Stress tests have previously been utilised as an assessment of general health or vigour in several aquatic species (Dhert et al., 1990a; Dhert et al., 1990b; Briggs, 1992; Boarder and Maguire, 1998; Samocha et al., 1998). Abalone are osmoconformers and possess only limited osmoregulatory capabilities, thus the effects of salinity fluctuations directly effect internal ionic composition (Brix, 1983; Somero and Bowlus, 1983). It has been shown previously that dietary history can directly effect resistance to low salinity in abalone (Boarder and Maguire, 1998).

This trial was conducted to assess the suitability of enriched Ulva rigida as a feed for Haliotis roei and to compare growth rates of this diet with growth rates on various artificial diets.

Materials and Methods

H. roei were collected from limestone reef platforms in the Perth Metropolitan area in March 1999. Abalone were gently removed from the reef-top and were immediately placed into aerated seawater for transport. All animals were tagged (Hallprint Pty Ltd; FPN shellfish tags) and then randomly distributed to 28 trial tanks after being weighed to the nearest 0.01 g (whole wet body weight; WWBW) and measured with manual calipers (0.1mm accuracy). The initial mean length and weight of the abalone were 31.9 ± 4.3 mm and 6.17 ± 2.28 g (mean ± SE) (n = 560).

Trial System

Diets used in this trial are listed in Table 1. All artificial diets were commercially available (with the exception of the FRDC2 control diet) and enriched Ulva was grown in high nutrient water (5 g N m^-2 day^-1; 0.6 g P m^-2 day^-1) on inorganic nutrients (NH₄Cl and Na₂HPO₄) in accordance with Shpigel et al., (1999). The abalone were fed 3% b.w./day, which is in excess of requirements for greenlip abalone, H. laevigata (Tom Coote, 1999 pers. comm.). Tanks were cleaned by siphon every second day. Feed rates for Ulva were calculated on a dry weight basis. This was based on enriched Ulva possessing a water content of 84%. The control diet, known as the FRDC2 formulation, was made available by the Fisheries Research and Development Council (FRDC). The dry ingredients included fishmeal, soyflour, semolina, sodium alginate and CaCO₃. The Deakin diet (Diet 6), and Adam and Amos (Diet 2) diet were both commercially available within Australia. Haliogro (Diet 5) was from New Zealand and AB-feed (Diet 4) from South Africa. It must be noted that none of the diets used in this trial were specifically formulated for H. roei.

Analysis of U. rigida

Proximate analysis of U. rigida was undertaken throughout the trial to quantify moisture, ash, protein (nitrogen x 6.25), fat and carbohydrate levels. Moisture content was determined by gravimetric weight loss on heating the samples to a constant weight at 105^oC (AOAC method number 950.46B). Ash was determined by gravimetric weight loss on ashing the samples in a muffle furnace at 550^oC (AOAC method number 920.153). Analysis of nitrogen content utilised the Kjeldahl method (AOAC method number 928.08) and fat/lipid levels were determined via acid hydrolysis and solvent extraction (AOAC method number 950.54).

Consumption analyses

Consumption was assessed twice during the trial by manually siphoning uneaten feed from tanks. Consumption was estimated by relating the dry weight of the uneaten food to the known dry weight of the feed provided. Consumption data were corrected for dry matter weight loss due to leaching by leaching all diets over a period of 48 hours and drying to a constant weight. Consumption estimates involved the collection of all uneaten food prior to cleaning on the second day of the feed/clean cycle, through manual siphon onto 100 µm mesh screen. Collected feed was carefully washed onto a circular filter mesh for drying and weighing. Feed was dried at 55-60^oC for a period of two days prior to weighing. This procedure was conducted on two occasions over the duration of the trial.

Salinity Stress Test

As measured by either whole weight (P<0.05) or by shell length (P<0.01), specific growth rate (SGR) was significantly affected by diet. Growth rates for H. roei fed inorganically enriched U. rigida (D3) were not significantly different (Tukey test, P>0.05) to growth achieved from the best performing artificial diets. The abalone grew best on the Adam and Amos (D2) diet for both length and weight (SGR-L = 0.161 ± 0.004; SGR-W = 0.495 ± 0.2). However, growth was not significantly higher than on the FRDC2 (SGR-L = 0.139 ± 0.006; SGR-W = 0.371 ± 0.035), Ulva (SGR-L = 0.142 ± 0.010; SGR-W = 0.379 ± 0.040) or AB-feed (SGR-L = 0.131 ± 0.006; SGR-W = 0.4113 ± 0.014) diets. This is shown in Figures 1 and 2, respectively.

Consumption analyses

Consumption rate was significantly affected (P<0.01) by diet for both consumption assessments. The two consumption analyses were also significantly different from each other (p<0.01), as shown in Figure 3. Diets 2, 5, 6 and 7 were all observed to be less water stable than Diets 1, 3 and 4 after two days within the treatment tanks.

