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The effect of dietary protein content on growth and biochemical composition of Chilean scallop Argopecten purpuratus (L.) postlarvae and spat
Aquaculture 180 Ž1999. 119–127 www.elsevier.nlrlocateraqua-online
The effect of dietary protein content on growth and biochemical composition of Chilean scallop Argopecten purpuratus žL. / postlarvae and spat Iker Uriarte ) , Ana Farıas ´ Facultad de Pesquerıas ´ y Oceanografıa, ´ UniÕersidad Austral de Chile, Casilla 1327, Puerto Montt, Chile Received 30 November 1998; received in revised form 31 March 1999; accepted 31 March 1999
Abstract This study shows the effect of three different levels of dietary protein content on the performance of postlarvae Ž1.8 mm. and spat Ž6.3 mm. of the Chilean scallop, Argopecten purpuratus. The postlarvae showed significant differences in growth and survival, with better growth when fed the high protein diet ŽN q .. The biochemical composition of the postlarvae showed only significant differences in the content of total carbohydrate, with the highest values in postlarvae fed the normal protein diet ŽN " . and the lowest values in starved postlarvae. There were no significant differences in growth nor survival of spat fed the test diets. The biochemical composition of the spat was significantly different in protein content, with the highest level in spat fed the N q diet, while the lipid content increased marginally significantly in spat fed the normal diet. The carbohydrate content in spat did not change. These results show that postlarvae and spat follow a different pattern of energy metabolism, because the diet N q only increased the growth in early postlarval stages and lost its effect in the later stages of development. This paper discusses the threshold age at which the metabolic pathway changes, coinciding with the change from the sessile life attached by the byssus to the typical unattached and free swimming life of A. purpuratus. It also highlights the efficiency and possible use in commercial hatcheries of high protein diets. q 1999 Elsevier Science B.V. All rights reserved. Keywords: Dietary protein; Bivalve nutrition; Scallop hatchery; Argopecten purpuratus
)
Corresponding author. Tel.: q56-65-277120; Fax: q56-65-255583; E-mail: [email protected]
0044-8486r99r$ - see front matter q 1999 Elsevier Science B.V. All rights reserved. PII: S 0 0 4 4 - 8 4 8 6 Ž 9 9 . 0 0 1 4 5 - 3
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I. Uriarte, A. Farıasr ´ Aquaculture 180 (1999) 119–127
1. Introduction The Chilean scallop, Argopecten purpuratus, is a subtidal bivalve that supports a considerable commercial aquaculture explaining more than 90% of total scallop production in Chile ŽServicio Nacional de Pesca, 1997.. Production of ‘seed’ scallops in hatcheries has proven to be a successful management tool for commercial grow-out as well as settlement in artificial collectors. Most modern hatcheries in the North of Chile produce algal feeds of known identity for rearing larval and young post-set scallops. Considering the commercial importance of A. purpuratus culture, there have been surprisingly few studies on nutrition for hatchery management. The typical works examining nutrition at hatcheries have compared the food value of microalgal species for larvae and postlarvae of bivalve ŽWebb and Chu, 1983; Enright et al., 1986; Whyte et al., 1990; O’Connor et al., 1992. where several algae were shown to support consistently rapid growth of scallops. The identification of the characteristics that differentiated the better algal diets from those that yielded poor growth has facilitated the development of nutrition in bivalves ŽEnright et al., 1986; Whyte et al., 1990; Wikfors et al., 1992.. The list of microalgal strains used in marine research institutions and commercial hatcheries is short, interest remains high for finding new and better algal feeds or algal supplements ŽCoutteau and Sorgeloos, 1992; Coutteau et al., 1994.. Live unialgal food culturing permits control over the nutritional quality of cells through various culture-management practices. These include among others: altering the nutrient composition by controlling the nutrient concentrations. Post-set oysters, Crassostrea Õirginica, Crassostrea gigas and Ostrea edulis, have been shown to grow more rapidly with a relatively high carbohydrate content when protein decreased under conditions of nitrogen deficiency in the unialgal food culturing ŽWikfors et al., 1984; Enright et al., 1986; Utting, 1986.. However, high protein diets, with relatively low carbohydrate content, have shown to promote rapid growth in post-set scallops A. purpuratus ŽUriarte and Farıas, ´ 1995., therefore information on nutritional responses of oysters cannot be applied directly to scallops. In the present study, experiments were conducted with a mixture of two different microalgae, a flagellate and a diatom, that were manipulated in their protein contents, and growth of post-set and juvenile scallops Žpostlarvae and spat, respectively. fed different algal qualities was compared. The relative importance of the major nutritional constituents—protein, carbohydrate and lipid—on the scallop tissue reserves were investigated.
