Uptake of C+0, and C glycine by
zooxanthellae and Anthopleura elegantissima
Richard M. Leonard*
Abstract--1. C+0, and C-glycine are both incorporated into the
free amino acids of Anthopleura elegantissima and its
zooxanthellae.
2. C+ is incorporated in greater concentration by
zooxanthellae than by Anthopleura elegantissima
Introduction
The uptake of C labelled substances and the subsequent
transference of C+ labelled photosynthetic products by endozoic
zooxanthellae to their coelenterate host has been demonstrated
in the Zoanthid Zoanthus flosmarinus (von Holt and von Holt 1968a).
In subsequent study if was shown that the endozoic zooxanthellae
were unable to synthesize their own glycine when given COg or when
given serine which is the immediate precursor in the glycine synthetic
pathway (C. von Holt 1968 ). Using " radio-tracer" methods it was
shown (C. von Holt 1968 ) that zooxanthellae preferentially
incorporated over other amino acids C
labelled glycine into their
amino acid pools, indicating a possible specific metabolic interaction
in the Zoanthus-zooxanthellae symbiotic relationship. These observations
prompted a study of the dynamics of CO, and glycine uptake in the
Actinaria Anthopleura elegantissima to see if there was any similarity
to the dynamics of Zoanthus flosmarinus.
Materials and Methods
Specimens of the experimental animal Anthopleura elegantissima
were collected from the intertidal region of Mussel Point in Pacific
Grove California. Oral discs of all the specimens were between 3 and
4cm in diameter at maximum expansion. Animals were kept in running
seawater prior to experiments. The isolation procedure of the
zooxanthellae from the coelenterate was that of von Holt and von Holt
(1968a).
*Present address: 2891 Dellwood Dr. Lake Oswego, Oregon 97034.
Glycine and Anthopleura elegantissima
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For incorporation experiments three organisms of
Anthopleura elegantissima were allowed to settle onto the inside of
lids of 50ml glass jars. The jars were each filled with 40ml of fresh
seawater, to which was added lmicroCurrie of respective C source
(either Nac-0, or C+-glycine (Universal Label-New England Nuclear
Corp.)). The lids containing the coelenterates were put on the jars
and the jars were inverted and placed under a bank of flourescence
lights (150 foot-candles) and incubated for four hours. After
incubation the animals were washed in fresh seawater and the algal
and animal cells separated as described by von Holt and von Holt
(1968a). After separation both the animal and the algal fractions
were boiled in 250ml of 85% ethanol for two hours. The insoluble portion
was removed via centrifugation, the soluble portion was mixed with
200ml of petroleum ether, and the aqueous layer was removed and
reduced in volume by boiling to 5ml. This I called the "amino acid
fraction! An aposymbiotic specimen of Anthopleura elegantissima was
also incubated under the same conditions and its "amino acid fraction
was similarly extracted.
Zooxanthellae isolated from three specimens of Anthopleura
elegantissima of the 3-4cm size were incubated in 40ml of fresh sea¬
water as described. An "amino acid fraction" was prepared in the
manner described.
250 microliters was taken from each of the "amino acid fractions
added to 10ml Aquasol universal liquid scintillation cocktail (New
England Nuclear Corporation,mfg.) and counted in a Unilux II
scintillation counter for one minute. 250microliters of each "amino
acid fraction" was dried and weighed. For each sample a ratio of C
activity to weight of dry "amino acid fraction " was determined,
expressed in units of microci/gram. This ratio was then used as a
means of comparing the C uptake of different " amimo acid fraction'
samples.
Results
The results are summarized in table 1. Anthopleura
elegantissima incubated in seawater in the presence of labelled carbonate
incorporates C+0, into the "amino acid fraction " of the algal cells
as well as the animal cells( Table 1,samples 1 and 2 ). In samples
1 and 2 the zooxanthellae incorporated more C+ than the host
Anthopleura elegantissima. C -glycine was also incorporated into
Glycine and Anthopleura elegantissima
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the algal cells and into the animal cells (Table 1, sample 3).
