The effect of egg quality on larval period and postlarval survival of an abalone Haliotis discus hannai.

ABSTRACT The effect of egg quality on larval period and postlarval survival was investigated for the abalone Haliotis discus hannai. Broodstock were conditioned for spawning at different effective accumulative temperatures (EAT) and the subsequent larvae were used in experiments. Larval period until spontaneous metamorphosis or death and postlarval survival without food were investigated. Larval period and postlarval survival of offspring from broodstock conditioned at 1,650 and 2,350[degrees]Cxdays EAT were greater than those of larvae from 1050 and 1150[degrees]Cxdays EAT conditioned broodstock. Biochemical composition and egg cytoplasm diameter were measured as egg quality from broodstock conditioned under different EAT treatments. There was a positive correlation between lipid and protein content in eggs. Protein and lipid content showed positive correlations with larval period and postlarval survival without food. The eggs from higher EAT tended to contain higher protein and lipid contents, but there was no significant difference in protein levels among broodstock conditioned at different EAT. No relationship was found between egg cytoplasm volume, which calculated using egg cytoplasm diameter and larval period or postlarval survival. These results suggest that the quality of eggs became higher as EAT increased, and that protein and/or lipid were important for larval and postlarval survivorship. For H. discus hannai, the biochemical composition of eggs may be more important than yolk volume for early development and survival of larvae and postlarvae.

KEY WORDS: abalone, Haliotis, egg quality, larval period, postlarval survival, effective accumulative temperature

INTRODUCTION

Abalone species have a lecithotrophic (nonfeeding) larval stage. The survival and development of larval abalone are largely influenced by the nutrient reserves in eggs (Litaay et al. 2001, Roberts & Lapworth 2001, Takami et al. 2002) and/or the availability of dissolved organic matter in seawater (Jaeckle & Manahan 1989a, Jaeckle & Manahan 1989b, 1992). Newly metamorphosed postlarvae of abalone depend on residual yolk reserves for initial growth in addition to particulate matter, which postlarvae start ingesting within a day of the velum being shed (Ohashi 1993, Kawamura et al. 1998, Roberts et al. 1999, Roberts & Lapworth 2001, Takami et al. 2000, 2002).

It has been reported that maternal conditions affect reproductive characteristics such as size and quality of eggs and hence affect the survival and growth of offspring in fishes (e.g., Marteinsdottir & Steinarsson 1998, Riveiro et al. 2000, Saillant et al. 2001) and marine invertebrates (e.g., George 1996, Ito 1997, Bertman & Strathmann 1998). However, the importance of maternal condition and egg characteristics for the fitness of larvae and newly metamorphosed postlarvae remains largely unknown for abalone species.

Haliotis discus hannai (Ino) has the highest commercial value of any abalone species in Japan. Seed production and reseeding of this species have been conducted to enhance the natural stocks in northern Japan. At present, H. discus hannai is cultured not only in Japan but also in many other countries, since the techniques for maturation and spawning were established by Uki and Kikuchi (1984). Gonad maturation of H. discus hannai is controlled by water temperature; the gonad index increases as the effective accumulative temperature (EAT) increases (Kikuchi & Uki 1974a). Kikuchi and Uki (1974a) found the relationship between the increase in gonad index (Y) and seawater temperature (T) was: Y = 0.0597T-0.04527. A value of T = 7.6[degrees]C was given as solving the equation for Y = 0 and was defined as the theoretical minimum temperature (biologic zero point) for gonad growth and development. Gonad maturation is initiated only when the water temperature rises above 7.6[degrees]C. EAT is the cumulative temperature above the biologic zero point 7.6[degrees]C (Kikuchi & Uki 1974a). Gametogenesis of wild H. discus hannai generally begins between winter and early spring, and then gonads become matured from early summer. Compared with other abalone species in Japan the spawning season of 14. discus hannai is long, from early summer to late autumn (Ino & Harada 1961). Large-scale spawnings of H. discus hannai seemed to be synchronized with typhoons or storm events once the EAT has reached the appropriate level in a population (Sasaki 1985, Sasaki & Shepherd 1995). Early in the spawning season, sudden temperature drops also trigger spawning events for H. discus hannai (Kawamura et al. 2002). These findings suggest that H. discus hannai can release gametes at variable EAT levels in the natural habitat, once they are stimulated by environmental spawning cues after they experience an appropriate EAT level. If egg quality varies with the EAT level then survival and growth of larvae and newly metamorphosed postlarvae may be influenced by the timing of spawning.

