Combined effects of light condition (constant illumination or darkness) and diatom density on postlarval survival and growth of the abalone Haliotis rufescens.
KEY WORDS: abalone postlarvae, survival, growth, darkness, diatom density
The red abalone (Haliotis rufescens) is the largest abalone species in the world, reaching up to 28 cm in shell length (Hahn 1989, Leighton 2000). It represents 95% of the abalone culture in California (USA) (Leighton 2000) and is also the most important species cultured in Baja California, Mexico (McBride 1998). Abalone culture is a high-risk activity and mortalities can reach up to 99% after 3 y (Hone et al. 1997). In the postlarval stage, which begins after settlement and metamorphosis, the highest mortality is usually reported (80% to 95%) (Searcy-Bernal 1996, Hone et al. 1997).
In abalone postlarvae, growth is influenced by several factors including type of food (Kawamura et al. 1998a, Roberts et al. 1999, Daume et al. 2000), quantity of food (Searcy-Bernal et al. 2001), starvation period (Roberts et al. 2001, Takami et al. 2000), temperature (Leighton 1974), and light intensity (Searcy-Bernal et al. 2003). For example, postlarvae of the blue abalone Haliotis fulgens grow better in low irradiances (6 [micro]E/[m.sup.2]/s) than in higher light intensities (24-75 [micro]E/[m.sup.2]/s), probably because of differences in ecologic conditions in the biofilms developed over culture surfaces (Searcy-Bernal et al. 2003). In the abalone farms of California and Baja California, postlarvae are usually maintained at irradiances from 3 to 200 [micro]E/[m.sup.2]/s under natural photoperiods or continuous artificial light (Searcy-Bernal et al. 2003). Although the effect of photoperiod on postlarval growth is unknown, diatoms need light for their growth, which is important to maintain a good density of cells to feed the postlarvae.
Juvenile and adult abalones have a circadian rhythm; they are more active and feed at night (Hahn 1989, Leighton 2000). Some studies have shown increased growth rates when these gastropods are cultured in darkness (Ebert & Houk 1984, Godoy-Corrales 1989). Research addressing this issue for postlarvae is lacking, although a preliminary study provided some evidence that postlarvae of H. fulgens increase their grazing rates at dusk (Velez-Espino 1999).
Recent studies have shown that the quantity of food available for postlarvae is an important factor determining their growth. Grazing and growth rates of H. fulgens postlarvae increase as the density of the diatom Navicula incerta increases from 500 to 4,000 cells/[mm.sup.2] (Searcy-Bernal et al. 2001) but studies for other species are lacking.
The objective of this work is to evaluate the combined effect of continuous light or darkness conditions and different densities of the diatom N. incerta on the survival and growth of red abalone (H. rufescens) postlarvae.
MATERIALS AND METHODS
Abalone larvae were provided by the commercial farm Abulones Cultivados (Erendira, B.C., Mexico) on March and May 2002, transported to the Instituto de Investigaciones Oceanologicas (IIO) laboratory and used in Experiments 1 and 2, respectively. Competent larvae were induced to settle by adding 1.5 [micro]M of gamma-aminobutyric acid (GABA) (Searcy-Bernal & Anguiano-Beltran 1998).
Monocultures of N. incerta, a benthic diatom widely used to feed abalone postlarvae, were provided by the IIO Microalgae Laboratory. Diatoms were inoculated in 250 mL Erlenmeyer flasks containing 150 mL of f/2 medium (Guillard 1975) and cultured under constant temperature (17 [+ or -] 1[degrees]C) and illumination (37 [micro]E/ [m.sup.2]/s, provided by fluorescent day-light lamps). After 3-4 days of culture, the flasks were immersed in an ultrasound bath (Fisher Scientific FS6) for 3 min to detach the diatoms, which were counted using a hematocytometer to estimate cellular density and to calculate diatom densities to be used in the experiments.