2. Materials and methods The postlarvae of A. purpuratus were produced under laboratory conditions and measured with a binocular stereomicroscope with an accuracy of "10 mm. The survival was checked on a weekly basis and the sizes were taken at the beginning and at the end of the experiment. The instantaneous growth rate G 30 was calculated according to
I. Uriarte, A. Farıasr ´ Aquaculture 180 (1999) 119–127
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Spencer Ž1988.. Several sizes of postlarvae were sampled in order to determine the relationship between shell height and live weight of scallop. The diets of different protein content consisted of a mixture of two species of microalgae: Isochrysis aff galbana ŽT-Iso strain. and Chaetoceros neogracile ŽVan Landengham, 1986. containing high, normal or low content of protein ŽN q , N " and N y , respectively.. The biochemical strain of each species was grown using Walne medium adjusted to three separate conditions containing the following nitrogen concentrations in accord with Uriarte et al. Ž1993.: 0.45, 1.18 and 3.53 mg-at N ly1 of sodium nitrate for conditions low-, normal- and high-protein, respectively. In order to generate a significant low protein strain of T-Isochrysis, it was necessary to reduce the nitrogen concentration to 0.24 mg-at N ly1 ŽUriarte et al., unpublished data.. Each strain was cultured in 5 l flasks at a temperature of 258C and under 24 h illumination. A dispensing pump dosed the diets at an equivalent concentration of 2% of the live weight per day. The mixture of 1:1 ŽT-Iso:Chaetoceros. was calculated on the basis of number of cells. The diets were analysed three times during the experiments. The replicates in the first experiment held 500 mg postlarvae with a mean size of 1800 Ž"83. mm. Each replicate contained an up-welling silo with a mesh wire of 500 mm at the bottom, which was suspended in a closed system of 20 l tanks with filtered and UV-sterilized seawater. The treatments consisted of three mixed diets with different levels of protein and one control without feeding. The latter served as a reference to demonstrate possible signs of malnutrition in the animals receiving the diets. Each treatment contained six replicates and the experiment was run at 19 " 0.28C for four weeks. In the second experiment each replicate contained 900 mg of spat of 6.3 Ž"0.1. mm. The experimental set-up was the same as for experiment 1. The treatments consisted of three mixed diets of the different protein levels. There were four replicates per treatment. At the end of the experimental period, the scallops were fasted for 24 h for gut clearance before being analysed for gross biochemical composition following the methodology used by Farıas ´ et al. Ž1998..
Fig. 1. Allometric relationship between the live weight and the size in height of the scallops after the metamorphosis. The curve was fit by regression Žmultiplicative model: Y s a= X b ., the real points are shown, r indicates the correlation coefficient with the model.
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Table 1 Gross biochemical composition of the microalgal mixture: Nq, N" and Ny, composed by a mixture of I. aff. galbana ŽT-Iso. and C. neogracile conditioned at media with high, normal and low nitrogen concentrations, respectively Microalgal quality
Protein mg 10 6 cellsy1
Lipid mg 10 6 cellsy1
Carbohydrate mg 10 6 cellsy1
Organic matter Žorganic wt.rdry wt..
Nq N" Ny
7.84"0.39 b 5.64"0.41ab 4.48"0.78 a
4.12"0.38 4.77"0.20 4.68"0.52
4.31"0.31a 5.28"0.26 a 7.36"1.31b
0.76"0.03 a 0.74"0.05a 0.72"0.06 b
Means in the same column sharing the same superscript letter were not significantly different as determined by Tukey test Ž P ) 0.05.. Organic weight calculated as the sum of protein, lipid and carbohydrate. Average"standard error.
3. Results The relation between live weight and size of post-metamorphic scallops ŽFig. 1. allowed us, through extrapolation, to calculate the initial number of scallops and to evaluate the percentage of weight that was administered as food on a daily basis. The microalgal mixtures showed a dry weight ŽDW. of 21.4 Ž"1.4. mg per million of cells for diets N q and N " , and 22.9 Ž"9.9. mg per million of cells for diet N y . The protein content showed highly significant differences among treatments ŽTable 1, F s 14.35; df s 2, 65, P - 0.00001. with a following decreasing order: N q ) N " ) N y ŽTukey’s test, P s 0.05.. There were significant differences in the carbohydrate content among all diets Ž F s 61.32; df s 2, 61, P - 0.00001., showing an opposite pattern to protein content ŽTukey’s P s 0.05.: N q - N " - N y . The lipid remained approximately constant among diets. During the 30 days of the first experiment, the postlarvae grew 170 Ž"124., 1085 Ž"106., 1148 Ž"123. and 1660 Ž"140. mm in the control and with diet N y , diet N " and diet N q , respectively. The quality of the food had a significant effect on the final weight of the animals Ž F s 16.902; df s 3, 23; P - 0.00001. and on G 30 Ž F s 37.56; df s 3, 23; P - 0.00001.. The highest final weights and G 30 were obtained with diet N q , while the lowest values were obtained in the controls where the animals were submitted to starvation ŽTable 2, Tukey’s test P s 0.05.. Table 2 Growth and survival of postlarva in the first experiment of nutrition with diets of three protein qualities: Nq, N" and Ny, and a starved control Microalgal diet
Final live weight Žmg.
G 30 ln ŽWf yWi .r Ždaysr30.
Survival Ž%.
Nq N" Ny control
5.92 Ž"1.10. c 3.81 Ž"0.73. b 3.52 Ž"0.61. b 1.21 Ž"0.17. a
1.83 Ž"0.16. c 1.39 Ž"0.17. bc 1.34 Ž"0.17. b 0.31 Ž"0.20. a
71.9 Ž"9.8. c 57.9 Ž"14.2. bc 46.7 Ž"12.3. b 17.8 Ž"3.3. a
Means in the same column sharing the same superscript letter were not significantly different as determined by Tukey test Ž P ) 0.05.. Average Ž"standard error.. N s6.
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Table 3 Biochemical composition of postlarva in the first experiment of nutrition. Nq, N", Ny are the high, normal and low diet in protein, control is the fasted treatment Microalgal diet
Protein mg mgy1
Lipid mg mgy1
Carbohydrate mg mgy1
Nq N" Ny Control
50.97"2.36 b 47.24"4.33 b 60.71"4.65 c 7.47"0.35a
3.05"0.69 3.80"0.79 4.35"1.18 0.37"0.00
4.33"0.71b 6.88"0.97 bc 4.88"0.56 b 3.05"1.01a
Means in the same column sharing the same superscript letter were not significantly different as determined by Tukey test Ž P ) 0.05.. Average"standard error.
