Glycerol Utilization
ABSTRACT
Oxygen respirometry proved to be a useful means of evaluation
for the effects of glycerol on the cells of Anthopleura
xanthogrammica. The utilization of glycerol was demonstrated
both qualitatively and quantitatively. An experiment using
(U-17C) glycerol did not show evidence for direct or inductive
utilization of glycerol. Comparisons made with Anthopleura
elegantissima using data from similar experiments performed by
Harvie indicated that the role of glycerol in both Anthopleura
was similar.
KEY WORDS: Anthozoa - Anthopleura xanthogrammica - 02
consumption - glycerol
Glycerol Utiliza
INTRODUCTION
Much of the work to date dealing with symbiotic sea anemones
concerns the role of symbiotic zooxanthellae in Anthopleura
elegantissima. These algae, thought to be locked among the
endodermal cells of the host, grow slowly and produce more photo¬
synthate than they can utilize. Some of the excess can be released
by the algae. Earlier experiments have shown that the zooxanthellae
of A. elegantissima fix +00 in vitro and release 50% of the
fixed 1 to the medium chiefly as (40) glycerol, (17) glucose,
and other organic acids (Muscatine, 1967; Trench, 1971,a,b).
Later experiments have shown that +002 is actually translocated
in vivo from the zooxanthellae to host tissue principally in the
form of animal lipid and protein. Deacylation of the lipid
C) glycerol (von Holt and von Holt, 1968a; Muscatine
yielded (
and Cernichiari, 1969; Trench, 1971a). This symbiotic role is
probably a nutritional one. Symbiotic A. elegantissima, when
starved in the dark, lose weight at a faster rate than controls
starved in light. Algal photosynthate translocation would
therefore seem to offset weight loss in the host (Muscatine, 1961a).
The studies in this paper deal specifically with the effects
of glycerol upon 0 consumption in symbiotic (and aposymbiotic)
A. xanthogrammica. Very little previous work has dealt with the
symbiotic role in this sea anemone. For a first time some effects
of glycerol upon the Og consumption by A. xanthogrammica are
revealed, and a comparison with similar studies on A. elegantissima
are made.
Glycerol Utilization
MATERIALS AND METHODS
I. Collection and maintenance of specimens
Large symbiotic A. xanthogrammica were collected without
damage from the lower intertidal zone at Point Joe along the 17
Mile Drive, Del Monte Forest, California. Aposymbiotic specimens
were collected from under the cannery in Pacific Grove next to
Hopkins Marine Station in California.
2. Cell preparation, protein and chlorophyll measurements
After four weeks from the time of collection, a longitudinal
section was cut from the animal. The section was then finely scis-
sor minced and suspended with 0.45 micron millipore sea water.
The supernatant, composed of whole cells and mucus, was centrifuged.
The centrifuge supernatant of suspended mucus was discarded.
The centrifuge residue of whole cells was kept. Coarse tissue
from the previous suspension was centrifuged and residues returned
to scissor mincing procedures. This was repeated until final whole
cell residue was of an adequate volume.
Next the residue was washed with the millipore sea water
and centrifuged three times to thoroughly remove particulate
food sources. The cell preparation was then placed in a depletion
bath. A Warburg respirometer was used as a bath set thermostat-
ically at 25° C and allowed to shake with a 1.5 cm stroke length
at a rate of 104 shakes per minute. After various periods of
incubation—- 48, 72, 96 hours, the stock of cells was washed
three times as before in order to remove lysed cell food sources.
Glycerol Utilization
The final stock was then ready to be used in part for Warburg
respirometry, protein, chlorophyll, and dry weight determinations.
The remaining stock was returned to the depletion bath until the
next period 24 hours later. The Lowry method for protein determin-
