Growth and survival of juvenile red abalone (Haliotis rufescens) fed with macroalgae enriched with a benthic diatom film.

ABSTRACT The objective of this work was to evaluate the growth rate of 150-day-old juvenile abalone Haliotis rufescens fed with blades of the seaweed Macrocystis pyrifera epiphyted with a film of the benthic diatom Navicula incerta. Five treatments were used as food for juvenile abalone: (1) blades of M. pyrifera washed for 10 min with fresh water to remove natural epiphytes; (2) blades of M. pyrifera without natural epiphytes and colonized with N. incerta at an initial density of approximately 500 cells [mm-.sup.2]; (3) blades of M. pyrifera with natural epiphytes; (4) blades of M. pyrifera with natural epiphytes and colonized with N. incerta; and (5) batch cultures of N. incerta without blades. All treatments were kept on 18-L plastic buckets with 10 L of "f/2" medium at 16[degrees]C and 100 [micro]E[m.sup.2][s.sup.-1] of continuous light. All treatments were kept under culture conditions for 2 days. Plastic buckets of 18 L filled with 10 L of filtered seawater at 16[degrees]C was used for the bioassay with the red abalone. The five feeding treatments were provided ad libitum to juvenile abalone and were removed after 4 days. The water exchange rate in the buckets was ~300% per day. Every 15 days, and for a period of 90 days, weight and growth (as shell length and width) were measured. At the end of the experiment mortality was determined. The results showed that the highest growth rates and weight were observed in treatments 2, 4, and 5 whereas the lowest growth rates and weight were observed in treatments 1 and 3. The highest average survival was recorded in treatment 5 (52.17%), whereas the lowest was observed in treatment 2 (34.20%). This study showed that the enrichment of seaweed blades improves growth and survival in abalone and thus has potential to be used in abalone farms to enhance productivity.

KEY WORDS: benthic diatoms, epiphytes, seaweed, Haliotis rufescens, Navicula incerta, Macrocystis pyrifera

INTRODUCTION

Commercial abalone culture in Mexico and California has traditionally been based on the supply of benthic diatoms as food for abalone postlarvae. When juvenile abalone reach a size of approximately 10 mm, the type of nourishment changes from benthic diatoms to seaweed. The main seaweed species used as food for juvenile and adult abalone are Macrocystis pyrifera, Nereocystis luetkeana, and Egregia menziesii, these and other brown seaweeds of the North Pacific coasts are abundant and easy to harvest from coastal waters (Hahn 1989, Buchal et al. 1998, McBride 1998).

The biochemical composition of seaweed varies among species and is related to the geographical zone, season, currents and wave exposure, nutrient availability, depth, temperature, and life stage of the algae (Cruz-Suarez et al. 2000). This variability in nutritional value produces heterogeneous growth rates and, in some cases, low growth in abalone (Bautista-Teruel & Millamena 1999).

The biochemical composition of the seaweed Macrocystis pyrifera is poor, ranging from 5% to 12% proteins, 0.5% to 1% lipids, and 46% to 50% carbohydrates (Cruz-Suarez et al. 2000). Benthic diatoms have a much better biochemical composition, 22% to 36% proteins, 13% to 58% lipids, and 5% to 23% carbohydrates (Renaud et al. 1999, Simental-Trinidad et al. 2001).

When the food for juvenile abalone is changed from benthic diatoms to seaweed it is believed to reduce growth and survival, and may be a factor involved in high mortalities of the organisms under culture conditions. The biochemical composition of seaweed can be enhanced by the performance of a selective colonization over the surface of the blade with benthic diatoms. The aim of this work is to improve the growth rate and survival of juvenile red abalone (Haliotis rufescens) by enhancing the biochemical composition of its food (Macrocystis pyrifera) with a selective epyphitation of the seaweed blades with the benthic diatom Navicula incerta.

