Growth and survival of post-larval abalone Haliotis diversicolor supertexta (lischke) using an alternative culture method in the light and dark.

ABSTRACT Stott's Abalone Postlarvae Production System (SAPPS) was developed as an alternative method for settling larvae and raising postlarval abalone. It allows continuous hatchery production of abalone without using live food. The new system was tested in a 28-day trial, both in the dark and light, on Haliotis diversicolor supertexta. Plates were sprayed with a mixture containing commercial, artificial micro particle diet 1 or 2, and a 1% agar solution and assigned a 10-L flow-through tank, either in the dark or light. The four treatments--Diet 1-dark (D1-D), Diet 1-light (D1-L), Diet 2-dark (D2-D), and Diet 2-light (D2-L) were replicated 3 times. The chemical composition of the two diets was similar although lipids were 21.05% in diet 1 compared with 13.74% in diet 2. Final survival was not significantly different (P > 0.05) among the treatments and ranged from 14.8% to 31.4%. The variable results for larval settlement and low postlarval survival in the first week was most likely due to unsatisfactory preconditioning of plastic plates with juvenile abalone trail mucus or weak settlement induction of this mucus. Although the light/dark factor was not significantly different (P > 0.05), abalone postlarvae fed diet type 2 (D2-D and D2-L) were significantly larger (P < 0.05) than those fed diet type 1 (D1-D and D1-L). The average growth rate per day was 43 [+ or -] 4 [micro]m, 44 [+ or -] 5 [micro]m, 51 [+ or -] 4 [micro]m, and 53 [+ or -] 4 [micro]m for D1-D, D1-L, D2-D, and D2-L respectively. Diet 2 may have been more nutritionally complete than diet 1. SAPPS was used successfully in this trial to culture H. diversicolor supertexta postlarvae, regardless of light and natural food.

KEY WORDS: SAPPS, larval settlement, post-larval nutrition, micro particle diet, abalone, Haliotis

INTRODUCTION

Several problems have been identified and documented in regards to the current method of raising abalone on diatom biofilms. Abalone hatcheries often suffer 90% to 100% mortality by 2 mo postsettlement (Searcy-Bernal et al. 1992). Management of these diatom biofilms is often challenging and Hahn (1989) and Seki (1997) suggest that control of the initial food supply is still one of the most critical problems in hatchery seed production. It is a difficult task to maintain a supply of readily ingestible and digestible food (diatoms) for growing postlarvae (Kawamura et al. 1998). Alternative methods to those utilizing living diatoms, for abalone postlarval production, maybe beneficial to industry.

A mass culture system for the green alga Ulvella lens on plastic plates has been developed to provide a settlement cue for larval abalone and provide a source of nutrition for the postlarvae. However, growth rates of postlarvae fed U. lens are significantly lower than favorable diatom species (Kawamura 1996, Seki 1997, Daume et al. 2000). Alternatively, Buchal et al. (1998) reported that postlarval Haliotis rufescens could be raised on dulse (Palmaria mollis) but direct utilization of dulse as a principle food source was only observed in abalone >3 mm in length.

Olin and McBribe (2000) suggested that artificial diets are sometimes used by commercial abalone hatcheries to feed postlarval abalone once they have reached 2 mm in shell length. Stott et al. (2002) reported that young postlarval Haliotis discus discus grew well on a micro particle diet that was bound within an alginate gel which had been thinly pasted onto plastic plates. In a follow-up study, Stott et al. (2003a) developed a method for settling larval H. diversicolor supertexta directly onto plastic plates coated with the micro particle diet/alginate gel. Growth and survival rate of abalone postlarvae using this method was comparable to that of post larvae grown using the diatom biofilm method. However, the alginate gel method would not be suitable for large-scale production of postlarval abalone (Stott et al. 2002, Stott et al. 2003b).

Consequently we focused our research on addressing this limitation and SAPPS was developed. This new method utilizes the temperature dependant setting properties of agar to cover plates, via spraying, with a micro particle diet/agar gel mixture. Commercial scale trials were previously conducted to test growth and survival rate of postlarval H. diversicolor supertexta using SAPPS compared with that using conditioned diatom biofilm (Stott et al. unpublished data). After 42 days abalone grown using SAPPS had reach an average size of 3.2 mm with a survival rate of 51.7% compared with a size of 2.2 mm with a survival rate of 10.6% for conditioned diatom biofilm.

The current trial was conducted to determine whether SAPPS could be used to culture H. diversicolor supertexta in small tanks. Two commercial, artificial micro particle diets were used and each was tested in the dark and light to determine if natural microalgal related food was affecting results.