ation was used (Lowry, 1951). An acetone extraction of
chlorophyll, followed by centrifugation, and cubette spectro¬
photometry was performed on each cell sample. The milligrams
of chlorophyll'a'present was determined according to procedures
obtained from personal communication with J. Phillips, professor
at Hopkins Marine Station. All quantitative measurements were made
from averages of three duplicates each in turn averaged from five
readings.
3. Warburg Respirometry
Millipored sea water was added to 0.5, 1.0, 1.5 ml of cell
preparation to bring all Warburg flasks to a constant volume of
1.5 ml. The flask side arm contained 0.2 ml of 1.4 x 10" M glycerol,
enough to allow for a period of utilization equal to 20 ul of
oxygen. Otherwise the side arm contained 0.2 ml of 1 M glycerol¬
essentially an unlimited amount. The center well contained 0.2 ml
of 208 KOH and a wick. Total volume for each flask was 1.9 ml.
The Warburg was set at the same shake rate and temperature as was
the depletion bath and an air gas phase used. After thermoequil-
ibration, readings were taken at 15 minute intervals for 8 hours
and the rate of oxygen consumption in microliters of 0 per
milligram protein per hour was calculated from the period during
which the rate was constant with time.
Glycerol Utilization
4. Utilization of (U-170) glycerol
Animal cells were depleted for 72 hours in the dark, washed
three times every 24 hours, and 1 ml samples used in the Warburg
flasks. Three duplicates of symbiotic cell preparations were
run with the light intensity kept constant. In order to prove
whether glycerol was directly or inductively used by the
animal cells, 0.2 ml of 1.4 x 10— M glycerol in the side arm was
spiked with 2000 DPM of (U-176) glycerol (50uCi of U-1 glycerol
was obtained from Amersham/Searle). A 0.07 ml amount of B-phenyl-
ethylamine, completely wick absorbed, was used as C0 absorbant.
After allowing four hrs. for the utilization of glycerol and return
to the endogenous rate, the wick was removed and placed in a
scintillation vial, aquasol was added, and counts were taken in a
scintillation counter.
5. Cell preparation microscopy
The cell preparation was examined under the microscope
under 450 power.
Glycerol Utilization
RESULTS AND DISCUSSION
The study was divided basically into a qualitative and quant-
itative approach. More experimention using final procedures and
techniques is necessary before all of the conclusions in this
paper can be statistically verified.
From first experiments the maximum non-depleted endogenous
rate of 0, consumption was estimated. The amount of protein used
in each Warburg flask had to be diluted down until the O2
consumption rate did not exceed the 0» liquid diffusion rate.
Using 90 ugm of protein a mean rate of 48 ul/hr/mg protein was
obtained. Comparisons were made with whole animal 0 consumption
rate estimates from two sources. One upper fugure for
A. xanthogrammica (symbiotic) is 2 x 10° ul/hr/mg wet wt. at 10'C
(F. Belick, 1968). This estimate was derived from Verduin methods
of respirometry believed to be 80% accurate (J. Verduin, 1963).
Using a 8- 2, wet wt./dry wt.- 5, protein/dry wt.= 0.1 a wet
wt. rate at 10'C can be converted into a protein wt. rate at 25°0.
Using Belick's estimated rate and these experimentally determined
conversions (Q, was assumed to be equal to 2), one obtains a
rate of 3 x 10° ul/mg protein/hr at 25 C. This figure is 60 times
the maximum rate for cell 0» consumption described in this paper.
R. Shaver, using the Warburg, obtained an upper rate of 0.2
ul/hr/mg dry wt. at 10'C. Similar conversions show that cell
consumption is 800 times the whole animal consumption rate. The
discrepancy remains until further work determines whether surface
area availability to 0 is the determing factor in the rate of
Glycerol Utilization
respiration or whether the integrity of the tissue is the determin-
ing factor.
The preparation of A. xanthogrammica was observed microscop-