Settlement was carried out in plastic containers (57 x 37 x 13 cm) containing 5 L of filtered (1 [micro]m) and UV-irradiated seawater. After metamorphosis, postlarvae were fed with N. incerta (250 cells/[mm.sup.2]) and cultured in these containers for 14 days. Seawater was changed every 2 days and diatoms were added every week.
The experiment was conducted in six-well tissue culture plates (Falcon 3046, 9.45 [cm.sup.2] bottom area) with 10 mL of 1 [micro]m-filtered UV-irradiated seawater per well. Water changes were performed daily.
Two light conditions (light and dark) and six diatom densities were tested following a split-plot experimental design (Steel at al. 1997). Three culture plates (whole units) were placed inside thick black polyethylene bags to obtain dark conditions, and three plates were in continuous light of between 24 and 33 [micro]E/[m.sup.2]/s (fluorescent day-light lamps). Six densities of N. incerta (250, 500, 750, 1,000, 2,000, and 3,000 cells/mm2) were randomly inoculated in the wells (subunits) of each plate. After 1 day, ten 14-day-old postlarvae (480 [micro]m average shell length) were placed in each well. Water changes were performed daily. The culture plates in the dark treatment were exposed daily to light for -30min during water changes.
After a week in these food densities all wells were cleaned of diatoms and feces with a brush and rinsed two or three times with seawater. Then, the wells were reinoculated with higher densities of N. incerta (500, 1,000, 2,000, 4,000, 8,000, and 10,000 cells/ [mm.sup.2]). These final diatom densities were not applied during the first days of the experiment, because previous experience had shown deleterious effects of high densities on early postlarvae. Every 4 days, the wells were cleaned as described earlier and were reinoculated with these final diatom densities of N. incerta.
The same experimental design and vessels were used, but some different procedures were followed. Competent larvae (13-15) were placed and settled with GABA in the wells of culture plates. Irradiance of the culture plates under continuous light was between 19 and 21 [micro]E/[m.sup.2]/s. Sea water quality (1 [micro]m-filtered, UV-irradiated) was further improved by sterilization (in an autoclave). Two days after metamorphosis induction postlarvae were fed with 6 densities of N. incerta (250, 500, 750, 1000, 2000, and 3000 cells/[mm.sup.2]). Every 4 days, the wells were cleaned and rinsed as described in Experiment 1 and reinoculated with these initial diatom densities. After 16 days (when postlarvae were 18-days old), reinoculation densities were increased to 500, 1,000, 2,000, 4,000, 8,000, and 10,000 cells/[mm.sup.2]. These differences between experiments are summarized in Table 1.
Both experiments were conducted at 17[degrees]C [+ or -] 1[degrees]C. Survival was determined by counting live postlarvae in each well. To determine shell length and growth rates, all surviving postlarvae were video-recorded with a high-resolution camera (Sony SSC-C374) in an inverted microscope (Meiji Techno). Images were digitalized in a computer and measurements of shell length were performed with the program Scion Image (4.0.2). Survival and shell lengths were evaluated at days 9 and 20 in Experiment 1, and at days 10, 18, and 32 in Experiment 2 (in this trial day 0 corresponded to the day of the first diatom addition, i.e., 2 days after metamorphosis induction).
Statistical analysis was performed using the program JMP (version 3.2.6, SAS Institute Inc.). Split-Plot analyses of variance (ANOVA) (Steel et al. 1997) were used to evaluate the effect of diatom densities, light condition and their interaction on postlarval survival, shell length, and growth rates. When significant interactions were found, separate ANOVAs and multiple comparisons (LSD tests) were performed to evaluate the effect of diatom density within each light condition. Percent survival data were transformed before analysis (arcsine square root). A value of [alpha] = 0.05 was chosen as the significance level.
In Experiment 1, survival was high (>80%) in all treatments during the experimental period of 20 days (Fig. 1) and there were no significant over-all effects of diatom density or light condition, although in the first 9 days a significant interaction was found (Table 2) reflecting a different effect of one factor in levels of the other. Separate ANOVA tests within each light condition showed a significant effect of diatom density only in the dark (F = 3.71, P = 0.029) where a significantly lower survival in 2,000 cells/ [mm.sup.2] was detected (LSD tests, Fig. 1).