The survival varied significantly among treatments Ž F s 14.42; df s 3, 23; P 0.0001. with the controls having the greatest mortality in the postlarvae, followed by those fed diet N y . Good survival was observed in animals fed diet N " and diet N q ŽTable 2; Tukey’s test, P s 0.05.. The biochemical composition ŽTable 3. showed a significant difference in protein composition of larvae Ž F s 33.48, df s 4, 26, P - 0.00001. between the control Ždecrease in protein content. and those fed diets N y , N " and N q ŽTukey’s, P s 0.05.. No significant differences were registered among the microalgal diets. A significant effect of the treatments on the postlarval carbohydrate was observed Ž F s 7.31, df s 4, 25, P s 0.0007. with the highest content in the postlarvae fed diet N " , an intermediate content in the postlarvae fed diet N q and N y , and the lowest carbohydrate content in the starved postlarvae. The total lipid content did not show a significant variation among the tested diets, although there was an apparent decrease in the lipid content of the starved animals Ž P s 0.06.. In the 50 days of the second experiment, the spat grew on average 3.25 Ž"0.64., 4.76 Ž"1.09. and 3.91 Ž"0.70. mm with diet N y , diet N " and diet N q , respectively. There was a tendency for better final weights and G 30 with diet N q ŽTable 4., however not significant. The survival as well did not vary significantly among treatments ŽTable 4., as it was superior to 80% in all treatments. The protein composition of the tissues in spat ŽTable 5. varied significantly with the diets Ž F s 4.73, df s 2, 11, P s 0.04. showing the highest value in spat fed diet N q .
Table 4 Growth and survival of spat in the second experiment of nutrition with diets of different protein content: Nq, N" and Ny Microalgal diet
Final live weight Žmg.
G 30 ln ŽWf yWi .r Ždaysr30.
Survival Ž%.
Nq N" Ny
246.6 Ž"84.5. 197.4 Ž"48.6. 205.9 Ž"74.2.
1.04 Ž"0.08. 1.02 Ž"0.02. 0.93 Ž"0.07.
82.0 Ž"7.0. 88.8 Ž"8.0. 91.8 Ž"3.0.
Average Ž"standard error.. N s 4.
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Table 5 Biochemical composition of spat in the second experiment of nutrition Microalgal diet
Protein mg mgy1
Lipid mg mgy1
Carbohydrate mg mgy1
Nq N" Ny
45.16"8.31ab 24.82"1.12 a 24.45"4.30 a
2.90"0.21a 6.08"0.67 b 2.00"0.34 a
6.00"0.85 6.21"0.46 4.23"0.10
Means in the same column sharing the same superscript letter were not significantly different as determined by Tukey test Ž P ) 0.05.. Nq, N", Ny are the high, normal and low diets in protein. Average"standard error.
The carbohydrates on the other hand were not affected by the diets. The total lipid content in spat depended significantly on diet quality Ž F s 17.15, df s 2, 11, P s 0.0009., with the highest values in spat fed diet N " .
4. Discussion The present results about the microalgal mixture of I. aff. galbana ŽT-Iso. and C. neogracile confirm previous studies on the influence of dissolved nitrogen on the proximate composition of algal diets N q , N " and N y . Farıas ´ et al. Ž1997. found that under all N-conditions, the energy content in the mixture was constant, then the relationship among dietary protein and energy was modified only by the differences in protein. The diets with different protein level affected significantly the postlarval stage of the A. purpuratus. The best results in growth and survival were obtained with diet N q and they got worse as the nutritive value of the diet decreased to diet N " and N y . The experiment with spat on the other hand did not lead to conclusive answers in relation to their response to the three different qualities of diets. The analysis of the biochemical composition of the postlarvae showed that the microalgal diets affected the total content of carbohydrate in postlarvae, while in spat they affected the protein and total lipid content, but not the carbohydrate. A higher protein content in the diet has associated with a significant increase of protein content in the spat, which in turn lead to a significant decrease in lipid. Carbohydrates were not affected. According to Hawkins and Bayne Ž1991., bivalves require more C than N in their natural environment and increasing the availability of protein through their diet can only be beneficial to the animals if they have enough energy to metabolize the protein. The growth and biochemical composition of postlarvae and spat confirms the different patterns in using nutrients. Postlarvae seem to behave according to the proposed model by Hawkins and Bayne Ž1991. and excrete more nitrogen or in their defect grow more when fed diets with high protein content, future work must be designed to measure the nitrogen balance of postlarvae to confirm this supposition. Spat do not behave in that way. They deposit protein in the tissues without changing growth
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rate. This result is opposite from the results obtained by Fidalgo et al. Ž1994. in juvenile mussels of about 9 mm. Future work must be designed to measure the net protein utilization of dietary protein ŽNPU. on juvenile bivalves to determine the percentage of ingested protein deposited as tissue protein and the percentage of endogenous protein losses. Farıas ´ et al. Ž1998. found a significantly higher level of protein in Chilean scallop spat of 60 days old than in postlarvae of 3 and 22 days old. They thought this was an artefact provoked by the bigger shell size, but our study leads to the same results and therefore may indicate that A. purpuratus spat indeed accumulate protein. This accumulation is not observed in early post-metamorphic larvae and suggests that the change in energy metabolism occurs after the first month of postmetamorphic life. This change involves the use of protein as an energy storage instead of carbohydrate as was demonstrated for spat of the flat oyster O. edulis ŽHolland and Hannant, 1974. and early post-metamorphic scallops ŽFarıas ´ et al., 1998.. Furthermore, the lipid content in spat had a tendency to decrease when fed the diets N y and N q , strengthening the hypothesis that