5. Lipid Dietary Dependencies in Zooplankton
95
monoolein was substituted for oleic acid and egg albumin was omitted. In some
tests, an equivalent amount of oleic acid was substituted for the two EFAs to make
a "non-EFA" microcapsule, or microcapsules were synthesized exclusively from
the triglyceride triolein. Lipids were encapsulated by the procedures of Langdon
and Waldock (1981). The lipid mixture was emulsified in a 4% solution of gum
arabic (40°C) with a high-speed homogenizer. After heating and adjusting pH, the
microcapsules were refrigerated to harden the coats, then centrifuged and rinsed
twice, and finally autoclaved. Microcapsule concentrations were determined by
hemacytometer counts. The capsules averaged 2 /-Lm in diameter. Samples of
microcapsules were analyzed and estimated to contain, on average, 1 pg of free
fatty acids, by thin-layer chromatography with an Iatroscan flame ionization
detector system (Parrish and Ackman, 1983).
Protein was usually added as microparticles of casein. Casein is commonly
used in aquaculture because of its wide complement of essential amino acids. To
make microparticles, casein suspended in distilled water was initially dissolved by
elevating pH to 11.5 with 0.1 Eq . L - 1 NaOH, precipitated by lowering pH to 4.8
(0.1 Eq . L -I HCl), and then homogenized with a high-speed homogenizer. Particles averaged -10 /-Lm in diameter but ranged from <1 to >50 /-Lm. Protein
concentrations of the microparticle slurry were determined by the modified Lowry assay (Sigma Kit) after sonication. A protein microcapsule was also prepared
by following the methods of Langdon (1989) and Langdon and DeBevoise (1990)
but with casein substituted for the protein mix suggested by Langdon (1989).
However, we were unable to use these because they retained a slight toxic effect
on the daphniids.
Prior experiments in our laboratory (C.E.G. and J.N.M.) had demonstrated that
daphniids would ingest lipid microcapsules containing either TAG, EFA, or only
non-EFA (oleic and palmitic acids). In preliminary experiments, laboratory animals did respond to EFA microcapsules by increased reproduction, but they did
not respond to the same microcapsule containing comparable amounts of nonEFAs alone. The lack of response to the non-EFA microcapsule that contained
both monoolein and the vitamin additions indicated that the original response
to the EFA microcapsule was not a response to the monoolein or the vitamin
additions.
5.2.2. Dietary Supplement Experiments
Dietary supplement experiments were performed in the summers of 1988-1989
using Daphnia catawba from mesotrophic Lake Lacawac and Daphnia pulicaria
and Daphnia laevis from eutrophic Lake Waynewood. Both lakes are located in
Wayne County, in the Pocono region of northeastern Pennsylvania. Spring
(April-May) and fall (late September-October) populations of daphniids in these
two lakes average 20-30 individuals per liter. Summertime densities seldom
exceed 2-5 individuals per liter in Lacawac due to size-selective predation or
food limitation, or both (Tessier, 1986).
95
monoolein was substituted for oleic acid and egg albumin was omitted. In some
tests, an equivalent amount of oleic acid was substituted for the two EFAs to make
a "non-EFA" microcapsule, or microcapsules were synthesized exclusively from
the triglyceride triolein. Lipids were encapsulated by the procedures of Langdon
and Waldock (1981). The lipid mixture was emulsified in a 4% solution of gum
arabic (40°C) with a high-speed homogenizer. After heating and adjusting pH, the
microcapsules were refrigerated to harden the coats, then centrifuged and rinsed
twice, and finally autoclaved. Microcapsule concentrations were determined by
hemacytometer counts. The capsules averaged 2 /-Lm in diameter. Samples of
microcapsules were analyzed and estimated to contain, on average, 1 pg of free
fatty acids, by thin-layer chromatography with an Iatroscan flame ionization
detector system (Parrish and Ackman, 1983).
Protein was usually added as microparticles of casein. Casein is commonly
used in aquaculture because of its wide complement of essential amino acids. To
make microparticles, casein suspended in distilled water was initially dissolved by
elevating pH to 11.5 with 0.1 Eq . L - 1 NaOH, precipitated by lowering pH to 4.8
(0.1 Eq . L -I HCl), and then homogenized with a high-speed homogenizer. Particles averaged -10 /-Lm in diameter but ranged from <1 to >50 /-Lm. Protein
concentrations of the microparticle slurry were determined by the modified Lowry assay (Sigma Kit) after sonication. A protein microcapsule was also prepared
by following the methods of Langdon (1989) and Langdon and DeBevoise (1990)
but with casein substituted for the protein mix suggested by Langdon (1989).
However, we were unable to use these because they retained a slight toxic effect
on the daphniids.
Prior experiments in our laboratory (C.E.G. and J.N.M.) had demonstrated that
daphniids would ingest lipid microcapsules containing either TAG, EFA, or only
non-EFA (oleic and palmitic acids). In preliminary experiments, laboratory animals did respond to EFA microcapsules by increased reproduction, but they did
not respond to the same microcapsule containing comparable amounts of nonEFAs alone. The lack of response to the non-EFA microcapsule that contained
both monoolein and the vitamin additions indicated that the original response
to the EFA microcapsule was not a response to the monoolein or the vitamin
additions.
5.2.2. Dietary Supplement Experiments
Dietary supplement experiments were performed in the summers of 1988-1989
using Daphnia catawba from mesotrophic Lake Lacawac and Daphnia pulicaria
and Daphnia laevis from eutrophic Lake Waynewood. Both lakes are located in
Wayne County, in the Pocono region of northeastern Pennsylvania. Spring
(April-May) and fall (late September-October) populations of daphniids in these
two lakes average 20-30 individuals per liter. Summertime densities seldom
exceed 2-5 individuals per liter in Lacawac due to size-selective predation or
food limitation, or both (Tessier, 1986).