MATERIALS AND METHODS

Blades of the seaweed Mucrocystis pyrifera (excluding pneumatocysts) and cultures of the benthic diatom Navicula incerta were used in this work. The seaweed M. pyrifera was harvested every 4 days from a mantle located at Punta Morro (31[degrees]51'30"North, 116[degrees]38"38"West), on the coastal waters of Bahia Todos Santos, Baja California (BC), Mexico. Every 4 days, from August to November 2002, seaweed was harvested at a depth of 2 m from the apical blades. The benthic diatom N. incerta was isolated from the same location by the staff of Universidad Autonoma de Baja California, and was also obtained by donation from the abalone farm "Abulones Cultivados" Ejido Erendira, BC, Mexico.

This study used 150-day-old juveniles of red abalone Haliotis rufescens, which were obtained from the earlier mentioned abalone farm. The abalone were selected to provide a group with an average shell length of 3.67 mm [+ or -] 0.32 and weight of 0.0055 g [+ or -] 0.0015. Organisms were acclimated to the experimental conditions for a period of 1 wk and fed M. pyrifera ad libitum.

Five treatments were used to provide food to juvenile abalone: (1) blades of M. pyrifera without natural epiphytes; (2) blades of M. pyrifera without natural epiphytes and colonized with N. incerta; (3) blades of M. pyrifera with natural epiphytes; (4) blades of M. pyrifera with natural epiphytes and colonized with N. incerta; and (5) batch cultures of N. incerta without blades.

All treatments were performed by triplicate and kept under controlled light irradiance (100 [micro]E[m.sup.2][s.sup.-1]). The temperature for the experimental containers was maintained at 16[degrees]C by using an electronic controller.

The seawater used for all experiments was passed through sand and cartridge filters of 10 [micro]m and 5 [micro]m, and finally irradiated with ultraviolet light. Cultures of N. incerta were prepared for inoculum in "f/2" medium (Guillard & Ryther 1962) in progressive volumes of 10 mL, 150 mL, 900 mL, and 10 L. The strain was acclimated to the culture conditions of temperature and continuous light previously described. For the production of the 10-L inoculum used in the bioassay with juvenile abalone, circular 18-L white plastic containers (30 x 35 cm) with a lexon acrylic lid at the top were used (Simental-Trinidad et al. 2001). An initial density of 150 organisms per container was used in the bioassay. Seawater was exchanged at approximately 300% per day.

For treatment 1, blades of M. pyrifera were washed with freshwater for 10 min to remove all the natural epiphytes. After the bath, sets of two blades were placed in the plastic container with 10 L of "f/2" medium. For treatment 2, blades of seaweed were treated similarly to those in treatment 1, and were inoculated with a 4-day-old culture of N. scerta at an initial density of 50,000 cells [ml.sup.-1] (obtaining approximately 500 cells [mm.sup.-2] on the blade surface). For treatment 3, the seaweed blades were placed into the containers without any previous washing or inoculation. For treatment 4, the blades of M. pyrifera without previous washing were inoculated with N. Incerta, similarly to the process described in treatment 2. Treatment 5 was a monospecific culture of N. incerta with the same cell density as the one used in treatments 2 and 4.

Two days after the setting of the 5 treatments, all were given as food to the abalone ad libitum and left for 4 days, after that time, excess food was removed and new treatments were added to the containers.

Every 15 days, and for a period of 90 days, weight and shell length were obtained by randomly sampling 15 organisms. Weight was measured with an electronic scale and shell length was measured by using a calibrated compound microscope. At the end of the experiment, survival of juvenile abalone was determined. Daily growth rates, in terms of weight (DGw) and shell length (DGsl), were evaluated as described by Capinpin & Corre (1996). Growth rate was calculated as follows:

DGw ([micro]g per day) = Gw/n;

and

DGsl ([micro]m per day) = Gsl/n;

where Gw is the increase in weight ([micro]g), Gsl is the increase in shell length ([micro]m) and n are the days of rearing.

To determine if there were significant differences in weight through time as a result of different treatments, a covariance statistical analysis was used. The same analysis was performed to determine differences in shell length, and shell width. To determine if there were significant differences in growth rates, a 1-way Anova was used. When differences were detected, a Tukey a posteriori test was performed. All the differences were evaluated at [alpha] = 0.05. For all statistical analysis, the software Statistica 5.0 for Windows was used.

