Ž .
Fig. 6. Relationship between total ny3 FA proportion as percentage of total identifiable fatty acids, TIFA , protein and carbohydrate content of R. reticulata and larval length achieved after feeding for 2 weeks on this
species. Protein content is shown colour-coded.
positive correlation between the 16:0 and SAFA, with growth and negative correlations Ž
. existing between PUFA and n y 3 FA with larval size Fig. 3 , with correlation values
of 0.97, 0.98, y0.98 and y0.97, respectively. Protein and carbohydrate content were not significantly correlated with larval growth, with correlation values of 0.19 and 0.57,
respectively. However, protein, carbohydrate and some fatty acids of R. reticulata were correlated with larval growth. Generally, lower correlation values were calculated with
0.73 and 0.48 for protein and carbohydrate content and 0.55, y0.91, 0.56, y0.6 for 16:0, 18:3n y 3, SAFA and n y 3 FA, respectively. Four-dimensional plots of larval
length, algal protein, carbohydrate and one of these algal fatty acids components are shown in Figs. 4–6 for the 16:0, 18:3n y 3 and n y 3 FA, respectively. Collectively,
these graphs, together with the correlation analysis, indicate that there is a positive correlation between algal 16:0 and SAFA with larval growth while algal 18:3n y 3 and
n y 3 FA were negatively correlated with larval growth. Despite careful examination of the data for C. muelleri and S. costatum, no consistent relationship could be established.
4. Discussion
Culturing live algae for food in hatcheries is essential, especially given the limited Ž
. success, up to now, of artificial feeds Laing, 1987 ; hence, optimizing the nutritional
value of any alga for a specific animal should be of critical importance. Environmental Ž
conditions have been shown to influence algal biochemical composition Leonardos and .
Lucas, 2000a . The optimization of the algal nutritional value in this investigation was found to be of significant importance, with algal culturing conditions increasing larval
size from 10 to 30, depending on the algal species.
S. costatum is a species used extensively in hatcheries mainly because of its ability to thrive in a variety of environments while also supporting good growth of animals
Ž .
O’Connor et al., 1992 . It was found to be the second highest-ranked diatom for Ostrea Ž
. edulis growth by Enright et al. 1986a among the species tested and one of the best
Ž .
diets for the hard clam, Mercenaria mercenaria Wikfors et al., 1992 . C. muelleri has Ž
been shown to have good potential in a subtropical greenhouse bivalve hatchery Nelson .
et al., 1992 . A good nutritional value record is also reported for this diatom for the Ž
. Sydney rock oyster, Saccostrea commercialis by O’Connor et al. 1992 and its high
nutritional value is further confirmed here with M. edulis larvae. It appears that this Chaetoceros species is no exception from the generally good nutritional value record
Ž that this genus has been shown to demonstrate Chu, 1989; O’Connor and Heasman,
. Ž
. 1997 . Of all the species tested here, R. reticulata previously Rhodomonas baltica is
the least studied, although other Cryptomonads, described as Rhodomonas species, have Ž
. been found to be good diets for O. edulis Enright et al., 1986a . On the basis of the
very high nutritional value found here, the apparent lack of extensive usage of this species in mariculture is difficult to substantiate. P. lutheri has an ambiguous record of
nutritional value in which it appears to be a very good food for larvae of several
Ž bivalves, e.g., doughboy scallops, Mimachlamys asperrima O’Connor and Heasman,
. Ž
1997 , American and European oysters, Crassostrea Õirginica and O. edulis Walne, .
1963; Chu and Dupuy, 1980 , but mediocre and poor for some others like the Japanese Ž
. scallop, Patinopecten yessoensis Thompson et al., 1994 and the Pacific oyster, C.