In this study, the effect of different EAT treatments on egg quality was examined for H. discus hannai in the laboratory. Egg qualities were assessed by its biochemical compositions and yolk volume. The effects of the egg qualities on larval period without settlement cues and postlarval survival without food were examined.

MATERIALS AND METHODS

Broodstock Rearing Conditions and Induction of Spawning

Adult H. discus hannai were obtained in June 2001 and April 2002 from Hirota Fisheries Cooperative, Iwate, Japan. Animals were transported to Tohoku National Fisheries Research Institute, Miyagi Prefecture, Japan and kept in 518 L (72 x 72 x 100 cm) V-shaped plastic tanks (V-tank, Kubota Industry Co., LTD., Japan) until experiments began. The seawater flow rate was 480 L/h throughout the experiment. Photoperiod was maintained at 12 h light/12 h dark. Initial broodstock conditions were: shell length 91.0 [+ or -] 0.6 mm, whole body wet weight 88.2 [+ or -] 2.5 g (mean [+ or -] SE, n = 24).

Rearing experiments were conducted from April to October 2002 in 6 V-tanks. Each V-tank was partitioned into four different rooms by plastic panels that did not disturb the water flow in the tank. The rearing temperature was regulated to simulate the water temperature that H. discus hannai would encounter in the coastal waters of Iwate (Kadonohama Bay), which is the center of their natural distribution, during the period of gametogenesis (from winter to autumn). Daily temperature was recorded automatically at noon. Temperature differences among aquaria never exceeded 0.3[degrees]C during the experimental period (Fig. 1). EAT was calculated using the formula defined by Kikuchi and Uki (1974a)

[FIGURE 1 OMITTED]

[Y.sub.n] = [summation over (i=1)] ([t.sub.i] - [theta])

where [Y.sub.n] ([degrees]Cxdays) = EAT, n (days) = number of days since water temperature rose above [theta], [t.sub.i] ([degrees]C) = daily water temperature in which the animal was reared and 0 ([degrees]C) = biologic zero point for gonad maturation (7.6[degrees]C for H. discus hannai; Kikuchi & Uki 1974a). For instance, if water temperature was 20[degrees]C, the effective temperature was 12.4[degrees]C (20 - 7.6) and then accumulated using the above formula for EAT. When EAT reached 500[degrees]Cxdays, the sex of broodstock abalone became discernable with the naked eye. Males and females were reared separately. Ten females were reared in each room of the V-tank (total 240 females), and about 100 males were reared in a different V-tank. All animals were fed ad libitum with fresh Laminaria japonica, but only animals that showed active feeding behavior were used in the spawning experiments. We did not measure the feeding rate of broodstock.

Spawning was induced at 850, 1,050, 1,150, 1,650, 1,900 and 2,350[degrees]Cxdays EAT using a combination of the desiccation and UV irradiation methods (Kikuchi & Uki 1974b). Three to five females in each EAT treatment were used for spawning. Spawning occurred from 2 h after the start of UV treatment.

Egg Measurements

Eggs were collected immediately after spawning and the cytoplasm diameter was measured. Eggs from 1,050, 1,150, 1,650 and 2,350[degrees]Cxdays EAT were used for rearing experiments of offsprings and those from 850, 1,050, 1,150, and 2,350[degrees]Cxdays EAT were used for biochemical analyses (Table 1).

The cytoplasm diameter of eggs was measured under a microscope to the nearest 1 [micro]m within 3 hours after spawning. Fifty to 80 eggs were measured from each female. Assuming a completely spherical shape, egg cytoplasm volume was calculated using the formula:

V = 4/3[pi][(d/2).sup.3]

where V = egg cytoplasm volume and d = diameter of egg cytoplasm.