RESULTS

Significant differences in weight among treatments were detected (F = 75.83, P < 0.00) (Fig. 1). The highest weights were detected in treatments 2 (0.100 g [+ or -] 0.005 SE) and 5 (0.080 g [+ or -] 0.004). The lowest abalone weights were obtained on treatments 1 (0.046 g [+ or -] 0.003 SE) and 3 (0.039 g [+ or -] 0.001 SE).

[FIGURE 1 OMITTED]

Shell length showed significant differences (F = 132.03, P < 0.00) (Fig. 2). The highest shell lengths were detected on treatments 2 (9.06 mm [+ or -] 0.09 SE) and 5 (8.81 mm [+ or -] 0.14 SE). The lowest shell lengths were detected on treatments 1 (6.76 mm [+ or -] 0.09 SE) and 3 (8.20 mm [+ or -] 014 SE).

[FIGURE 2 OMITTED]

Significant differences were obtained for shell width (F = 46.06, P < 0.00) (Fig. 3). A maximum value of shell width was recorded on treatment 2 (6.17 mm [+ or -] 0.010 SE). The lowest values were detected on treatments 1 (4.57 mm [+ or -] 0.07 SE) and 5 (5.66 mm [+ or -] 0.08 SE).

[FIGURE 3 OMITTED]

The average daily growth rate for the 5 treatments in terms of weight and shell length of abalone for 0-30 days, 30-60 days, and 60-90 days are shown in Table 1. DGw showed significant differences (F = 23.4, P < 0.00) on days 30-60 and 60-90. Abalone fed on treatments 2, 4, and 5 produced the highest growth rates in terms of weight. Regarding growth rate expressed as DGsl, a decrease was observed from day 30 until the end of the experiment. There were significant differences only during the 30-60 day period, when abalone were fed on treatments 2, 4, and 5 and where the highest growth rates in terms of shell length were recorded.

Minimum and maximum survival is presented in Table 2. There were significant differences between repetitions, with a minimum survival value recorded on treatment 2 (9.56%) and a maximum survival value on treatment 4 (82.60%).

DISCUSSION

In natural populations, abalone are nonselective feeders (Leighton & Boolootian 1963). In Mexican farms, where commercial abalone production is performed, food supply is based on seaweed instead of formulated diets. The seaweed is used mainly because of its availability, easy harvesting methods, and low cost. The main species used are Macrocystis pyrifera and Gracilaria spp. (Carbajal-Miranda pers. com. 2003). Juvenile begin to eat macroalgae at about 10 mm in length and will eat from 10% to 30% of their whole-body wet weight in algae each day. The high feeding rate of macroalgae is due to the high water content and relative low protein content of fresh macroalgae (Hahn 1989).

Usually, the protein content of benthic diatoms, such as Navicula sp. under culture conditions, varies according to the age and culture conditions from 20% to 30%; whereas carbohydrate content ranges from 10% to 15%, and lipid content ranges between 15% to 20% (Flores-Vergara 1998, Correa-Reyes et al. 2001, Carbajal-Miranda 2002, Simental-Trinidad et al. 2001, Simental & Sanchez-Saavedra 2003).

In Mexican abalone farms, the change in nourishment, from microalgae to seaweed, is initiated when juvenile abalone are 3 mo old (Vazquez-Moreno pers. com. 2002) or when they are close to 10 mm in length. The daily seaweed portion that juvenile abalone receive ranges from 10% to 30% of their whole-body wet weight (Hahn 1989). According to Kawamura et al. (2001), the structure of the radula and digestive abilities for the use of seaweed as food is ready at 2-4 mm shell length in H. discus hannai, thus, the juvenile H. rufescens used in this experiment (3.67 [+ or -] 0.32 mm) were fully able to feed on seaweed.

In an experiment performed with deep-sea water and a continuous and simultaneous culture of the benthic diatom Nitzschia sp., used as food for 7-mo-old juvenile abalone Haliotis sieboldi (12.4 mm [+ or -] 0.2 Sd.), daily growth rate of the organisms was 50 to 110 [micro]m [day.sup.-1. Such results show that continuous cultivation of Navicula sp. with deep seawater promotes growth of juvenile abalone, without the need for providing supplementary seaweed or artificial diets (Fukami et al. 1997). The best growth rate in length and weight of H. rufescens was obtained from treatments with selective epyphitation or enriched with the benthic diatom Navicula incerta. Juvenile abalone retains the ability to digest diatoms after developing the ability to digest macroalgae. A combined diet of benthic diatoms and macroalgae provides greater nourishment and faster growth rate.