ically to be mostly whole cells 7u in diameter, which were stained
with methylene blue. A diameter measurement of 12u was observed
for zooxanthellae.
The next set of experiments were concerned with the depletion
of cell reserves. The temperature of the bathing medium was
chosen to produce the highest metabolic rate, therefore depletion
rates, without harming the tissues symbiotically (V. Buchsbaum, 1968).
It was suspected that with a lower endogenous rate of consumption
the effect of glycerol addition would be more noticeable. Before
48 hours the endogenous rate was too high to notice the effect
of glycerol addition, and after 96 hours most of the cells were
dead. The percent increase in the rate of 02 consumption after
the addition of 0.2 ml of 1 M glycerol was used as an index of
relative depletion. As seen from Table 1 and Graph 1 the most
rapid depletion occurs between 48 and 72 hours.. This depletion
curve is very similar to one made by Harvie for symbiotic A.
elegantissima. Depletion could have been accelerated if animal
cells were kept in the dark. Throughout most of the experiments
the light was not controlled.
A small enough amount of glycerol was added such that the
previous endogenous rate returned after substrate utilization.
From the balanced equation of the oxidation of glycerol into
COand H,O it was calculated that 0.2 ml of 1.4 x 10- M glycerol
Glycerol Utilization
would be needed in order for the utilization of glycerol by the
animal tissues to require about 20 ul of 0». Experimentation
demonstrated the expected effect of glycerol well (see Graph 2).
More duplicates of this effect are needed. There appears to be a
lag time after the addition of glycerol until the time of utili-
zation. This lag time of about 1-1.5 hours is too long a time
to account for diffusion of the substrate into the cells. A
better hypothesis is that the effect of glycerol may be an inductive
one. One should also note from Graph 2 that not only are the
endogenous rates the same before and after the utilization of
glycerol, but also the percent increase in 0, consumption from the
endogenous rate to the glycerol utilization rate is about 200%.
This figure is the same figure obtained for 96 hour depletion
figure in Table 1.
Graph 2 compares quite closely with a very similar graph
made by Harvie using A. elegantissima. The mean endogenous rate
Harvie obtained for A. elegantissima, depleted 96 hours, was
compared with the mean endogenous rate for A. xanthogrammica,
also depleted 96 hours. The two rates were standardized for
protein and zooxanthellae amounts, the latter evaluated by
chlorophyll A measurements. For A. elegantissima the result
was 3.9 ul 0/hr/mg protein/mg chlorophyll A. For A. xanthogrammice
the result was 4.3 ul 0/hr/mg protein/mg chlorophyll A. These
two figures suggest that these two Anthopleura consume 02 at
nearly the same rate after 96 hours depletion.
glycerol Utilization
Aposymbiotic hosts occur naturally in dark environments.
After seven weeks in darkness, A. elegantissima algae became
degenerate and plasmolyzed and decreased in number by 80%
(Muscatine, 1974a).
A few experiments were run using aposymbiotic A. xanthogrammica.
It was determined from chlorophyll A/protein ratios that the
bleached specimens contained about 50 times fewer zooxanthellae
than the pigmented specimens used in earlier experiments.
Chlorophyll A is directly related to zooxanthellae counts, because
chlorophyll A is one of the diagnostic pigments in zooxanthellae
and not in zoochlorellae (Muscatine, 1974).
Qualitative respirometry demonstrates that glycerol has a
similar effect with symbiotic cell preparations as with apo¬
symbiotic cell preparations. Depletion rates suggest that the
aposymbiotic issue deplete sooner and faster than symbiotictissues,
but more work needs to be done. Work with aposymbiotic A.
elegantissima has not been done to this effect yet.