Sample 3 also incorporated more label into the algal cells than into
the animal cells.
Discussion
Endozoic zooxanthellae are widely distribued in marine
organisms particularly in coelenterates (Droop, 1963). That the
zooxanthellae release organic material into the incubation medium
under in vitro conditions has been demonstrated by the work of
von Holt and von Holt (1968a,b) and by Muscatine( see Muscatine 1974).
In von Holt and von Holts work with Zoanthus flosmarinus (1968a,b)
the in vivo incorporation of photosynthetically assimilated C
into the animal matrix was evidence that the coelenterate host
utilized products released by the zooxanthellae. My results may
indicate similar in vivo utilization by Anthopleura elegantissima.
Of particular interest is the comparison of C 0, and C-
glycine uptake. In samples 1 and 2 the ratio of C incorporation
in the algal cells and animal cells is approximately 1.4:1 whereas
in sample 3 the ratio is approximately 5:1. The greatly increased
ratio resulting from C-glycine uptake may result from the
zooxanthellae in Anthopleura elegantissima being unable to synthesize
their own glycine and thereby incorporating it in large concentration
when available. CO, enters through the animal tissue, goes to the
algal cells where it is acted upon photosynthetically and some of
the labelled photosynthetic products are released back to the
coelenterate host. When Cglycine is the
source the photosynthetic
abilities of the algal cells are not needed for conversion and
inorporation of(
" into the algal and animal cells. This fact, the
fact that the C source must go through animal tissue to be
incorporated by the algal cells, and the 5:1 C+-glycine uptake ratio
previously eluded to may indicate that glycine occupies an important
role in Anthopleura elegantissima symbiosis.
The metabolic relationship between Anthopleura elegantissima
and its endozoic zooxanthellae is obviously complex. Moreover there
may be important differences in the metabolic activities of the
zooxanthellae isolated from different Anthopleura elegantissima, as
well as metabolic differences in the host. The dependence of the
algal metabolism on glycine from Anthopleura elegantissima would play
an important role in the symbiosis of Anthopleura elegantissima and
its zooxanthellae.
Glycine and Anthopleura elegantissima
-4-
References
Droop M.R. (1963) Algae and invertebrate in symbiosis
Symp. Soc. gen. Microbiol. 13, 171-199
Muscatine L. (1974) Coelenterate Biology Reviews and New Perspectives
pp. 376-389, Academic Press, New York.
von Holt C. and von Holt M. (1968a) Transfer of photosynthetic
products from zooxanthellae to coelenterate hosts. Comp. Biochem.
Physiol. 24,83-92
von Holt C. and von Holt M. (1968b) The secretion of organic
compounds by zooxanthellae isolated from various types of Zoanthus.
Comp. Biochem. Physiol. 24,73-81
von Holt C. (1968) Uptake of glycine and release of nucleoside¬
polyphosphates by zooxanthellae. Comp. Biochem. Physiol. 26,
1071-1079
Acknowledgements
Thanks to everyone involved with 175H, it was a great quarter.
Special thanks to Robin Burnett for his help and patience.
sample +
Glycine and Anthopleura elegantissima
Table 1
cell source C
source C+ activity
cell type
.o026
animal
symbiotic
co
.0038
co
plant
symbiotic
00
animal
symbiotic
.0015
plant
co
.0020
symbiotic
0044
animal
symbiotic
glycine
symbiotic
plant
glycine
0213
isolated
from
plant
00
.0032
symbiotic
co
aposymbiotic
.O010
animal
isolated
from
plant
symbiotic
co
.0033
isolated
from
.0107
glycine
plant
symbiotic
e
Table 1.
Glycine and Anthopleura elegantissima
-6-
Caption for table
+ activity of Anthopleura elegantissima
and zooxanthellae (microCurries/gram of
dried amino acid fraction").
0