spat mobilize lipid reserves. In the same diets the lipids in postlarvae stayed without modification. According to Holland and Hannant Ž1974., the explanation for the post-metamorphic changes in energy metabolism of bivalves is related to the change from a pelagic larval life to benthic life. Before metamorphosis, lipids are used as energetic substrate and energy reserve, while glycogen is used after metamorphosis. However this pattern for bivalves is based on the analysis of the flat oyster, O. edulis, and demonstrates a metabolism highly valuable to bivalves that suffer the typical anaerobic conditions of intertidal environments. Pectinids, in general, have lower levels of glycogen than other bivalves and it is related with their low resistance to emersions and their swimming behaviour ŽBricelj and Shumway, 1991; Duncan et al., 1994.. It would be useful to investigate more in depth the metabolic pathways in the life history of bivalves, as suggested in this work. Postlarvae of A. purpuratus are sedentary, attached by byssus to filamentous substrate until they reached 5 mm height. Then spat acquires a free life as swimmers, looking for suitable substrate but maintaining its swimming capacity to escape from predators, as is common in other scallops ŽFranklin et al., 1980.. It is highly probable that these animals undergo a change in energetic metabolism in order to increase its performance, for example the main end product accumulated in muscle tissue as a result to extract energy from glycogen is different between swimming and sessile bivalves. Besides, several scallop species are known to utilize protein as an energy source during gametogenesis as well as during periods of negative energy balance compared with other groups of bivalves ŽBricelj and Shumway, 1991.. Therefore, the response to different levels of dietary protein would be dependent on the size or age of the juvenile scallops. Further studies should be conducted to demonstrate whether the biochemical changes in A. purpuratus during the transition of postlarvae to spat are an exception or whether if there is a basic difference in the metabolism between intertidal and subtidal bivalves orrand between sessile and swimming bivalves after metamorphosis. The results of Uriarte Ž1996. demonstrates that the protein quality of the diet influences significantly the growth and the survival of the postlarva up to 2 mm of the Chilean scallop, A.
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purpuratus, and the sweet clam, Gari solida; but has no effect on the Japanese oyster, C. gigas, or on the common clam, Venus antiqua. This supports the idea that the protein quality of a diet can improve growth and shorten the rearing period of postlarvae only in some species ŽUriarte and Farıas, ´ 1995. and only till a certain size because bivalves have at least 2 different sources of energy after metamorphosis.
Acknowledgements This work was supported by IFS A2075 and FONDECYT 1970807 grants of the first author. We are grateful for the technical support of P. Varas and P. Chavez. We also acknowledge useful corrections and comments by N. Nevejah ŽBelgium. and two anonymous reviewers. We would like to acknowledge FONDAP of Marine Biology and Oceanography for the valuable discussion on the topics of this manuscript, and DIDUACH for the facilities that supported this project.
References Bricelj, V.M., Shumway, S., 1991. Physiology: energy acquisition and utilization. In: Shumway, S. ŽEd.., Scallop Biology and Culture. Int. Pect. Workshop 1989, pp. 305–346. Coutteau, P., Sorgeloos, P., 1992. The use of algal substitutes and the requirement for live algae in the hatchery and nursery rearing of bivalve molluscs: an international survey. J. Shellfish Res. 11, 467–476. Coutteau, P., Hadley, N.H., Manzi, J.J., Sorgeloos, P., 1994. Effect of algal ration and substitution of algae by manipulated yeast diets on the growth of juvenile Mercenaria mercenaria. Aquaculture 120, 135–150. Duncan, P., Spicer, J., Taylor, A., Spencer, P., 1994. Acid–base disturbances accompanying emersion in the scallop Pecten maximus ŽL... J. Exp. Mar. Biol. Ecol. 182, 15–25. Enright, C.T., Newkirk, G.F., Craigie, J.S., Castell, J.D., 1986. Growth of juvenile Ostrea edulis L. fed Chaetoceros gracilis Schutt ¨ of varied chemical composition. J. Exp. Mar. Biol. Ecol. 96, 15–26. Farıas, ´ A., Uriarte, I., Varas, P., 1997. Estudio de los requerimientos nutricionales del ostion ´ del norte Argopecten purpuratus ŽLamarck, 1819. durante el acondicionamiento reproductivo ŽNutritional study on the broodstock conditioning of the Chilean scallop, Argopecten purpuratus ŽLamarck, 1819... Revista de Biologıa ´ Marina y Oceanografıa ´ ŽValparaıso ´ . 32, 127–136. Farıas, ´ A., Uriarte, I., Castilla, J.C., 1998. A biochemical study of the larval and postlarval stages of the Chilean scallop Argopecten purpuratus. Aquaculture 166, 37–47. Fidalgo, J.P., Cid, A., Lopez, I., Abalde, J., Herrero, C., 1994. Growth and biochemical profile of juvenile mussels Ž Mytilus galloproÕincialis Lmk. fed on different algal diets. J. Shellfish Res. 13, 67–75. Franklin, A., Pickett, G.D., Connor, P.M., 1980. The scallop and its fishery in England and Wales. Lab. Leafl., MAFF Direct. Fish. Res., Lowestoft, 51. Hawkins, A.J.S., Bayne, B.L., 1991. Nutrition of marine mussels: factors influencing the relative utilization of protein and energy. Aquaculture 94, 177–196. Holland, D.L., Hannant, P.J., 1974. Biochemical changes during growth of the spat of the oyster, Ostrea edulis L. J. Mar. Biol. Assoc. UK 54, 1007–1016. O’Connor, W.A., Nell, J.A., Diemar, J.A., 1992. The evaluation of twelve algal species as food for juvenile Sydney rock oysters Saccostrea commercialis ŽIredale and Roughley.. Aquaculture 108, 277–283. Servicio Nacional de Pesca, 1997. Anuario Estadıstico de Pesca ŽFishery Statistical Annuary., SERNAP, ´ Ministerio de Economıa, ´ Fomento y Reconstruccion, ´ Santiago-Chile. Spencer, B.E., 1988. Growth and filtration of juvenile oysters in experimental outdoor pumped upwelling systems. Aquaculture 75, 139–158.