MATERIALS AND METHODS

Methodology for SAPPS

Two experimental versions of the commercially available artificial microparticle diet "Plate Powder" were supplied by Adam and Amos Pty Ltd (Mt Barker, Australia). The diets were sieved and only particles of 53-[micro]m or less were used in the trial. The original particle size of diet 1 was quite large and less than 10% of the diet passed through the sieve. However, the original particle size of diet 2 was smaller than diet 1, with over 70% of the particles passing through the sieve. Examination of both diets under a compound microscope at x400 magnification revealed that many of the ingredients were not finely ground during the initial grinding process when the diets were produced. Large clumping of individual ingredients was observed among particles that failed to pass through the sieve.

A solution of agar was prepared by dissolving 10 mg/mL of agar powder (Kokusan Chemicals; Tokyo, Japan) in boiling, deionized water. Once the temperature of the solution had fallen to 60[degrees]C, 100 mg/mL of micro particle diet was added to the solution and the contents were thoroughly mixed. Results from pretrials confirmed that agar by itself (without microparticulate diet) was inappropriate for supporting high growth and survival of postlarval H. discus discus.

The resulting solution was then transferred to a MK-7 fine mist sprayer (Calmar; NY, USA) and the temperature was maintained at approximately 50[degrees]C. Each plastic plate (dimensions of 22.5 cm x 11.5 cm) was sprayed (individually) after being removed from the experimental tank and allowed to drain for approximately 10 sec. Plastic plates were sprayed by holding them 30 cm away from the sprayer. The entire surface of the plate was subsequently sprayed with solution contained in the MK-7 fine mist sprayer. Pretrials confirmed that the SAPPS methodology did not compromise postlarval survival. Results from pretrials also confirmed that newly metamorphosed postlarvae did not incur mortality via drowning from the fine mist produced by the sprayer and the temperature of the agar/diet solution dropped to safe levels during the process of spraying (as the solution moves through the air and contacts with the wet plastic plates). Each side of the plastic plate was sprayed twice (approximately 0.4 mL of solution per side). Once the spray process was complete, the plate was quickly placed back into the experimental tank.

Spawning and Larval Settlement

Broodstock H. diversicolor supertexta, at a ratio of two females to one male, were induced to spawn at Banda Marine Laboratory, Tokyo University of Fisheries, using UV light and temperature shock. After hatching, larvae were reared at three larvae/mL in a 100-L flow-through system with a flow rate of 400 mL/min and a temperature of 23[degrees]C. Larvae were deemed competent for settlement when the third tubule on the cephalic tentacle appeared. At this stage larvae were concentrated, counted, and then transferred to the laboratory ready for assignment to the experimental tanks.

Seven days before placing the plastic plates in the experimental tanks, they were preconditioned with juvenile abalone mucus to act as an attachment and settlement inducer. This was achieved by spraying the appropriate diet onto the plastic plates using the method stated earlier. The plates were then placed vertically into a tank with a small number of juvenile H. discus discus (20-50 mm) and the tank covered with black plastic to prevent the growth of microalgae. The juvenile abalone were encouraged to graze the diet coated plates and the food was reapplied via the spray system every second day for a period of 5 days.

Two conditioned plates (with the appropriate diet) were hung vertically in each 10-L experimental tank and approximately 2,000 larvae were introduced. Larvae were allowed to settle for 24 h, and then the plastic plates were removed and the number of attached larvae was enumerated lbr each tank. In this study, abalone larvae were considered to have completed metamorphosis when they had dropped their velum and showed evidence of peristomal shell growth.

Unattached larvae in the experimental tanks were discarded along with the seawater when the tanks were cleaned. All tank surfaces were scrubbed with a scrubbing brush and washed with freshwater to remove any larvae that had attached. The tanks were then refilled with fresh seawater, ready for the postlarval grow-out phase of the trial.

Post-larval Grow-out

The trial consisted of 4 treatments: Diet 1-dark (D1-D), Diet 1-light (D1-L), Diet 2-dark (D2-D), and Diet 2-light (D2-L), each replicated three times (total of 12 experimental units). Independent tanks were used for replicates. Each experimental unit was assigned a 10-L flow-through tank that was supplied with charcoal filtered water at 250 mL/min. The tank's outer surfaces were painted with black paint and 150 [micro]m was glued over the outlet of the tank to stop dead larvae from being flushed out of the tanks. The tanks to be placed in the dark, D1-D and D2-D, were covered with black plastic. The tanks remaining in the light, D1-L and D2-L, were left uncovered and exposed to a natural lighting regimen (approximately 7,000 LUX during daylight hours).