Egg samples obtained prior to fertilization from animals in the 850, 1,050, 1,150 and 2,350[degrees]Cxdays EAT treatments were washed with filtered (0.2 [micro]m pore size) ammonium acetate (3.4% w/v, isotonic with seawater) following the method of Jaeckle and Manahan (1989b). Washed samples were freeze-dried for at least 72 h, and then 3 mg of samples were sonicated in 2.0 mL of deionized distilled water using ultrasonic disruption (Branson Model 250 sonifier, USA). Total lipids were quantified gravimetrically using the method of Mann and Gallager (1985). Protein content was measured using the method of Jaeckle and Manahan (1989b), with an alternation: protein was assayed using the Bicinchoninic Acid Protein Assay Kit (Sigma Diagnostics, St. Louis), adapted from the method of Smith et al. (1985). Carbohydrate was extracted as described by Mann and Gallager (1985) and content was determined by the anthrone-[H.sub.2]S[O.sub.4] method. Chemical contents were presented as the weight percentage of dry egg weight.

Larval Period and Postlarval Survival

Eggs obtained for rearing experiments of offspring were fertilized using sperm from males kept at the ambient seawater temperature. Fertilized eggs were rinsed with filtered seawater to remove excess sperm, incubated at 20[degrees]C and larvae were reared separately. Five days after fertilization competent veliger were placed individually into polystyrene 24 well tissue culture plates (Coming, USA). They were cultured at 20[degrees]C in 1 mL of 0.45 [micro]m-filtered seawater (FSW) containing 150 [micro]g/mL of Penicillin G sodium and Streptomycin sulfate BP, respectively, without the addition of metamorphosis cues. Metamorphosed or dead larvae were quantified by inspecting the floor and walls of the assay chamber using inverted and dissecting microscopes. Larval period was defined as the time between hatching and the time when larvae metamorphosed or died.

The second group of 5-day-old veliger larvae obtained from the same batch of each EAT above were placed into tissue culture plates with 1 mL of FSW containing antibiotics described earlier. They were induced to metamorphose with the addition of 1 [micro]M [gamma]-aminobutyric acid (GABA). Five days after the addition of GABA, larvae that had not metamorphosed were removed, and only metamorphosed postlarvae were used for the experiment. Postlarvae were reared without food and were inspected every 2-3 days to record the number of dead individuals. We defined dead individuals as those with no heartbeat during 10 sec of observation (Takami et al. 2000). In this study, we defined survival of individuals that were induced metamorphosis with GABA as postlarval survival.

Statistical Analysis

Data normality was assessed visually and with normal probability plots. Percentage data were arcsin transformed prior to the statistical analysis to obtain homogeneous variances. The effects of the EAT treatments on cytoplasm volume, lipid content and protein content of eggs per female abalone (mothers) were tested using nested analysis of variance (nested ANOVA). When the significant differences were found among EAT treatments, data were compared by Tukey-Kramer multiple comparison tests. We also examined relationships between egg quality (egg cytoplasm volume, lipid content, protein content) and both days, larval period and postlarval survival using Pearson correlation coefficient and a linear regression. All of the statistical analyses were carried out using the JMP software (ver. 5, SAS Institute Inc. USA).

RESULTS

Biochemical Contents and Volume of Eggs

Significant difference was found in egg cytoplasm volume among eggs from broodstock in different EAT treatments (nested ANOVA, P < 0.001, Table 2) and in the same EAT treatment (nested ANOVA, P < 0.001, Table 2). Eggs from 1,050[degrees]Cxday treatment had a significantly larger cytoplasm volume than those from other EAT treatments (Tukey-Kramer multiple comparison test, P < 0.01). However there was no clear relationship between egg cytoplasm volume and EAT level (Fig. 2).