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Uriarte, I., 1996. Effect of larvae diet on hatchery production of the intertidal Ž Venus antiqua, Gari solida. and subtidal Ž Argopecten purpuratus, Crassosstrea gigas . bivalve species. Unpublished report to International Foundation for Science, IFS-Sweden. Uriarte, I., Farıas, ´ A., 1995. Effect of broodstock origin and postlarval diet on postlarval growth and physiological performance of the Chilean scallop Argopecten purpuratus. In: Lavens, P., Jaspers, E., Roelants, I. ŽEds.., European Aquaculture Society Special Publication 24, pp. 75–78. Uriarte, I., Farıas, ´ A., Hawkins, A.J.S., Bayne, B.L., 1993. Cell characteristics and biochemical composition of Dunaliella primolecta Butcher conditioned at different concentrations of dissolved nitrogen. J. Appl. Phycol. 5, 447–453. Utting, S.D., 1986. A preliminary study on growth of Crassosstrea gigas larvae and spat in relation to dietary protein. Aquaculture 56, 123–138. Van Landengham, 1986. Webb, K.L., Chu, F.L.E., 1983. Phytoplankton as food source for bivalve larvae. In: Pruder, G.D., Langdon, C., Conklin, D. ŽEds.., Proceedings of the Second International Conference on Aquaculture Nutrition: Biological and Physiological Approaches to Shellfish Nutrition. Louisiana State University Press, Baton Rouge, LA, USA, pp, 272–291. Whyte, J.N.C., Bourne, N., Hodgson, C.A., 1990. Nutritional condition of rock scallop, Crassadoma gigantea ŽGray., larvae fed mixed algal diets. Aquaculture 86, 25–40. Wikfors, G.H., Twarog, J.W., Ukeles, R., 1984. Influence of chemical composition of algal food sources on growth of juvenile oysters, Crassosstrea Õirginica. Biol. Bull. 167, 251–263. Wikfors, G.H., Ferris, G.A., Smith, B.C., 1992. The relationship between gross biochemical composition of cultured algal foods and growth of the hard clam, Mercenaria mercenaria ŽL... Aquaculture 108, 135–154.
The effect of dietary protein content on growth and biochemical composition of Chilean scallop Argopecten purpuratus žL. / postlarvae and spat Iker Uriarte ) , Ana Farıas ´ Facultad de Pesquerıas ´ y Oceanografıa, ´ UniÕersidad Austral de Chile, Casilla 1327, Puerto Montt, Chile Received 30 November 1998; received in revised form 31 March 1999; accepted 31 March 1999
Abstract This study shows the effect of three different levels of dietary protein content on the performance of postlarvae Ž1.8 mm. and spat Ž6.3 mm. of the Chilean scallop, Argopecten purpuratus. The postlarvae showed significant differences in growth and survival, with better growth when fed the high protein diet ŽN q .. The biochemical composition of the postlarvae showed only significant differences in the content of total carbohydrate, with the highest values in postlarvae fed the normal protein diet ŽN " . and the lowest values in starved postlarvae. There were no significant differences in growth nor survival of spat fed the test diets. The biochemical composition of the spat was significantly different in protein content, with the highest level in spat fed the N q diet, while the lipid content increased marginally significantly in spat fed the normal diet. The carbohydrate content in spat did not change. These results show that postlarvae and spat follow a different pattern of energy metabolism, because the diet N q only increased the growth in early postlarval stages and lost its effect in the later stages of development. This paper discusses the threshold age at which the metabolic pathway changes, coinciding with the change from the sessile life attached by the byssus to the typical unattached and free swimming life of A. purpuratus. It also highlights the efficiency and possible use in commercial hatcheries of high protein diets. q 1999 Elsevier Science B.V. All rights reserved. Keywords: Dietary protein; Bivalve nutrition; Scallop hatchery; Argopecten purpuratus
)
Corresponding author. Tel.: q56-65-277120; Fax: q56-65-255583; E-mail: [email protected]
0044-8486r99r$ - see front matter q 1999 Elsevier Science B.V. All rights reserved. PII: S 0 0 4 4 - 8 4 8 6 Ž 9 9 . 0 0 1 4 5 - 3
120
I. Uriarte, A. Farıasr ´ Aquaculture 180 (1999) 119–127
1. Introduction The Chilean scallop, Argopecten purpuratus, is a subtidal bivalve that supports a considerable commercial aquaculture explaining more than 90% of total scallop production in Chile ŽServicio Nacional de Pesca, 1997.. Production of ‘seed’ scallops in hatcheries has proven to be a successful management tool for commercial grow-out as well as settlement in artificial collectors. Most modern hatcheries in the North of Chile produce algal feeds of known identity for rearing larval and young post-set scallops. Considering the commercial importance of A. purpuratus culture, there have been surprisingly few studies on nutrition for hatchery management. The typical works examining nutrition at hatcheries have compared the food value of microalgal species for larvae and postlarvae of bivalve ŽWebb and Chu, 1983; Enright et al., 1986; Whyte et al., 1990; O’Connor et al., 1992. where several algae were shown to support consistently rapid growth of scallops. The identification of the characteristics that differentiated the better algal diets from those that yielded poor growth has facilitated the development of nutrition in bivalves ŽEnright