Food was sprayed onto the plastic plates using the earlier mentioned method, on day 1 (after the 24 h settlement period) and every second day thereafter. Tanks were siphoned every second day and the siphoned water samples were examined and the number of dead postlarvae tallied to calculate percentage survival. Postlarvae from the siphoned water samples were transferred to a glass slide with a pipette and examined to confirm if they were dead. This process was also used to determine the relative proportion of mortality of larvae (no peristomal shell formation) and postlarvae (peristomal shell formation). Tanks were also thoroughly scrubbed twice per week to remove any attached microalgae, etc. At the end of the experiment the total number of postlarvae remaining in each tank was enumerated. This number was compared with previously recorded survival data to validate the accuracy of the methodology.

Post-larvae were sampled twice per week by randomly removing 10 animals from plastic plates in each tank using a soft brush. The size (maximum standard length) of postlarvae was taken using a compound microscope fitted with an ocular eyepiece. Water temperature was measured using a standard thermometer and averaged 25.3[degrees]C [+ or -] 0.9[degrees]C.

Approximate Analysis and Particle Size Determination of the Diets

Proximate analysis of the diets for moisture, crude protein, crude lipid, and crude ash was performed using the methodology outlined in Hernandez et al. (1995).

The particle size of the artificial diets was determined by randomly extracting 40 particles and measuring the size under a compound microscope fitted with an ocular eyepiece, at xl00 magnification.

Statistical Analysis

Statistical analysis was conducted using the computer package SYSAT (SPSS Inc., IL USA). Before conducting statistical ANOVAs, normality was confirmed using normal probability plots and homogeneity of variance was validated utilizing Levene's test. Data was transformed where necessary and a one-way ANOVA performed. Differences in means were determined using Tukey's test.

RESULTS

The mean composition of the diet 1 and diet 2 were similar, with exception to crude lipids (Table 1). Crude lipid was 21.0% for Diet 1 compared with 13.8% for Diet 2. The particle size of diet 1 was slightly larger than diet 2; 34 [+ or -] 11 [micro]m compared with 31 [+ or -] 6 [micro]m.

Attachment of larvae in the different treatments was variable and patchy on the plates in the experimental tanks. Final mean settlement of larvae was 241 [+ or -] 142, 173 [+ or -] 87, 333 [+ or -] 56 and 195 [+ or -] 60 in D1-D, D1-L, D2-D, and D2-L respectively (Table 2).

Mortality of postlarvae in the different treatments was high in the first week, ranging from 55.4% to 74.8% (Fig. 1). The majority of these mortalities were observed to have no peristomal shell growth. Thus, a high percentage of the mortalities in the first week were larvae that had attached but failed to complete metamorphosis. Very little mortality occurred after the first week and final mean mortality of postlarvae in the different treatments ranged between 14.8% and 31.4%.

[FIGURE 1 OMITTED]

The daily growth rate of postlarvae in all treatments increased in the ensuing weeks (Fig. 2). The final mean size of post larvae was significantly higher (P < 0.05) in D2-D (1,695 [+ or -] 148 [micro]m) and D2-L (1752 [+ or -] 134 [micro]m) compared with that of D1-D (1,488 [+ or -] 153 [micro]m) and DI-L (1512 [+ or -] 181 [micro]m).

[FIGURE 2 OMITTED]

DISCUSSION

H. diversicolor supertexta postlarvae grew well using SAPPS, regardless of light, suggesting that they can grow without the requirement of light or photosynthetic live food. Plastic plates in D1-D and D2-D were kept in the dark to have been relatively free of microalgae. Additional measures to limit microalgal growth in the experimental tanks were also included (e.g., supplying tanks with charcoal filtered water and scrubbing them twice weekly). Thus, postlarvae were able to gain adequate nutrition via SAPPS and those fed diet 2 grew at over 50 [micro]m/day.

Post-larvae that were grown in the dark may have gained exogenous nutrition from the artificial diet, bacteria that proliferated on the plates and mucus deposited by the juvenile abalone. Takami et al. (2000) found that H. discus hannai shift from yolk energy source to particulate food by the time they reach 500 [micro]m. Takami et al. (1997) reported that all H. discus hannai larvae that were raised on an unsuitable diatom biofilm died before they reach 500 [micro]m and all those raised exclusively on abalone mucus died before they attained a size of just over 700 [micro]m. Postlarvae over 1 mm in length require high levels of digestion of diatoms in addition to any extracellular substances for favorable growth (Kawamura 1996). Thus, in this study, postlarval abalone fed diet 2 grew to about 1,700 [micro]m and the growth rate doubled from week 1 to week 4 (approx. 30 [micro]m to 60 [micro]m). Therefore, postlarvae were most likely gaining a high level of nutrition from the artificial micro diet. Visual observations also confirmed that food was in the gut and mortality after week 1 was minimal. However, the influence of bacteria on growth and survival of postlarval abalone was not determined.