[FIGURE 2 OMITTED]

Although protein content was highest in eggs from the highest EAT treatment (2350[degrees]Cxday), there was no significant difference in protein content among eggs from different EAT treatments (nested ANOVA, P > 0.05, Table 2). No significant difference was found in those from broodstock in the same EAT treatments, either (nested ANOVA, P > 0.05, Table 2). A significant difference in the lipid content was observed among different EAT treatments (nested ANOVA, P < 0.001, Table 2), though there was no significant difference in the lipid content within the same EAT treatments (nested ANOVA, P > 0.05, Table 2). Eggs from 2,350[degrees]Cxdays EAT treatment contained significantly higher lipid content than those from the other EAT treatments (Tukey-Kramer multiple comparison test, P<0.05, Fig. 3). No carbohydrate content was detected in any eggs measured. Eggs that contained higher percentages of protein contained higher percentages of lipid ([r.sup.2] = 0.72, P < 0.001, Fig. 4).

[FIGURES 3-4 OMITTED]

Larval Period and Postlarval Survival

Larval survival and spontaneous metamorphosis without cues depended on the level of EAT. Survival and metamorphosis of larvae from all EAT treatments were compared at day 29. Larval survival from lower EAT treatments (1,050 and 1,150[degrees]Cxdays; 59% to 66%) was significantly lower than for larvae from higher EAT treatments (1,650 and 2,350[degrees]Cxdays; > 91% survived) (Tukey-Kramer multiple comparison test, P < 0.05, Fig. 5A). Metamorphosis rates of larvae at day 29 from lower EAT treatments were also significantly lower than those from higher EAT treatments (Tukey-Kramer multiple comparison test, P < 0.05, Fig. 5B). However, the metamorphosis of larvae from lower EAT treatments (1,050 and 1,150[degrees]Cxdays) was faster than larvae from higher EAT treatments (1,650 and 2,350[degrees]Cxdays); a marked increase in metamorphosis rate was observed after day 13-14 for larvae from the lower EAT treatments, whereas after day 19 for larvae from the higher EAT treatments (Fig. 5B). Survival rates of postlarvae without food declined more rapidly for postlarvae from the lower EAT treatments (1,050 and 1,150[degrees]C-days) than those from the higher EAT treatments (1,650 and 2,350[degrees]Cxdays) (Fig. 6).

[FIGURES 5-6 OMITTED]

Batches with higher percentages of protein and lipid in the eggs showed significantly longer larval period (protein: [r.sup.2] = 0.69, P < 0.05; lipid: [r.sup.2] = 0.77, P < 0.05, Fig. 7A) and longer survival period of postlarvae without food (protein: [r.sup.2] = 0.68, P < 0.05; lipid: [r.sup.2] = 0.74, P < 0.01, Fig. 7B). There was no correlation between egg cytoplasm volume and larval period ([r.sup.2] = 0.22, P = 0.21), and postlarval survival ([r.sup.2] = 0.22, P = 0.21).

[FIGURE 7 OMITTED]

DISCUSSION

The results of this study show that the egg quality of an abalone H. discus hannai changes depending on conditions of broodstock. Both of the individuals that had a longer larval period and a longer postlarval survival were hatched from eggs with higher percentages of protein and lipid. This result suggests that protein and/or lipid are important energy sources for larval and postlarval survivorship of H. discus hannai. It has been reported that protein was the major endogenous energy reserve used by larvae of H. rufescens (Jaeckle & Manahan 1989b, Vavra & Manahan 1999). In contrast, lipid was reported to be a major energy source for larval H. rubra (Litaay et al. 2001) and H. fulgens (Moran & Manahan 2003). In this study, survivorship of larvae and postlarvae related to both protein and lipid composition, and the eggs that contained higher levels of protein showed a higher percentage of lipid. Therefore, we cannot conclude which protein or lipid is more important as an initial energy source for H. discus hannai. Protein and lipid in eggs may be used as important energy sources for larvae and early postlarvae.

Dissolved organic matter (DOM) taken up from the ambient seawater may be an important initial energy source for larval abalone (Jaeckle & Manahan 1989a, 1989b, 1992). The seawater used in this experiment was passed through a sand filter and therefore a considerable amount of DOM may have been removed (Manahan & Stephens 1983). As a result of the low DOM concentration in the rearing seawater, individuals may largely depend on egg yolk reserves. Thus, larval period may be dependent on the amount of DOM in the ambient seawater and the biochemical contents of eggs.