et al., 1986; Whyte et al., 1990; Wikfors et al., 1992.. The list of microalgal strains used in marine research institutions and commercial hatcheries is short, interest remains high for finding new and better algal feeds or algal supplements ŽCoutteau and Sorgeloos, 1992; Coutteau et al., 1994.. Live unialgal food culturing permits control over the nutritional quality of cells through various culture-management practices. These include among others: altering the nutrient composition by controlling the nutrient concentrations. Post-set oysters, Crassostrea Õirginica, Crassostrea gigas and Ostrea edulis, have been shown to grow more rapidly with a relatively high carbohydrate content when protein decreased under conditions of nitrogen deficiency in the unialgal food culturing ŽWikfors et al., 1984; Enright et al., 1986; Utting, 1986.. However, high protein diets, with relatively low carbohydrate content, have shown to promote rapid growth in post-set scallops A. purpuratus ŽUriarte and Farıas, ´ 1995., therefore information on nutritional responses of oysters cannot be applied directly to scallops. In the present study, experiments were conducted with a mixture of two different microalgae, a flagellate and a diatom, that were manipulated in their protein contents, and growth of post-set and juvenile scallops Žpostlarvae and spat, respectively. fed different algal qualities was compared. The relative importance of the major nutritional constituents—protein, carbohydrate and lipid—on the scallop tissue reserves were investigated.
2. Materials and methods The postlarvae of A. purpuratus were produced under laboratory conditions and measured with a binocular stereomicroscope with an accuracy of "10 mm. The survival was checked on a weekly basis and the sizes were taken at the beginning and at the end of the experiment. The instantaneous growth rate G 30 was calculated according to
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Spencer Ž1988.. Several sizes of postlarvae were sampled in order to determine the relationship between shell height and live weight of scallop. The diets of different protein content consisted of a mixture of two species of microalgae: Isochrysis aff galbana ŽT-Iso strain. and Chaetoceros neogracile ŽVan Landengham, 1986. containing high, normal or low content of protein ŽN q , N " and N y , respectively.. The biochemical strain of each species was grown using Walne medium adjusted to three separate conditions containing the following nitrogen concentrations in accord with Uriarte et al. Ž1993.: 0.45, 1.18 and 3.53 mg-at N ly1 of sodium nitrate for conditions low-, normal- and high-protein, respectively. In order to generate a significant low protein strain of T-Isochrysis, it was necessary to reduce the nitrogen concentration to 0.24 mg-at N ly1 ŽUriarte et al., unpublished data.. Each strain was cultured in 5 l flasks at a temperature of 258C and under 24 h illumination. A dispensing pump dosed the diets at an equivalent concentration of 2% of the live weight per day. The mixture of 1:1 ŽT-Iso:Chaetoceros. was calculated on the basis of number of cells. The diets were analysed three times during the experiments. The replicates in the first experiment held 500 mg postlarvae with a mean size of 1800 Ž"83. mm. Each replicate contained an up-welling silo with a mesh wire of 500 mm at the bottom, which was suspended in a closed system of 20 l tanks with filtered and UV-sterilized seawater. The treatments consisted of three mixed diets with different levels of protein and one control without feeding. The latter served as a reference to demonstrate possible signs of malnutrition in the animals receiving the diets. Each treatment contained six replicates and the experiment was run at 19 " 0.28C for four weeks. In the second experiment each replicate contained 900 mg of spat of 6.3 Ž"0.1. mm. The experimental set-up was the same as for experiment 1. The treatments consisted of three mixed diets of the different protein levels. There were four replicates per treatment. At the end of the experimental period, the scallops were fasted for 24 h for gut clearance before being analysed for gross biochemical composition following the methodology used by Farıas ´ et al. Ž1998..
Fig. 1. Allometric relationship between the live weight and the size in height of the scallops after the metamorphosis. The curve was fit by regression Žmultiplicative model: Y s a= X b ., the real points are shown, r indicates the correlation coefficient with the model.
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Table 1 Gross biochemical composition of the microalgal mixture: Nq, N" and Ny, composed by a mixture of I. aff. galbana ŽT-Iso. and C. neogracile conditioned at media with high, normal and low nitrogen concentrations, respectively Microalgal quality
Protein mg 10 6 cellsy1
Lipid mg 10 6 cellsy1
Carbohydrate mg 10 6 cellsy1
Organic matter Žorganic wt.rdry wt..
Nq N" Ny
7.84"0.39 b 5.64"0.41ab 4.48"0.78 a
4.12"0.38 4.77"0.20 4.68"0.52
4.31"0.31a 5.28"0.26 a 7.36"1.31b
0.76"0.03 a 0.74"0.05a 0.72"0.06 b
Means in the same column sharing the same superscript letter were not significantly different as determined by Tukey test Ž P ) 0.05.. Organic weight calculated as the sum of protein, lipid and carbohydrate. Average"standard error.