Post-larval abalone cultured in the dark grew as well as those grown in the light. Photosynthetic food that may have grown on the plates, provided postlarvae no advantages in terms of growth and survival. It seems that abalone in this trial had no requirement for light. However, it is also necessary to investigate the opposite scenario because complete, continuous darkness could potentially offer postlarval abalone advantages in terms of growth and survival. Adult and juvenile Haliotis spp. mainly teed at night (Ino 1943, Sakai 1962, Uki 1981) and grow better when cultured in the dark (Ebert & Houk 1984). However Velez-Espino (1999) found that there was no evidence of nocturnal feeding habits in postlarvae. Abalone postlarvae remained attached to the plastic plates but become increasingly sensitive to light after they develop their first respiratory pore (Hahn 1989). Thus, postlarval feeding most likely takes place regardless of light or time of day. Darkness at this trial provided no advantages in terms of growth and survival.

The final mean size of postlarvae fed diet 2 was greater than that of postlarvae fed Diet 1. Proximate composition of the diets was similar, apart from a higher percentage of crude lipids in Diet1, compared with Diet 2. Uki et al. (1985) found that growth performance of H. discus hannai was maximized at 5% crude lipid inclusion in an artificial diet. Other species such as H. tuberculata have an optimum growth rate at levels as low as 3.11% crude lipid (Mai et al. 1995). In the current trial, the artificial diet was supplied by a commercial manufacturer and no information was made available regarding the ingredients or binders incorporated into the diet. The only obvious difference after proximate analysis was lipid content. Total crude lipid in diet 1 was 21% compared with only 13.8% in diet 2 and this high inclusion of lipid, although not conclusive, may have negatively affected postlarval growth. Processing the artificial micro particle diet for application via SAPPS may have affected the original gross composition or nutritional balance. The sieving process resulted in selection of small particles (<53 [micro]m), whereas larger particles were excluded. The original particle size of diet I (before sieving) was much larger than diet 2 and this resulted in high particle retention when sieving with a 53-[micro]m sieve. This may have caused higher selection of particles that contained high levels of lipids. However, because the crude lipid content of the diets before sieving was not determined and a list of ingredients and methods used to make the diet were not available, the exact reason for such a high level of lipids in the diet remains unclear.

In future, it is recommended that ingredients incorporated into diets that are applied using SAPPS, be thoroughly ground. Additionally, after processing the final diet, if the particle size of the diet is over 53 [micro]m, the diet should be ground down further to create a finer particle size before sieving. Artificial abalone diets that have been made for juvenile or adult abalone can also be used in SAPPS provided they are ground down to a fine powder.

Extremely high postlarval mortality in the first week of the trial in all treatments was due to a high rate of unsuccessful metamorphosis of larvae. Many of the larvae attached to the plates, dropped their vellum but failed to deposit peristomal shell. These larvae died before the end of the first week. However, larvae that completed metamorphosis were in high densities in small patches on the plates. Thus, it is possible that the plates were not adequately conditioned with mucus due to the fact that the number of juvenile abalone that were placed into the tanks to condition the plates with mucus for larval settlement, were too low. It was also observed that food sprayed on the plates for juvenile abalone during the conditioning period was not heavily grazed. Mucus was only deposited on the regions of the plastic plates where juvenile abalone grazed. Other regions of the plastic plates (with no mucus) larvae attached at lower densities and most of these larvae failed to complete metamorphosis. In a previous trial, H. discus discus larvae attached to blank plastic plates at a rate of up to 20%, but successful metamorphosis occurs in less than 5% of the larvae (Stott et al. In press). In the same study, attachment and metamorphosis was increased to about 80% by spraying freeze-dried algal powders (Spirulina platensis or Chlorella vulgaris) onto plastic plates and then conditioning them with abalone mucus by allowing juvenile H. discus discus to graze the plates. Takami et al. (1997) reported that H. discus hannai settled on abalone mucus conditioned plastic slides at a rate of 48.7% and that all postlarvae died by the end of the third week. This documents a much higher rate of attachment than the current trial but no larvae completed metamorphosis (and only 10% attached) in a control group that was offered no settlement stimulus. Additionally, as with our previous trial, the same species of juveniles as the larvae were used to condition plates in the mucus treatment.