A common practice on abalone farms is the washing of the seaweed blades to avoid the introduction of other algae, microorganisms, and bacteria. However, research has shown that the epiphytes can contribute to an increase in the chemical composition of seaweed (Simental-Trinidad 2003). When abalone were fed with blades of Laminaria religiosa with bryozoa (Membranipora membranacea) on the surface, they grew faster than those fed with the same species without bryozoa. These are evidences that bryozoa probably supply complementary nourishment to the abalone (Uki 1981). Research suggests that abalone also can use bacteria, yeast, and other micro-organisms associated with diatoms as food (McBride & Conte 2001).

Benthic diatoms are the main diet for small abalone (Voltolina 1985, 1994, Daume et al. 1997, Daume et al. 1999, 2000, Kawamura 1996, Kawamura et al. 1998a, Kawamura et al. 1998b; Roberts 2000; Searcy-Bernal et al. 2000). Some diatoms in feces were observed to have cytoplasm and nuclear material and were therefore, assumed not to have been completely digested. In the gut content of Haliotis asinina collected from the natural environment, it was found that the main nourishment appears to be composed of benthic diatoms. Dominant genera in gut content were Nitzschia, Amphora, Navicula, and Cocconeis, which were the predominant floral elements in the local environment (Sawatpeera et al. 1998). Relatively few diatom strains are ruptured when eaten by abalone postlarvae. Postlarvae <1 mm in shell length can grow using extracellular foods such as mucus from adult abalone. Postlarvae > 1mm require the cell content of diatoms for rapid growth. The natural succession of diatom communities can be controlled, to some extent, by light and grazing pressure, thus, favoring useful diatoms (Roberts et al. 2000).

The nutritional value of monospecific cultures of eight benthic diatom species was determined for abalone postlarvae of Haliotis rufescens. The best growth rate, measured as shell length, was obtained when organisms were fed with Amphiprora paludosa var. hyalina, Nitzschia thermalis var. minor, and Navicula incerta (Correa-Reyes et al. 2001, Correa-Reyes 2002).

The growth rate obtained in this work varied from 9.15-63.32 [micro]m [day.sup.-1]. However, Kawamura et al. (1995) determined growth rates from 13.6-50.1 [micro]m [day.sup.1] in H. discus hannai fed with several benthic diatoms. Meanwhile, Neori et al. (2000) reported growth rate values of 66.6 [micro]m [day.sup.-1] in H. discus hannai fed with Ulva lactuca or Gracilaria conferta. In this experiment we obtained better growth rates when abalone were fed with blades of seaweed containing benthic diatoms. Another important consideration is that both species (M. pyrifera and N. incerta) are commonly used on abalone farms in Mexico.

The use of seaweed as a food source on abalone farms has some other advantages due to the fact that they can also act as biologic filters and can be used as a coculture of abalone and seaweed (Evans & Langdon 2000). Thus, establishing the possibility for the use of systems with low water exchange or closed systems for the culture of abalone without the problem of loosing stability of formulated diets, which in some cases loose as much as 24% of dry matter in their pellets (Guzman & Viana 1998). Consequently, decreasing problems of waste removal and aeration, which are the main limiting factors, considered by many farmers, when using artificial diets (Fleming et al. 1996). Eutrophication by nitrogen can reduce the diversity of communities, and produce low water quality (Hillebrand & Sommer 2000). With seaweeds such as Gracilaria used as biofilters, 90% of the nitrogen present in the water can be removed (Ryther et al. 1982).

CONCLUSION

A significant increase in weight and shell length for juvenile abalone was determined over time when the organisms were fed with the algae Macrocystis pyrifera, selective epiphytes with cultures of the benthic diatom Navicula incerta, and with the monocultures of N. incerta. Growth rate and survival of juvenile abalone (Haliotis rufescens) can be increased by using the seaweed Macrocystis pyrifera and selective epiphytes with cultures of the benthic diatom Navicula incerta as feed.