Ž .
gigas Langdon and Waldock, 1981 . The present work classifies this species as an excellent food for M. edulis larvae since in almost all of its biochemical compositions, it
performed better than the control diet. This control two-species diet was expected to be of superior value, because it was considered to contain the greater diversity of
biochemical constituents to satisfy most nutritional requirements for growth than any
Ž .
one monospecific diet Widdows, 1991 . Ž
. Thompson et al. 1996 correlated positively the proportion of dietary 16:0 and
negatively the dietary 20:5n y 3 of P. lutheri with larval mortality in C. gigas; both these results were unequivocally confirmed here with the use of correlation analysis. It
appears that in PUFA and n y 3 FA, in general, rather than specifically, the 20:5n y 3 was negatively correlated with larval growth. Hence, it is not clear whether the positive
and negative correlations of 16:0 and 20:5n y 3, respectively, are due to their specific role or is a reflection of the positive and negative correlations of SAFA and n y 3 FA,
generally.
The use of a multidimensional model enables a more comprehensive inclusion of dietary components that have been shown in the past to be important in determining the
algal nutritional value, overcoming the problematic usage of multilinear regression Ž
. analysis Wikfors et al., 1992 . Although the FA content appears to be the main factor
influencing algal nutritional value in some cases, the protein and carbohydrate content is also modifying the alga’s dietary value; increased carbohydrate and protein content
within an algal species enhances its nutritional value. As shown in the four dimensional graphs, optimal larval growth is achieved when the alga contains the highest proportion
of 16:0 and SAFA as well as increased protein and carbohydrate content; singly any of these components cannot sufficiently explain the resulting variation of larval growth.
Again, the decrease of nutritional value was found when higher proportions of 18:3n y 3 and n y 3 fatty acids, in general, were observed. The descriptive nature of this approach
does not provide an exact measure by which the contributions to the nutritional value of any algal component can be determined, but it provides a means of demonstrating how
Ž dietary components interact to determine the overall dietary value
Enright et al., .
Ž .
1986a,b . However, the comments of Dickey-Collas and Geffen 1992 and Thompson Ž
. et al. 1996 about the positive effects of increased 16:0 and SAFA and the negative
effects of increased PUFA and more specifically, the n y 3 fatty acids are generally confirmed here. It would seem that in the cases where such a clear-cut relationship is
found, with dietary fatty acid being the main factor in determining nutritional value, the other biochemical components are of minor importance. In other cases, there is a
positive effect of increased protein and carbohydrate content, with fatty acids also contributing to the nutritional value.
Ž These results are apparently in conflict with the widely publicized held view e.g., Su
. et al., 1988 that increased dietary PUFA enhance the dietary value of an algal diet, not
least because larval PUFA have been shown to be a positive growth index for M. edulis Ž
. Ž
. Leonardos and Lucas, 2000b . Langdon and Waldock 1981 demonstrated the impor-
tance of PUFA, especially the 20:5n y 3, to C. gigas. Many researchers in the aquacultural field, largely because of this strong correlation of the larval 20:5n y 3 fatty
acid and PUFA, in general, with growth, use the occurrence and quantity of these Ž
. compounds as ‘‘a rule of thumb’’ guide to rank algal diets Su et al., 1988 . Other
workers have indirectly further substantiated this relationship by trying to explain the nutritional deficiency of particular algal species in terms of the lack of some essential
Ž .
fatty acids, such as some of the n y 3 groups De Pauw et al., 1984 . From the current findings, this is a misleading overgeneralization to make. A clear distinction should be
made between the nutritional elements in the diet and the way that they influence larval growth and development on one hand and the biochemical composition of the larvae
themselves. A low level or lack of one dietary component, e.g., a particular fatty acid, will not necessarily be reflected by a deficiency in the same component in the larvae,
since the animals may transform almost all of the components that they digest, through their metabolic pathways; a deficiency in the input element will translate into a
deficiency of the end product inside the animal cells. Therefore, it is not entirely correct to substantiate the correlation of a larval fatty acid with larval growth by observing the
effect that it has if it is excluded or inadequately present in the diet, or vice versa. The sole significance is related to the nutritional quality of the diet for that given organism
and not as to the importance as a growth index for the animals themselves.