It is possible that mucus from juvenile H. discus discus was not a strong attachment and metamorphosis inducer for larval H. diversicolor supertexta. Seki and Kanno (1981) found that larval H. discus hannai settled on mucus from 4 different Haliotiodes but not on mucus from a number of mollusc from different genus to Haliotis. However, in that study, percentage settlement was not determined and H. diversicolor supertexta was not tested. H. diversicolor supertexta is a small species of abalone that spawns sporadically over the summer period whereas H. discus discus is a relatively large species of abalone that spawns usually once in autumn (Hahn 1989). Attachment and metamorphosis rates of larval abalone may be improved by using juvenile abalone from the same species for conditioning plates.

After the first week, postlarval survival was very high. Even though the average water temperature was over 25[degrees]C, the use of artificial diets did not seem to adversely affect culture conditions. Hahn (1989) suggests that most abalone farmers stop using artificial diets when water temperatures rise above 24[degrees]C. The methodology for SAPPS results in the artificial micro particle diet being coated with agar. Stott et al. (2003b) found that nitrogen leaching from an artificial micro diet after 12 h immersion was nearly reduced by half when binding the diet in an alginate gel compared with adding the diet directly to the water column. Although this was not quantified in this trial, it is likely that binding the diet in an algal gel would protect it from bacterial degradation and leaching that may potentially adversely affect water quality.

There is a need for greater research in this area before modifying SAPPS for use in industry. It will be necessary to determine optimum concentration of agar as well as feeding frequency. A major disadvantage of SAPPS is that it is an extremely intensive method and spraying the plates requires much effort that may translate into high labor costs. It would be beneficial to spray plates as infrequently as possible without adversely affecting postlarval growth and survival. SAPPS allows application of artificial diet to wet plates but some of the diet is lost when replacing plates into the tanks.

In the near future, commercial trials of SAPPS will be conducted using state of the art spraying technology and incorporating new larval settlement technology.

TABLE 1.

The size and composition of the two test diets.

                                              Diet

                                       1                     2

Mean particle size              34 [+ or -] 11         31 [+ or -] 6
  ([micro]m) [+ or -] SD
Moisture (%)                         11.1                   9.8
                            On dry matter basis (%)
Crude protein                        31.5                  30.8
Crude lipid                          21.0                  13.8
Crude ash                             7.5                   7.0

TABLE 2.

Results of a 4-week feeding trial for post-larval H. diversicolor
supertexta fed two different artificial micro particle diets in the
light and dark.

                                           Final Mean Measurements

               Mean Number Settled
Treatments         [+ or -] SD           Size [+ or -] SD ([micro]m)

Diet 1 (D)       241 [+ or -] 142         1488 [+ or -] [153.sup.a]
Diet 1 (L)       173 [+ or -] 87          1512 [+ or -] [181.sup.a]
Diet 2 (D)       333 [+ or -] 56          1695 [+ or -] [148.sup.b]
Diet 2 (L)       195 [+ or -] 60          1752 [+ or -] [134.sup.b]

                              Final Mean Measurements

                Daily Growth Rate
Treatments    [+ or -] SD ([micro]m)    Survival Rate [+ or -] SD (%)

Diet 1 (D)        43 [+ or -] 4               18.8 [+ or -] 3.7
Diet 1 (L)        44 [+ or -] 5               20.4 [+ or -] 14.8
Diet 2 (D)        51 [+ or -] 4               31.4 [+ or -] 1.2
Diet 2 (L)        53 [+ or -] 4               14.8 [+ or -] 5.1

Different superscripts in the same column denote difference in means
(p < 0.05)

ACKNOWLEDGMENTS

The authors thank the staff from Banda Marine Laboratory for their assistance throughout the trial. Many thanks to Joel at Adam and Amos Pty. Ltd. (Mt Barker, Australia) for supplying the artificial diets ("Plate Powder") used in this trial. Finally, thanks to Ronald Mitchell and anonymous colleagues for reviewing the manuscript.

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ADRIAN E. STOTT, (1) TOSHIO TAKEUCHI (1) * AND YASUYUKI KOIKE (2)

(1) Laboratory of Fish Culture, Department of Aquatic Biosciences, Tokyo University of Fisheries, 4-5-7 Konan, Minato, Tokyo 108-8477, Japan; (2) Banda Marine Laboratory, Tokyo University of Fisheries, Banda, Tateyama, Chiba 227-0036, Japan

* Corresponding author. Email: [e-mail]