Salinity Stress Test

Abalone held in full strength seawater and 25‰ salinity exhibited 100% survival at the conclusion of the 96 hour trial, with the exception of abalone fed on Diet 3 which exhibited 92% survival over the same period in 25‰. Detachment rates for both salinities were negligible for all diets at the higher salinities.

Diet significantly affected survival at 20‰ (P<0.05). No abalone fed Diet 3 (enriched Ulva) survived in 20‰ after 96-h (Figure 4). Survival was significantly the same for all other diets (Tukey test; P>0.05), with an average survival of 40.28 ± 4.33%. Diet 3 was not included in the ANOVA due to zero variance caused by 0% survival. Mortality on Diet 3 was also observed earlier than all other diets (Table 2). Fifty percent of all animals fed enriched Ulva died after 68 hours, compared with an average of 9.73 ± 2.06% for abalone fed other diets over the same time period. Most other diets reached 50% mortality after approximately 85 hours, almost 20 hours later than the Ulva fed abalone (Table 2). Abalone fed Diet 3 were also observed to detach earlier than other diets.

Discussion

Growth rates of H. roei fed enriched U. rigida were not significantly different from growth rates achieved on the three best performing artificial diets (p>0.05). Previous growth trials with Ulva sp. as a feed for abalone species have utilised "wild" Ulva or Ulva that has not been enriched (Mai et al., 1994; Mai et al., 1996; Simpson and Cook, 1998). These growth trials have shown the effectiveness of Ulva as a feed to be totally dependent on the abalone species to which it is being fed. Mai et al. (1996) found that, although wild U. lactuca was a "moderately good diet" for H. tuberculata, it was the worst of five diets for H. discus hannai. Corazani and Illanes (1998) showed that H. discus hannai grew significantly faster on Ulva rigida than on other macroalgal diets and the authors recommend juvenile abalone of this species be fed a diet of Ulva sp. in combination with an artificial diet. They felt that the artificial component of the diet was a supplement to the Ulva sp. and should be fed at levels no higher than 1% of total body weight day^-1.

Simpson and Cook (1998) investigated the growth of H. midae grown on various natural algal diets. They found that after 4 months on trial diets, abalone fed on a diet of wild Ulva spp. were significantly shorter in shell length than abalone fed on most other macroalgal diets. Ulva fed animals were also found to have the lowest wet weight to shell length ratios. However, when the animals were fed Ulva as a proportion of a mixed diet they exhibited excellent growth rates suggesting that Ulva provided essential nutrients not found in the other algae fed.

The increase in protein content of U. rigida observed within this trial is supported by previous research in which Ulva spp. have been utilised as macroalgal biofilters (Tenore, 1976; Neori, 1996; Shpigel et al., 1996a; Shpigel et al., 1996b). In the current trial, wild U. rigida was found to have a protein content of 13% (% dry weight), compared with 32.2±1.5% (% dry weight) for Ulva which had been enriched on inorganic nutrients. The high protein content of the Ulva throughout the trial could explain the differences observed between the growth achieved on wild Ulva spp. in other research and the growth achieved within this trial. Fleming (1995) found that the intake of digestible nitrogen directly influences the growth rates of H. rubra. This suggests that nitrogen may be a limiting factor for growth in Haliotis spp. Britz and Hecht (1997) support this view by stating that maximum growth can only be achieved when sufficient protein, in the correct proportions of amino acids, is supplied in the feed. Shpigel et al. (1996a; 1999) state that the good growth of H. tuberculata and H. fulgens when fed enriched U. lactuca was due to consistent supply of high protein diet.

The consumption results in this study do not adequately reflect the trends observed within the tanks. Some diets were observed to be more water stable than others and thus were easier to fragment upon siphoning for collection. Diets 1 and 4 (FRDC2 and AB-feed) were both extremely water stable within the tank and thus held together well through the siphoning collection process. Diets 2, 5 and 6 (Adam & Amos, Haliogro and Deakin) were less water stable, but were still firm after two days. These diets fragmented when they were siphoned, as did Diet 7, which was very soft after 2 days within the tanks. The data was potentially biased against diets 1, 3 and 4 because of the fragmentation of other diets causing some loss of material during analysis. This would have reduced the amount of feed actually collected thus increasing the apparent consumption. Differences in the various diets water stability may be due to the diets being formulated for colder water species.

Stress determination for aquatic animals usually involves a quantitative evaluation of a variable that is directly affected by the stressor. The application of an environmental stress allows an assessment of general health or vigour for animals from different treatments. Salinity stress tests have been used previously to determine the quality of prawn post-larvae (Briggs, 1992; Samocha et al., 1998), fish larvae (Dhert et al., 1990a; Dhert et al., 1990b) and the effects of diet on abalone robustness (Boarder and Maguire, 1998). Boarder and Maguire (1998) found that dietary vitamin levels directly determined the survival of H. laevigata at 23‰ salinity. Animals fed on twice the normal dietary inclusion level of vitamin mix exhibited over 90% survival after 96 hours, compared with approximately 50% survival for animals fed the normal dietary vitamin level.