3. Results The relation between live weight and size of post-metamorphic scallops ŽFig. 1. allowed us, through extrapolation, to calculate the initial number of scallops and to evaluate the percentage of weight that was administered as food on a daily basis. The microalgal mixtures showed a dry weight ŽDW. of 21.4 Ž"1.4. mg per million of cells for diets N q and N " , and 22.9 Ž"9.9. mg per million of cells for diet N y . The protein content showed highly significant differences among treatments ŽTable 1, F s 14.35; df s 2, 65, P - 0.00001. with a following decreasing order: N q ) N " ) N y ŽTukey’s test, P s 0.05.. There were significant differences in the carbohydrate content among all diets Ž F s 61.32; df s 2, 61, P - 0.00001., showing an opposite pattern to protein content ŽTukey’s P s 0.05.: N q - N " - N y . The lipid remained approximately constant among diets. During the 30 days of the first experiment, the postlarvae grew 170 Ž"124., 1085 Ž"106., 1148 Ž"123. and 1660 Ž"140. mm in the control and with diet N y , diet N " and diet N q , respectively. The quality of the food had a significant effect on the final weight of the animals Ž F s 16.902; df s 3, 23; P - 0.00001. and on G 30 Ž F s 37.56; df s 3, 23; P - 0.00001.. The highest final weights and G 30 were obtained with diet N q , while the lowest values were obtained in the controls where the animals were submitted to starvation ŽTable 2, Tukey’s test P s 0.05.. Table 2 Growth and survival of postlarva in the first experiment of nutrition with diets of three protein qualities: Nq, N" and Ny, and a starved control Microalgal diet
Final live weight Žmg.
G 30 ln ŽWf yWi .r Ždaysr30.
Survival Ž%.
Nq N" Ny control
5.92 Ž"1.10. c 3.81 Ž"0.73. b 3.52 Ž"0.61. b 1.21 Ž"0.17. a
1.83 Ž"0.16. c 1.39 Ž"0.17. bc 1.34 Ž"0.17. b 0.31 Ž"0.20. a
71.9 Ž"9.8. c 57.9 Ž"14.2. bc 46.7 Ž"12.3. b 17.8 Ž"3.3. a
Means in the same column sharing the same superscript letter were not significantly different as determined by Tukey test Ž P ) 0.05.. Average Ž"standard error.. N s6.
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Table 3 Biochemical composition of postlarva in the first experiment of nutrition. Nq, N", Ny are the high, normal and low diet in protein, control is the fasted treatment Microalgal diet
Protein mg mgy1
Lipid mg mgy1
Carbohydrate mg mgy1
Nq N" Ny Control
50.97"2.36 b 47.24"4.33 b 60.71"4.65 c 7.47"0.35a
3.05"0.69 3.80"0.79 4.35"1.18 0.37"0.00
4.33"0.71b 6.88"0.97 bc 4.88"0.56 b 3.05"1.01a
Means in the same column sharing the same superscript letter were not significantly different as determined by Tukey test Ž P ) 0.05.. Average"standard error.
The survival varied significantly among treatments Ž F s 14.42; df s 3, 23; P 0.0001. with the controls having the greatest mortality in the postlarvae, followed by those fed diet N y . Good survival was observed in animals fed diet N " and diet N q ŽTable 2; Tukey’s test, P s 0.05.. The biochemical composition ŽTable 3. showed a significant difference in protein composition of larvae Ž F s 33.48, df s 4, 26, P - 0.00001. between the control Ždecrease in protein content. and those fed diets N y , N " and N q ŽTukey’s, P s 0.05.. No significant differences were registered among the microalgal diets. A significant effect of the treatments on the postlarval carbohydrate was observed Ž F s 7.31, df s 4, 25, P s 0.0007. with the highest content in the postlarvae fed diet N " , an intermediate content in the postlarvae fed diet N q and N y , and the lowest carbohydrate content in the starved postlarvae. The total lipid content did not show a significant variation among the tested diets, although there was an apparent decrease in the lipid content of the starved animals Ž P s 0.06.. In the 50 days of the second experiment, the spat grew on average 3.25 Ž"0.64., 4.76 Ž"1.09. and 3.91 Ž"0.70. mm with diet N y , diet N " and diet N q , respectively. There was a tendency for better final weights and G 30 with diet N q ŽTable 4., however not significant. The survival as well did not vary significantly among treatments ŽTable 4., as it was superior to 80% in all treatments. The protein composition of the tissues in spat ŽTable 5. varied significantly with the diets Ž F s 4.73, df s 2, 11, P s 0.04. showing the highest value in spat fed diet N q .
Table 4 Growth and survival of spat in the second experiment of nutrition with diets of different protein content: Nq, N" and Ny Microalgal diet
Final live weight Žmg.
G 30 ln ŽWf yWi .r Ždaysr30.
Survival Ž%.
Nq N" Ny
246.6 Ž"84.5. 197.4 Ž"48.6. 205.9 Ž"74.2.
1.04 Ž"0.08. 1.02 Ž"0.02. 0.93 Ž"0.07.
82.0 Ž"7.0. 88.8 Ž"8.0. 91.8 Ž"3.0.
Average Ž"standard error.. N s 4.
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Table 5 Biochemical composition of spat in the second experiment of nutrition Microalgal diet
Protein mg mgy1
Lipid mg mgy1
Carbohydrate mg mgy1
Nq N" Ny
45.16"8.31ab 24.82"1.12 a 24.45"4.30 a
2.90"0.21a 6.08"0.67 b 2.00"0.34 a
6.00"0.85 6.21"0.46 4.23"0.10
Means in the same column sharing the same superscript letter were not significantly different as determined by Tukey test Ž P ) 0.05.. Nq, N", Ny are the high, normal and low diets in protein. Average"standard error.