It has been reported that postlarval H. discus hannai and H. iris without food grew at comparable rates to those with food during the first several days after metamorphosis, but they could not grow well after 1 wk without food (Takami et al. 2000, Roberts et al. 2001). The early development of postlarvae seems to be supported by residual yolk reserves and/or DOM uptake. For H. rufescens, the metabolic rate of postlarvae increased 3-5-fold within a few days of metamorphosis (Shilling et al. 1996), and it was suggested that the amount of energy initially present in the oocyte may be related to postlarval survivorship, rather than to energy metabolism during larval development (Jaeckle & Manahan 1989b, Litaay et al. 2001). The results of the present study show that quantity and quality of the egg yolk reserves affect both maximum larval period and postlarval survival of H. discus hannai.

Kikuchi and Uki (1974a) reported that H. discus hannai gradually started being responsive to spawning cues between 5001-500[degrees]C x days EAT and most of the eggs obtained were fertilized regardless of the EAT. However, the results of this study suggest that consecutive larval and postlarval survivorship were affected by EAT. Larvae from lower EAT treatments (1,050 and 1,150[degrees]C x days) had a shorter larval period without settlement cues, and postlarvae from lower EAT treatments also had a shorter survival period without food than those from higher EAT treatments (1,650 and 2,350[degrees]C x days). In wild H. discus hannai the EAT has been calculated to increase from 1,000-2,500[degrees]C x days during the natural spawning season. Our results suggest that egg quality improves as the spawning season progresses. The biochemical composition of eggs seemed to affect the survival of larvae and postlarvae. The ratios of protein and lipid to dry egg weight tended to be higher in eggs from higher EAT treatments.

Larval H. discus hannai become competent from 4 days after fertilization at 20[degrees]C (Seki & Kanno 1977), and competent larvae delay metamorphosis if they fail to contact with an appropriate environmental stimulus under laboratory conditions (Takami et al. 2002). This study showed that larval period, without settlement cues, for individuals from lower EAT treatments was shorter than those from higher EAT treatments. Abalone larvae swim by cilial beating but disperse as passively transported particles because their swimming ability is poor. In the natural habitat, the dispersal area and thus the chance of contacting with appropriate substrata may be more limited for larvae from the eggs spawned at lower EAT than from higher EAT broodstock, because of their shorter larval period.

There was no correlation between cytoplasm volume and both maximum days of larval period and postlarval survival period in this study. Amount of energy sources in eggs is unlikely to depend on egg cytoplasm volume in an abalone H. discus hannai. Biochemical composition of eggs may be more important than cytoplasm volume for the early development and survival of larval and postlarval abalone. Similar result was reported for a starfish, Pteraster tesselatus; egg size was not a reflection of energetic content (McEdward & Coulter 1987).

Hahn (1994) observed that a slight increase in the mean oocyte volume in the ovary of H. discus hannai occurred between 600 and 900[degrees]C x day EAT and that the volume rapidly increased from 900-1500[degrees]C x day EAT. Whereas, in this study, even at lower EAT treatments such as 850[degrees]C x day, the mean cytoplasm volume of spawned eggs was comparable to those at higher EAT. These results suggest that spawned egg volume is independent of the oocyte volume in the ovary.