[FIGURE 1 OMITTED]
In Experiment 2, survival after the 32-day experimental period was below 70% (Fig. 2). Treatment effects were not significant during the first 10 days; however, after 18 days a significant interaction was detected (Table 3). Survival in the dark was lowest (45%) at the lowest diatom density (500 cells/[mm.sup.2]), increased as density increased up to 2,000 cells/[mm.sup.2] and remained more or less constant at higher densities (ca. 75%). In the light treatment survival was higher at intermediate diatom densities up to 8,000 cells/ [mm.sup.2] (ca. 60%) but decreased drastically to 24% at the highest density (10,000 cells/[mm.sup.2]) (Fig. 2). This survival was significantly lower than in the dark (76%) at the same diatom density (F = 23.27, P = 0.008). Similar survival patterns were observed at the final evaluation (32 days) (Fig. 2) when significant diatom and interaction effects were detected (Table 3). This interaction implied a significant effect of diatom density only in the dark (F = 3.34, P = 0.040), with the lowest survival (6%) at the lowest diatom density and more or less constant survival (ca. 60%) at densities above 2,000 cells/[mm.sup.2] (LSD tests). In this experiment survival was higher in dark conditions for most diatom densities. At the highest density (10,000 cells/[mm.sup.2]), final survival averages were 66% and 13% for the dark and light treatments, respectively, and this difference was significant (F = 24.99, P = 0.007).
[FIGURE 2 OMITTED]
Shell Length and Growth Rates
In both experiments, initial postlarval shell lengths were not significantly different among treatments. However, postlarval lengths and growth rates were significantly higher in darkness conditions at the end of the experimental periods.
In Experiment 1, final average shell lengths between 1,250 and 1,290 [micro]m were obtained at the four higher diatom densities under darkness, corresponding to growth rates of 37-40 [micro]m/d. In the light treatment the maximum length attained was 792 [micro]m at 4,000 cells/[mm.sup.2] (15 [micro]m/d) (Fig. 3, Fig. 4). This effect of light condition was already significant since the first evaluation (0-9 days, postlarval ages: 14-23 days) (Table 2). Diatom density did not have a significant impact on postlarval growth during the first 9 days but significant treatment and interaction effects were detected thereafter (Fig. 4, Table 2). At the end of the 20-day experimental period, diatom effects were significant only in the dark (F = 23.60, P < 0.001), showing an asymptotic behavior of growth after 2,000 cells/[mm.sup.2], whereas the slow growth (12-16 [micro]m/d) in the light treatment was unaffected by diatom density (F = 2.14, P = 0.130) (Fig. 4).
[FIGURES 3-4 OMITTED]
In Experiment 2, the effect of light condition on postlarval growth was significant at the first evaluation and thereafter (Fig. 5, Table 3). A maximum final length of 1,084 [micro]m (corresponding to a growth rate of 24 [micro]m/d) was obtained in darkness at the highest diatom density (10,000 cells/[mm.sup.2]) and shell length decreased as diatom density decreased. In the light treatment a maximum final length of 546 [micro]m was observed at 8,000 cells/[mm.sup.2] (7 [micro]m/d) (Fig. 5, Fig. 6). Diatom density and interaction effects on growth rate were also significant from the first evaluation onwards (Table 3). At the end of the 32-day experimental period diatom effects were significant in both light conditions (F = 11.96, P < 0.001 and F = 6.16, P = 0.005 for darkness and light, respectively), following a similar pattern than in Experiment 1 with a marked suppression of growth below 2,000 cells/[mm.sup.2] in the dark and little effect of diatom density in the light treatment (Fig. 6).