The optimal nutritional value was obtained at low-light, nitrogen-limited or non- limited nutrient cells for S. costatum and C. muelleri, for R. reticulata at high-light
conditions under phosphorus limitation or low-light without nutrient limitation and for P. lutheri at high-light conditions under phosphorus limitation. Given the simplicity of
the modification of the culture conditions, this approach is a cost-effective way of increasing the nutritional value of the cultured algae and should be further explored for
other algae. Although algal fatty acids were shown to be of particular importance in determining the algal nutritional value, overall, they were found to be species-specific.
Therefore, future research not only should be directed towards examining the way in
which environmental conditions determine the nutritional value of an alga, but the range of species and clones investigated must also be widened. Further research should aim at
enlightening the interactive way in which discrete algal biochemical components deter- mine the nutritional value of the species, with emphasis given to their fatty acid profile.
Acknowledgements
The first author would like to thank Andy Beaumont for his helpful comments throughout this work and J. East for his invaluable help with the fatty acid analyses. The
present work is part of a PhD research thesis funded by the Greek State Scholarship Ž
. Foundation I.K.Y. whose assistance is acknowledged.
References
Baars, J.W.M., 1981. Autecological investigations on marine diatoms: II. Generation time of 50 species. Hydrobiol. Bull. 15, 137–151.
Bayne, B.L., 1965. Growth and the delay of metamorphosis of the larvae of Mytilus edulis L. Ophelia 2, 1–47.
Chu, K.H., 1989. Chaetoceros gracilis as the exclusive feed for the larvae and postlarvae of the shrimp Metapenaeus ensis. Aquaculture 83, 281–287.
Chu, F.L.E., Dupuy, J.L., 1980. The fatty acid composition of three unicellular algal species used as food Ž
. sources for larvae of the American oyster Crassostrea Õirginica . Lipids 15, 356–364.
De Pauw, N., Morales, J., Persoone, G., 1984. Mass cultures of microalgae in aquaculture systems: progress and constraints. Hydrobiologia 116r117, 121–134.
Dickey-Collas, M., Geffen, A.J., 1992. Importance of the fatty acids 20:5v3 and 22:6 v3 in the diet of plaice Ž
. Pleuronectes platessa larvae. Mar. Biol. 113, 463–468.
Enright, C.T., Newkirk, G.F., Craigie, J.S., Castell, J.D., 1986a. Evaluation of phytoplankton as diets for juvenile Ostrea edulis L. J. Exp. Mar. Biol. Ecol. 96, 1–13.
Enright, C.T., Newkirk, G.F., Craigie, J.S., Castell, J.D., 1986b. Growth of juvenile Ostrea edulis L. fed Chaetoceros gracilis Schutt of varied chemical composition. J. Exp. Mar. Biol. Ecol. 96, 15–26.
Epifanio, C.E., 1979. Growth in bivalve molluscs: nutritional effects of two or more species of algae in diets Ž
. fed to the American oyster Crassostrea Õirginica Gmelin and the hard clam Mercenaria mercenaria L.
Aquaculture 18, 1–12. Gallager, S.M., Mann, R., 1981. The effect of varying carbonrnitrogen ratio in the phytoplankter T.
Ž
3
. pseudonana
H on its food value of the bivalve Tapes japonica. Aquaculture 26, 95–105. Laing, I., 1987. The use of artificial diets in rearing bivalve spat. Aquaculture 65, 243–249.
Langdon, C.J., Waldock, M.J., 1981. The effect of algal and artificial diets on the growth and fatty acid composition of Crassostrea gigas spat. J. Mar. Biol. Assoc. UK 61, 431–448.