It was reported that in H. discus hannai gametes developed in unison in the ovary for a single cohort (Hahn 1994). Most individuals, which spawned without any artificial spawning cues at 1450[degrees]C x day EAT, released normal gametes again by artificial spawning induction at 1,650 or 2,350[degrees]C x day EAT in this study. These observations suggest that H. discus hannai has a capacity to spawn multiple times during a spawning season; some proportion of larger oocytes in a single cohort may be gradually released in the early period of the spawning season. The second cohort of oogonia may begin to develop rapidly after the first spawning and the size of oocytes may increase dramatically to be spawned at a later time as reported for tropical abalone species H. asinina (Jebreen et al. 2000, Counihan et al. 2001). This study suggested that the amount of energy sources in eggs for larvae and early postlarvae increases toward the end of the spawning season. If H. discus hannai has the ability to undertake multiple spawnings, lipid and protein might be at a higher concentration in eggs held for a longer period in the gonad than eggs released early in the spawning season. On the other hand, if H. discus hannai shows a spawning pattern similar to H. asinina, after the spawning of eggs in the first cohort, eggs of better quality might develop in later cohorts. More detailed studies on the process of oogenesis in wild populations are needed to develop a better understanding of the reproductive cycle of H. discus hannai.

ACKNOWLEDGMENTS

The authors thank Hiroyuki Kawakami and Takashi Igarashi, Yamagata Aquaculture Fishery Association, for technical assistance. They also thank Yutaka Kurita, Tohoku National Fisheries Institute, Yoh Yamashita, Kyoto University, and Christopher B. Clarke for their valuable comments.

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

HIROTATSU FUKAZAWA, (1) * HIDEKI TAKAMI, (2) TOMOHIKO KAWAMURA (1) 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

TABLE 1.
Experimental information detailing broodstock characteristics
and subsequent data collected from gametes/offspring.

                                    Maternal Characteristics

                                                   Whole
EAT                                 Shell           Wet
([degrees]         Spawning        Length          Weight
day)                 Date           (mm)            (g)

850                17/6/2002        91.4            91.7
                                    90.6            93.8
                                    92.7            87.1
                                    93.2            87.9
1,050              2/7/2002         91.4           100.1
                                    98.0           107.5
                                    90.6            91.6
                                    89.4            77.9
                                    91.7            92.7
1,150              8/7/2002         91.1            84.9
                                    88.2            68.0
                                    95.0            79.7
                                    84.4            71.3
1,650              14/8/2002        90.0            71.1
                                    92.9            90.4
                                    91.1            96.0
                                    91.1            96.7
1,900              2/9/2002         92.5           109.9
                                    88.5            89.5
                                    91.7            76.3
                                    91.1            99.8
2,350             18/10/2002        84.5            65.2
                                    91.4            86.3
                                    92.6           100.8
                     mean           91.0            88.2
                      SE             0.6            2.5

                 Determination
EAT                 of Egg       Biochemical   Larval Period
([degrees]         Cytplasm       Analyses     and Postlarval
day)                Volume         of Eggs        Survival

850                   --             --              nd
                      --             --              nd
                      --             --              nd
                      --             --              nd
1,050                 --             --              --
                      --             --              nd
                      --             --              --
                      --             --              nd
                      --             --              --
1,150                 --             --              --
                      --             --              --
                      --             --              --
                      --             nd              nd
1,650                 --             nd              --
                      --             nd              --
                      --             nd              --
                      --             nd              --
1,900                 --             nd              nd
                      --             nd              nd
                      --             nd              nd
                      --             nd              nd
2,350                 --             --              --
                      --             --              --
                      --             --              --

nd: not determined
--: determined

TABLE 2.
Nested analysis of variance for egg cytoplasm volume, percentage of
protein and lipid in eggs from different EAT treatments Protein and
lipid data were arcsine transformed prior to statistical tests.

                 Source          DF    MS

Egg cytoplasm
  volume         EAT               5   1.2 x [10.sup.-5]
                 Mother [EAT]     18   1.1 x [10.sup.-6]
                 Error          1148   8.6 x [10.sup.-8]
Protein          EAT               3   43.4
                 Mother [EAT]     12   10.8
                 Error            28   7.4
Lipid            EAT               3   176.3
                 Mother [EAT]     12   12.6
                 Error            31   10.5

                 Source           F    P

Egg cytoplasm
  volume         EAT            10.9   <0.001
                 Mother [EAT]   12.4   <0.001
                 Error
Protein          EAT             4.0   >0.05
                 Mother [EAT]    1.4   >0.05
                 Error
Lipid            EAT            14.0   <0.001
                 Mother [EAT]    1.2   >0.05
                 Error