[FIGURE 6 OMITTED]
Effects of Light or Dark Condition
Although a significant effect of light condition on postlarval survival of H. rufescens was not detected, the survival pattern in Experiment 2 (Fig. 2) suggests adverse effects of light at high diatom densities. Survival also decreased slightly at the highest diatom density in light conditions in Experiment 1 (Fig. 1) but this effect was not as sharp as in Experiment 2 probably because post-larvae were older and presumably more resistant to environmental stress. A similar pattern was observed in an experiment with H. fulgens where postlarval survival after 1 mo was 90% at 6 [micro]E/[m.sup.2]/s and only 3% at 47 [micro]E/[m.sup.2]/s) (Searcy-Bernal et al. 2003). This might be related to oxygen supersaturation conditions in the boundary layer (postlarval microhabitat) at high diatom densities when light is available for photosynthesis.
Oxygen conditions in the boundary layers over diatom films can change dramatically according to light conditions. Searcy-Bernal (1996) detected 140% and 50% oxygen saturation over Nitzschia sp. films at 59 and 4 [micro]E/[m.sup.2]/s, respectively, and Roberts et al. (2000) report oxygen saturation values of 400% and 60% over Achnanthes longipes films under light and dark conditions. Although preliminary evidence suggests the abalone postlarvae can tolerate high oxygen concentrations (Loipersberger 1996), the concentrations reached at the highest diatom density in Experiment 2 under light conditions, might have been above the tolerance limits of early H. rufescens postlarvae.
On the other hand, the high survival and growth of postlarvae at high diatom densities under darkness, suggest that they can tolerate very low oxygen concentration (expected under such conditions because of oxygen consumption by diatoms) without adverse effects.
The effect of light conditions on postlarval growth was dramatic in both experiments. In Experiment l, the average growth rate (over all diatom densities) in darkness was 2.4 higher than in light conditions (34.7 and 14.4 [micro]m/d, respectively). In Experiment 2, average growth in darkness was 3.0 times higher than under constant illumination (14.4 and 4.8 [micro]m/d, respectively). Searcy-Bernal et al. (2003) also report a positive effect of low irradiances, because postlarval growth of H. fulgens postlarvae was higher at 6 [micro]E/[m.sup.2]/s than at 47 [micro]E/[m.sup.2]/s (37 and 21 [micro]m/d, respectively). This might be explained by differences in postlarval feeding behavior and/or ecological conditions of biofilms.
Adults and juvenile abalones have nocturnal feeding habits. Commonly during daylight hours abalones remain relatively quiet and tend to aggregate in darkened locations. Grazing activity begins about half hour before sunset and continues throughout the night to satiation (Leighton 2000). Several studies have shown an increase in feeding and growth rates when abalones are cultured in darkness (Ebert & Houk 1984, Godoy-Corrales 1989, Kim et al. 1997).
This pattern has not been documented for postlarvae, which are generally assumed to feed all day because they do not seem to have the same negative phototactic behavior as juveniles and adults. However, this may not be true and nocturnal feeding habits might be established during the postlarval stage. Velez-Espino (1999) evaluated grazing rates of H. fulgens postlarvae during a 24-h cycle and found increased feeding about an hour before sunset in 15- and 30-day-old postlarvae, which is consistent with the feeding behavior of larger abalones. In the experiments reported here, a higher fecal production was qualitatively observed in the darkness treatments during the cleaning procedures, which is consistent with the hypothesis of increased feeding under dark conditions, which would in turn increase postlarval growth.
Light conditions might have an impact on ecologic conditions or nutritional value of the biofilm affecting postlarval growth. An important nutritional source for abalone postlarvae is provided by the mucus produced by diatoms and bacteria in the biofilm (Kawamura & Takami 1995, Kawamura et al. 1998a). This mucus is composed of extracellular polymeric substances (EPS) (Decho 1990), which are produced at a higher rate in darkness than under light conditions by the benthic diatom Navicula perminuta (Smith & Underwood 2000). This suggests that an increased nutritional value of diatom films under darkness might help to explain the higher growth rates of abalone postlarvae. On the other hand, ecologic conditions in the boundary layer in the light treatment might have limited postlarval growth (Searcy-Bernal 1996).