The carbohydrates on the other hand were not affected by the diets. The total lipid content in spat depended significantly on diet quality Ž F s 17.15, df s 2, 11, P s 0.0009., with the highest values in spat fed diet N " .
4. Discussion The present results about the microalgal mixture of I. aff. galbana ŽT-Iso. and C. neogracile confirm previous studies on the influence of dissolved nitrogen on the proximate composition of algal diets N q , N " and N y . Farıas ´ et al. Ž1997. found that under all N-conditions, the energy content in the mixture was constant, then the relationship among dietary protein and energy was modified only by the differences in protein. The diets with different protein level affected significantly the postlarval stage of the A. purpuratus. The best results in growth and survival were obtained with diet N q and they got worse as the nutritive value of the diet decreased to diet N " and N y . The experiment with spat on the other hand did not lead to conclusive answers in relation to their response to the three different qualities of diets. The analysis of the biochemical composition of the postlarvae showed that the microalgal diets affected the total content of carbohydrate in postlarvae, while in spat they affected the protein and total lipid content, but not the carbohydrate. A higher protein content in the diet has associated with a significant increase of protein content in the spat, which in turn lead to a significant decrease in lipid. Carbohydrates were not affected. According to Hawkins and Bayne Ž1991., bivalves require more C than N in their natural environment and increasing the availability of protein through their diet can only be beneficial to the animals if they have enough energy to metabolize the protein. The growth and biochemical composition of postlarvae and spat confirms the different patterns in using nutrients. Postlarvae seem to behave according to the proposed model by Hawkins and Bayne Ž1991. and excrete more nitrogen or in their defect grow more when fed diets with high protein content, future work must be designed to measure the nitrogen balance of postlarvae to confirm this supposition. Spat do not behave in that way. They deposit protein in the tissues without changing growth
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rate. This result is opposite from the results obtained by Fidalgo et al. Ž1994. in juvenile mussels of about 9 mm. Future work must be designed to measure the net protein utilization of dietary protein ŽNPU. on juvenile bivalves to determine the percentage of ingested protein deposited as tissue protein and the percentage of endogenous protein losses. Farıas ´ et al. Ž1998. found a significantly higher level of protein in Chilean scallop spat of 60 days old than in postlarvae of 3 and 22 days old. They thought this was an artefact provoked by the bigger shell size, but our study leads to the same results and therefore may indicate that A. purpuratus spat indeed accumulate protein. This accumulation is not observed in early post-metamorphic larvae and suggests that the change in energy metabolism occurs after the first month of postmetamorphic life. This change involves the use of protein as an energy storage instead of carbohydrate as was demonstrated for spat of the flat oyster O. edulis ŽHolland and Hannant, 1974. and early post-metamorphic scallops ŽFarıas ´ et al., 1998.. Furthermore, the lipid content in spat had a tendency to decrease when fed the diets N y and N q , strengthening the hypothesis that spat mobilize lipid reserves. In the same diets the lipids in postlarvae stayed without modification. According to Holland and Hannant Ž1974., the explanation for the post-metamorphic changes in energy metabolism of bivalves is related to the change from a pelagic larval life to benthic life. Before metamorphosis, lipids are used as energetic substrate and energy reserve, while glycogen is used after metamorphosis. However this pattern for bivalves is based on the analysis of the flat oyster, O. edulis, and demonstrates a metabolism highly valuable to bivalves that suffer the typical anaerobic conditions of intertidal environments. Pectinids, in general, have lower levels of glycogen than other bivalves and it is related with their low resistance to emersions and their swimming behaviour ŽBricelj and Shumway, 1991; Duncan et al., 1994.. It would be useful to investigate more in depth the metabolic pathways in the life history of bivalves, as suggested in this work. Postlarvae of A. purpuratus are sedentary, attached by byssus to filamentous substrate until they reached 5 mm height. Then spat acquires a free life as swimmers, looking for suitable substrate but maintaining its swimming capacity to escape from predators, as is common in other scallops ŽFranklin et al., 1980.. It is highly probable that these animals undergo a change in energetic metabolism in order to increase its performance, for example the main end product accumulated in muscle tissue as a result to extract energy from glycogen is different between swimming and sessile bivalves. Besides, several scallop species are known to utilize protein as an energy source during gametogenesis as well as during periods of negative energy balance compared with other groups of bivalves ŽBricelj and Shumway, 1991.. Therefore, the response to different levels of dietary protein would be dependent on the size or age of the juvenile scallops. Further studies should be conducted to demonstrate whether the biochemical changes in A. purpuratus during the transition of postlarvae to spat are an exception or whether if there is a basic difference in the metabolism between intertidal and subtidal bivalves orrand between sessile and swimming bivalves after metamorphosis. The results of Uriarte Ž1996. demonstrates that the protein quality of the diet influences significantly the growth and the survival of the postlarva up to 2 mm of the Chilean scallop, A.
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purpuratus, and the sweet clam, Gari solida; but has no effect on the Japanese oyster, C. gigas, or on the common clam, Venus antiqua. This supports the idea that the protein quality of a diet can improve growth and shorten the rearing period of postlarvae only in some species ŽUriarte and Farıas, ´ 1995. and only till a certain size because bivalves have at least 2 different sources of energy after metamorphosis.
Acknowledgements This work was supported by IFS A2075 and FONDECYT 1970807 grants of the first author. We are grateful for the technical support of P. Varas and P. Chavez. We also acknowledge useful corrections and comments by N. Nevejah ŽBelgium. and two anonymous reviewers. We would like to acknowledge FONDAP of Marine Biology and Oceanography for the valuable discussion on the topics of this manuscript, and DIDUACH for the facilities that supported this project.
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