Little success was obtained using (U-170) glycerol.
Accounting for background counts, only a 10% recovery of the
total spike counts was measured from the wicks of three sarples.
Now it seems obvious that the base saturated wicks could not
absorb from the air C carbonate ions known to be in the liquid
medium. Acid has to be added to drive off the hot carbonate ions
as 0, before recovery counts can be measured.
Experiments need to be done to assess the contribution or
e
Glycerol Utilization
consumption of 09 by the zooxanthellae. Until such data are
collected the effects of glycerol on 0» consumption by the
animal cells are not complete.
10
Glycerol Utilization
11
ACKNOVLEDGEMENT
I would like to thank Dr. Phillips and Dr. Fuhrman for
their invaluable assistance in conducting the research outlined
within this paper. Above all, thanks goes to Chris Harvie for
his help in making the Warburg happier. I would also like to thank
the entire class and faculty for making my experience at Hopkins
such a rewarding one.
12
Glycerol Utilization
REFERENCES
Belick, F. P. (1968). Metabolic rates in certain sea
anemones. Turtox News, 46: 178-181.
Buchsbaum, V. M. (1968). Behavioral and physiological
responses to light by the sea anemone Anthopleura
elegantissima as related to its algal endosymbionts.
Ph. D. Thesis, Stanford University.
Dixon, M. (1951). Manometric Methods, 3rd ed., Cambridge
University Press.
Lowry, O. H. (1951). Protein measurements with folin phenol
reagent. J. Biol. Chem., 193: 265-275.
Muscatine, L. (1961a). Some aspects of the relationship
between a sea anemone and its symbiotic algae, Ph. D.
Thesis, University of California, Berkeley.
Muscatine, L. (1967). Glycerol excretion by symbiotic algae
from corals and Tridacna and its control by the host.
Science, 156: 516-519.
Muscatine, L., and Lenhoff, H. M. (1974). Coelenterate
Biology Review and New Perspectives, Academic Press,
Inc., 376-389.
Trench, R. K. (1971a). The physiology and biochemistry of zoo-
xanthellae symbiotic with marine coelenterates. I. The
assimilation of photosynthetic products of zooxanthellae
by two marine coelenterates. Proc. Roy. Soc., Ser B 17
225-235.
Trench, R. K. (1971b). The physiology and biochemistry of
zooxanthellae symbiotic with marine coelenterates. 11.
Liberation of fixed 140 by zooxanthellae. Proc. Roy. Soc.,
Ser. B 177: 237-250.
Umbreit, W. W., Burris R. H. and Staufer, J. F. (1972)..
Manometric and Biochemical Techniques, 5th ed.
Eurgess Publ. Co., Minn.
e
13
Glycerol Utilization
Verduin, J. (1963). Simple measurements of respiration and
photosynthesis in aquatic organisms. Turtox News, 41:
234-237.
von Holt, C. and von Holt, M. (1968a). Transfer of photosynthetic
products from zooxanthellae to coelenterate hosts.
Comp. Biochem. Physiol. 24: 73-81.
Glycerol Utilization
FIGURE CAPTIONS
Table 1. Depletion in symbiotic Anthopleura xanthogrammica.
These figures represent the mean of several samples
which were treated with 0.2 ml of I M glycerol.
Depletion in symbiotic Anthopleura xanthogrammica.
Graph I.
The percentage increase in the 0 consumption rate
seen in Table 1 was used as an index for relative
depletion.
Graph 2.
Quantitative addition of glycerol to symbiotic
Anthopleura xanthogrammica after 96 hours depletion.
This is an expanded portion of a graph and does not
contain all of the data points recorded over a 10 hr.
period. Complete utilization of glycerol by the tissues
requires 20 ul of 09. The numbers along the three
segments of each line represent ul/hr/mg protein.
The three lines from bottom to top represent 0.5, 1.0,
and 1.5 ml samples.
14
C
Glycerol Utilization
Table 1
RATE OF O2 CONSUMPTION
(ul/hr/mg
After
HOURS
Before
Glycerol
Glycerol
48
10
2.4
72
6.4
4.2
13
PERCENTAGE
INCREASE
10%
160%
200g
Glycerol Utilization
Graph 1
200
100
o.
50

25
50
HOURS
75
100
110
100
90
80
70
60
k
50
40
30
20
10
124
R

e


2846



20p1 /35
140
150
88
HOURS


20u

1.5m

1K
Mim

O.5m
12
0