Effects of Diatom Density
Diatoms should be available in enough quantities to produce good growth of abalone postlarvae, but very ,high diatom densities may have a negative impact on postlarval growth because of extreme oxygen concentrations and/or other ecologically adverse conditions (Ebert & Houk 1984, Searcy-Bernal 1996). Therefore, higher survival would be expected at intermediate diatom densities as observed in Experiment 2 under illumination (Fig. 2). However, high diatom densities under darkness did not have a negative impact on postlarval survival as discussed earlier (Fig. 2). High sur viral (>60%) has been reported for postlarvae of H. discus hannai and H. iris fed different diatom species at densities between 1,000 and 4,000 cells/[mm.sup.2] (Kawamura & Takami 1995, Kawamura et al. 1998a).
Although the effect of diatom density on initial postlarval growth was not significant in Experiment 1 (Fig. 4, Table 2) where the initial postlarval age was 14 days; a significant effect was found in Experiment 2 (Fig. 6, Table 3), where the initial postlarval age was 2 days. Growth of these newly settled postlarvae increased most markedly as diatom density increased up to 1,000 cells/[mm.sup.2], and this effect was stronger under darkness. Probably this reflects the benefit of increased diatom mucus and associated bacteria in the biofilms as diatom density increases, since early postlarval growth does not depend on the direct digestion of diatom cells (Kawamura et al. 1998b).
The growth of older postlarvae responded to higher diatom densities following similar patterns in both trials. The second growth period evaluated in Experiment 1 (Fig. 4) started with 23-day-old postlarvae and is comparable to the third evaluation period (Fig. 6) in Experiment 2 that started with 20-day-old postlarvae. In both cases growth rates in darkness increased sharply as diatom density increased up to 2,000 cells/[mm.sup.2] and leveled off at higher densities. This suggests a possible optimum diatom density to feed H. rufescens postlarvae of these ages under darkness.
Under continuous illumination very little effect of diatom density on postlarval growth was observed in both experiments. This contrasts with the results of Searcy-Bernal et al. (2001) who showed that 15- and 30-day-old postlarvae of H. fulgens kept at between 17[degrees]C to 19[degrees]C and -50 [micro]E/[m.sup.2]/s, increase their growth as N. incerta density increases up to 2,000 cells/[mm.sup.2]. This difference might be due to several factors including experimental conditions, postlarval, or interspecific differences.
Survival and postlarval growth in Experiment 2 were lower than in Experiment 1, probably because of differences between larval batches or experimental conditions. Variability in abalone growth is common even within the same batch (Hahn 1989) and differences in postlarval growth of H. rufescens between batches similar to those observed here have been reported in other studies (Martinez-Ponce & Searcy-Bernal 1998). On the other hand Experiment 1 started with older postlarvae cultured under optimal conditions before the trial was conducted.
The main conclusion of this work is that the growth of abalone postlarvae might be dramatically improved if they are cultured in darkness. Large-scale trials under hatchery conditions are required to determine if this result can be extrapolated to production. The potential implications of these findings to postlarval culture would depend on a reliable source of cultured diatoms to reinoculate tanks as required, which is not currently a technical problem (Roberts et al. 2000).
