Chlorophyceae
    Cladophorajaponica            1         1
  Phaeophyceae
    Eisenia bicyclis             10         2
    Pachydictyon coriaceum        1         3
    Undaria pinnatida             6         4
  Rodophyceae
    Gelidium elegans              3         5
    Grateloupia lanceolata        1         6
    Pterocladiella tenuis         1         7
    Benthic microalgae            9         8
Abalone
  Haliotis discus
    hannai (l.9-6.9 mm SL)       12

TABLE 6.
Carbon distribution made by formulated pellets in Groups 1-3.

                % FP Carbon (a)

                         Group 1

Rearing                              P <0.05
  Days     n    Mean (%)     SD        (b)

    7      5      22.2       8.7        *
   14      3      41.8       0.9        *
   17
   26      4      38.3      13.9        *
   42      5      67.1      19.9        *

                % FP Carbon (a)

                         Group 2

Rearing                              P <0.05
  Days     n    Mean (%)     SD        (b)

    7      5       4.1       6.6
   14      4      33.3       9.5
   17
   26      4      29.3      18.0
   42      4      60.2      11.9        *

                % FP Carbon (a)

                         Group 3

Rearing                              P <0.05
  Days     n    Mean (%)     SD        (b)

    7      6      20.7       5.6
   14
   17      9      23.7      18.7
   26
   42      9      39.8      22.8        *

(a) Simple mixing equation was used: [%C.sub.FP-derived] =
([delta][sup.13][C.sub.abalone]--[delta][sup.13]
[C.sub.initial food]--I) / ([delta][sup.13][C.sub.FP]--
[delta][sup.13][C.sub.initial food]) X 100 where I is the average
postphotosynthetic fractionation of [delta][sup.13]C per trophic
level, provided as l.6 [per thousand] from our fractionation value
results.

(b) See Table 3 for details.