The poor survival at 20‰ for abalone fed U. rigida within this trial may be due to the osmoregulatory mechanisms utilised by Haliotis spp. rather than a general lack of health. Abalone species are known ionic conformers and thus blood osmolality and ionic concentration closely resembles that of the external environment (Somero and Bowlus, 1983). The ability to regulate cell volume is also an important ability in soft-bodied osmoconforming marine animals (Tarr, 1976; Burton, 1983) as this prevents cells rupturing upon exposure to low salinities. Cell volume regulation within some osmoconforming molluscs is controlled by organic solutes, such as amino acids (Burton, 1983; Mai et al., 1994). At low salinities, amino acids are excreted from the cell along with osmotically obligated water thus restoring cell volume (Pierce and Amende, 1981). Pierce and Amende (1981) state that there is a possibility that the physiological response of osmoconforming molluscs to low salinity may be directly related to the ability to maintain and control a large intracellular free amino acid pool.

The most important free amino acids for cell volume regulation (dependant on abalone species) are taurine, glycine, alanine and proline with an emphasis on taurine (Burton, 1983). Biosynthesis of the amino acid taurine from methionine is known to occur in gastropod molluscs (Mai et al., 1994). Mai et al. (1994) found that the levels of taurine in the viscera of H. discus hannai were significantly lower in abalone fed wild U. lactuca than abalone fed on all other diets. The authors also found that the viscera methionine levels within the abalone fed U. lactuca were extremely high, indicating some physiological inhibition in the biosynthesis of taurine from methionine. Toxic substances contained within U. lactuca, as detailed by Borowsky and Borowsky (1990), were thought to cause this inhibition. Johnson and Welsh (1985) found that an exudate excreted from U. lactuca killed 100% of estuarine crab larvae within 24 hours. The authors also report that the bactericidal and fungicidal properties of Ulva spp. exudate have been known for some time.

As taurine is one of the most important amino acids for cell volume regulation, inhibition of taurine production from methionine could significantly effect the cell volume regulation ability of abalone fed solely on Ulva spp. The comparatively good growth observed in H. roei fed U. rigida within this trial indicates that there was no feeding inhibition caused by anti-nutritional compounds or toxic substances contained within the seaweed. Fleming (1995) found that anti-nutritional compounds within some algal species did not prevent preferences for algae containing high levels of digestible nitrogen. This suggests that although the abalone fed on enriched U. rigida were capable of good growth, they were not capable of adequate cell volume regulation when exposed to hypo-osmotic conditions because of a lack of taurine within their free amino acid pool. It is important to note that all other health parameters (including growth) were excellent for abalone fed solely on enriched U. rigida.

The results of this trial indicate the salinity tolerance of H. roei to be between 25 and 20 ‰. This correlates closely with the limited literature on Haliotid salinity tolerances, which indicate short-term survival is possible at salinities around 20 ‰ (Singharaiwan et al., 1992; Jarayabhand and Phapavisit, 1996; pers. comm. T. McCormick, 1997; Boarder and Maguire, 1998). The 96-h LS50, the salinity at which 50% of the test animals survive after 96-h, is relatively close to 20 ‰ for H. roei. Most diets displayed 50% survival at just under 96-h within this trial. This compares with only 10% survival after 96-h for H. diversicolor supertexta juveniles transferred from 35 ‰ to 20 ‰ at similar temperature (Chen and Chen, 2000).

In conclusion, enriched U. rigida is a suitable feed for H. roei, producing comparable growth rates to several commercially available manufactured feeds. Diet directly effects survival of abalone under hypo-osmotic stress and U. rigida may impair the ability to cope with this stress by effecting cell volume regulatory mechanisms.

ACKNOWLEDGEMENTS

We thank the Natural Heritage Trust – Coasts and Clean Seas, South Metropolitan College of TAFE, Fisheries WA and the Aquaculture Development Fund for funding this project. We also thank Mr. Arron Strawbridge for technical assistance and Mr. Gavin Partridge for reviewing the manuscript.

References

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Note: feed manufacturers are listed in italics.

Figure 1. Effect of diet on specific growth rate for length (mm) for juvenile Haliotis roei (n=4).
Note: Diets sharing the same letters are not significantly different (Tukey test, P>0.05).

Figure 3. Effect of diet on consumption rate (grams dry feed consumed/gram tank biomass) for juvenile Haliotis roei at two separate sampling times (n=4).
Note: Diets sharing the same letters are not significantly different (Tukey test, P>0.05).

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