TABLE 1. Differences between experiments. Both were conducted at 17 [+ or -] 1[degrees]C in sterile six-well tissue culture plates (10-15 postlarvae per well). Seawater was changed daily and reinoculations of diatoms at the experimental densities were performed every 4 days. Procedure Experiment 1 (March 2002) Settlement (GABA, 1.5 [micro]M) In 51 containers Seawater quality 1 [micro]m-filtered, UV-irradiated Irradiance at continuous light treatment 24-33 [micro]E/[m.sup.2]/s Initial age of postlarvae (after metamorphosis induction) 14 d Low range of diatom densities (250-3,000 cells/[mm.sup.2]) At postlarval ages: 14-21 d High range of diatom densities (500-10,000 cells/[mm.sup.2]) At postlarval ages: 21-34 d Procedure Experiment 2(May 2002) Settlement (GABA, 1.5 [micro]M) In wells Seawater quality 1 [micro]m-filtered, UV-irradiated, sterilized Irradiance at continuous light treatment 19-21 [micro]E/[m.sup.2]/s Initial age of postlarvae (after metamorphosis induction) 2 d Low range of diatom densities (250-3,000 cells/[mm.sup.2]) At postlarval ages: 2-18 d High range of diatom densities (500-10,000 cells/[mm.sup.2]) At postlarval ages: 18-34 d TABLE 2. Results of split-plot ANOVAS for survival and growth rate of H. rufescens postlarvae in Experiment 1. Light Condition (df = 1, 4) Days in Postlarval Treatment Age (d) F P Survival 9 23 0.025 0.382 20 34 3.361 0.141 Growth rate 0-9 14-23 614.57 2 x [10.sup.-5] 9-20 23-34 131.03 3 x [10.sup.-4] 0-20 14-34 278.08 8 x [10.sup.-5] Diatom Density (df = 5, 20) Days in Treatment F P Survival 9 1.272 0.314 20 0.960 0.465 Growth rate 0-9 0.82 0.547 9-20 51.38 9 x [10.sup.-11] 0-20 20.78 3 x [10.sup.-7] Interaction (df = 5, 20) Days in Treatment F P Survival 9 4.307 0.008 20 1.039 0.422 Growth rate 0-9 2.03 0.118 9-20 46.71 2 x [10.sup.-10] 0-20 17.23 1 x [10.sup.-6] TABLE 3. Results of split-plot ANOVAS for survival and growth rate of H. rufescens postlarvae in Experiment 2. Light Condition (df = 1, 4) Days in Postlarval Treatment Age (d) F P Survival 10 12 0.900 0.396 18 20 3.029 0.157 32 34 3.833 0.122 Growth rate 0-10 2-12 25.37 0.007 10-18 12-20 26.46 0.007 18-32 20-34 29.24 0.006 0-32 2-34 91.66 6 x [10.sup.-4] Diatom Density (df = 5, 20) Days in Treatment F P Survival 10 2.552 0.061 18 1.957 0.129 Growth rate 0-10 5.97 0.001 10-18 21.39 2 x [10.sup.-7] 18-32 7.34 4 x [10.sup.-4] 0-32 11.79 2 x [10.sup.-5] Interaction (df = 5, 20) Days in Treatment F P Survival 10 1.401 0.266 18 2.568 0.060 32 4.55 0.006 Growth rate 0-10 4.10 0.01 10-18 8.80 1 x [10.sup.-4] 18-32 2.61 0.056 0-32 4.53 0.006
The authors thank the commercial farm Abulones Cultivados (Erendira, B.C., Mexico) for the donation of abalone larvae, Enrique Valenzuela (I.I.O.) for providing the monocultures of N. incerta, Casandra Anguiano-Beltran for her technical support, Carmen Paniagua-Chavez, and two anonymous reviewers for their critical comments on the manuscript. This study was partially funded by the University of Baja California (grants 4040 and 4403) and the Mexican Government (CONACYT grant 37461-B and SNI grant 5532). This paper is part of the doctoral dissertation of E. Gorrostieta-Hurtado supported by a CONACyT scholarship.
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ERENDIRA GORROSTIETA-HURTADO (1) AND RICARDO SEARCY-BERNAL (2) *
(1) Centro de Investigacion Cientifica y de Educacion Superior de Ensenada, Km 107 Carretera Tijuana-Ensenada, Ensenada, B. C. Mexico, 22800; (2) Instituto de Investigaciones Oceanologicas, Universidad Autonoma de Baja California, Km 103 Carretera Tijuana-Ensenada, Apartado Postal 453. Ensenada, B. C. Mexico, 22860
* Corresponding author. E-mail: firstname.lastname@example.org
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|Publication:||Journal of Shellfish Research|
|Date:||Dec 15, 2004|
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