Leonardos, N., 1998. Environmental effects on the growth and biochemical composition of four microalgae, in relation to their use as food for Mytilus edulis larval rearing. PhD thesis, University of Wales, Bangor, 248
pp. Leonardos, N., Lucas, I.A.N., 2000a. Effects of environmental parameters in the growth and biochemical
composition, with emphasis in fatty acid content, of four microalgae. J. Appl. Algae, submitted. Leonardos, N., Lucas, I.A.N., 2000b. The use of larval fatty acids as an index of growth in Mytilus edulis L.
larvae. Aquaculture, in press. Loosanoff, V.L., Davis, H.C., 1963. Rearing of bivalve molluscs. Adv. Mar. Biol. 1, 1–136.
Nelson, J.R., Guarda, S., Cowell, L.E., Heffernan, P.B., 1992. Evaluation of microalgal clones for mass culture in a subtropical greenhouse bivalve hatchery: growth rates and biochemical composition at 308C.
Aquaculture 106, 357–377.
O’Connor, W.A., Heasman, M.P., 1997. Diet and feeding regimens for larval doughboy scallops, Mimach- lamys asperrima. Aquaculture 158, 289–303.
O’Connor, W.A., Nell, J.A., Diemar, J.A., 1992. The evaluation of twelve algal species as food for juvenile Ž
. Sydney rock oysters Saccostrea commercialis Iredale and Roughley . Aquaculture 108, 277–283.
Sargent, J.R., Whittle, K.J., 1981. Lipids and hydrocarbons in the marine environment. In: Longhurst, A.R. Ž
. Ed. , Analysis of Marine Ecosystems. Academic Press, London, pp. 491–533.
Sokal, R.R., Rohlf, F.J., 1987. Introduction to Biostatistics, 2nd edn. Freeman, New York. Su, H.M., Lei, C.H., Liao, I.C., 1988. The effect of environmental factors on the fatty acid composition of
Ž . Skeletonema costatum, Chaetoceros gracilis and Tetraselmis chuii. J. Fish. Soc. Taiwan 15 1 , 21–34.
Thompson, P.A., Guo, M.-X., Harrison, P.J., 1993. The influence of irradiance on the biochemical composi- Ž
tion of three phytoplankton species and their nutritional value for larvae of the Pacific oyster Crassostrea .
gigas . Mar. Biol. 117, 259–268. Thompson, P.A., Guo, M.-X., Harrison, P.J., 1994. Influence of irradiance on the nutritional value of two
Ž .
phytoplankton species fed to larval Japanese scallops Patinopecten yessoensis . Mar. Biol. 119, 89–97. Thompson, P.A., Guo, M.-X., Harrison, P.J., 1996. Nutritional value of diets that vary in fatty acid
Ž .
composition for larval Pacific oysters Crassostrea gigas . Aquaculture 143, 379–391. Walne, P.R., 1963. Observations on the food value of seven species of algae to the larvae of Ostrea edulis: I.
Feeding experiments. J. Mar. Biol. Assoc. UK 43, 767–784. Watanabe, T., Kitajima, C., Fujita, S., 1983. Nutritional values of live organisms used in Japan for mass
propagation of fish: a review. Aquaculture 34, 115–134. Webb, K.L., Chu, F.L.E., 1983. Phytoplankton as a food source for bivalve larvae. In: Pruder, G.D., Langdon,
Ž .
C., Conklin, D. Eds , Proceedings of the 2nd International Conference on Aquaculture Nutrition.
Biochemical and Physiological Approaches to Shellfish Nutrition. World Mariculture Society Special Publication, Vol. 2, pp. 272–291.
Widdows, J., 1991. Physiological ecology of the mussel larvae. Aquaculture 94, 147–163. Wikfors, G.H., Twarog, J.R.W., Ukeles, R., 1984. Influence of chemical composition of algal food sources on
growth of juvenile oysters, Crassostrea Õirginica. Biol. Bull. 167, 251–263. Wikfors, G.H., Ferris, G.E., Smith, B.C., 1992. The relationship between gross biochemical composition of
cultured algal foods and growth of the hard clam, Mercenaria mercenaria L. Aquaculture 108, 135–154.