TABLE 1.

Average daily growth rates in terms of weight (DGw) and shell length
(DGsl) of red abalone Haliotis rufescens fed with different
treatments * using the seaweed Macrocystis pyrifera and the benthic
diatom Navicula incerta.

                     DGw ([micro]g [day.sup.-1])

Treatment *         Day 0-30             Day 30-60

     1          323.53 a (7.41)      189.07 ab (53.12)
     2         338.8942 a (67.66)    503.39 b (90.10)
     3          206.50 a (57.97)      142.64 a (8.28)
     4          298.97 a (66.07)     249.12 ab (65.38)
     5          645.91 a (76.93)      208.06 a (51.1)

                 DGw ([micro]g        DGsI ([micro]g
                 [day.sup.-1])         [day.sup.-1])

Treatment *        Day 60-90             Day 0-30

     1          234.81 a (95.48)      38.47 a (5.88)
     2         824.53 b (203.84)      52.24 a (9.27)
     3          208.43 a (87.05)      33.36 a (9.08)
     4         577.34 ab (88.16)      35.68 a (1.78)
     5         579.85 ab (194.62)     63.32 a (5.66)

                    DGsI ([micro]g [day.sup.-1])

Treatment *        Day 30-60             Day 60-90

     1          19.17 ab (2.13)        9.15 a (4.10)
     2           29.94 b (5.06)       25.01 a (3.90)
     3           11.92 a (3.20)       12.09 a (6.01)
     4          24.33 ab (2.95)       24.39 a (2.88)
     5          15.29 ab (4.58)       27.14 a (7.19)

* Treatments were: (1) Macrocystis pyrifera without natural epiphytes;
(2) M. pyrifera without natural epiphytes and with a layer of Navicula
incerta; (3) M. pyrifera with natural epiphytes; (4) M. pyrifera with
natural epiphytes and with a layer of N. incerta; (5) Culture of N.
incerta without blades. The standard error is included in parenthesis.

The different letters on the side of the quantities indicate
significant differences (1-way Anova and Tukey a posteriori test.
([alpha] = 0.05): a < b.

TABLE 2.

Average survival and minimum and maximum values due to
replicates (expressed in percentages) of juvenile red abalone Haliotis
rufescens, after twelve weeks of feeding with different treatments *
using the seaweed Macrocystis pyrifera and the benthic diatom Navicula
incerta.

                               Survival
Treatment *    Average (%)    Maximum (%)    Minimum (%)

     1            48.40          57.39          38.26
     2            34.20          67.82           9.56
     3            46.95          55.65          40.00
     4            52.17          82.60          30.43
     5            50.86          53.04          48.69

* Treatments were: (1) M. pyrifera without natural epiphytes: (2) M.
pyrifera without natural epiphytes and with a layer of N. incerta; (3)
M. pyrifera with natural epiphytes: (4) M. pyrifera with natural
epiphytes and with a layer of N. incerta; (5) Culture of N. incerta
without blades.

ACKNOWLEDGMENTS

The authors thank M. Carbajal-Miranda, M. Pacheco-Vega, C. Cruz-Fraga and S. Fierro-Resendiz for technical assistance, and L. Salinas-Flores and M. Segovia-Quintero for manuscript revision. The authors also thank Consejo Nacional de Ciencia y Tecnologia (CONAC y T) for the Ph. D scholarship to the first author. This work was supported by economic grants of Centro de Investigaci6n y de Educacion Superior de Ensenada (CICESE, Project 6554) and Consejo Nacional de Ciencia y Tecnologia (CONAC y T, Project 33016B).

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JORGE ARTURO SIMENTAL, MARIA DEL PILAR SANCHEZ-SAAVEDRA, * AND NORBERTO FLORES-ACEVEDO

Laboratorio de Biologia y Cultivo de Microalgas. Departamento de Acuicultura, Centro de lnvestigacion Cientifica y de Educacion Superior de Ensenada. Apdo. Postal 2732, C.P. 22860 Ensenada, Baja California, Mexico.

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