0
ABSTRACT
Elysia hedgepethi were starved until free of
chloroplasts. As feeding resumed, re-establishment of the
animal-chloroplast symbiosis was monitered and found to rebound
to normal levels after five to seven days of feeding on Codium
fragile. E. Hedgepethi fed seven days on a diet of isolated C.
fragile chloroplasts did not accumulate chloroplasts
intracellularly to any significant extent.
INTRODUCTION
Prolonged retention of functional chloroplasts in
digestive gland tissues is a common phenomenon among the
Elysiid sacoglossan opisthobranchs (Trench,1975). Sacoglossans
feed on siphonaceous algae, slitting the algal surface with
specially adapted radulae and sucking the cytoplasm out fron
the long, multinucleated coenocytic filaments. Chloroplasts
are phagocytosed into the digestive epithelial cells, then
the phagocytic membranes break down and the chloroplasts
migrate towards the back of the cell, fully contiguous with
the animal cytoplasm (McLean,1976). Indications are that
whereas little or no protein or lipid synthesis occurs in
the translocated chloroplasts, the chloroplasts continue to
produce and release photosynthate into the animal cytoplasm
for extended periods (Trench,1975).
In contrast to many algal-animal symbioses, there is a
turnover of these chloroplasts in the animal, and continued
ingestion is necessary to maintain a standing crop
intracellularly. Hinde and Smith (1972) have shown Elysia
viridis to maintain functional chloroplasts through 60
days' starvation in the light, while Elysia hedgepethi
has been reported to be free of chlorophyll after 10 days
without food (Greene,1970b). How quickly can starved
chloroplast utilizers rebound to normal photosynthetic
capacity? Can the starved slugs regain photos
heti
on a diet of isolated Codium fragile chloroplasts
MATERIALS AND METHODS
Collection
Codium fragile containing Elysia hedgepethi was
collected subtidally near Santa Barbara, California.
C. fragile for feeding animals and for isolation of
chloroplasts was collected intertidally at Hopkins Marine
Station, Pacific Grove, California.
Starvation
E. hedgepethi maintained in a running sea water
aquarium (16°C) were starved for 18 days. For the first
15 days, their tank received indirect natural light. To
accelerate the loss of chloroplasts from the animal tissue,
flourescent lights were placed near the tank and kept on
continuously for the next 3 days.
Feeding
After starvation, twelve slugs of approximately the same
vellowish color were transferred to a beaker containing
filtered sea water and freshly collected fronds of C.
fragile. The feeding tank was aerated with a bubbling
stone and thermostated in a water bath at 16°0. The
water was replaced daily and the C. fragile semi-daily.
Three starved slugs were placed in a dish with filtered
sea water and chloroplasts isolated from 5 grams of C.
fragile by the method of Shephard, et. al., 1968. The dish
was kept in a 16° room and loosely covered with slitted
plastic wrap to minimize evaporation while permitting gas
exchange.
Assay of Photosynthetic Competence and Chlorophyll Content
Feeding slugs were removed from the C. fragile,
allowed 30 minutes to finish digesting their meal, and then
were blotted and weighed. Each animal was then incubated
for 2 hours in 1 ml of filtered sea water containing 10
uc; of +C-sodium bicarbonate at 16° under
flourescent lighting sufficient to saturate photosynthesis.
Slugs were then removed from the incubation medium and
homogenized in 1 ml methanol. The homogenate was bathed in
approximately 50° water for 10 minutes to extract the
chloroplasts fully, then centrifuged for 2 minutes in an
Eppendorf centrifuge at 12,000 g. The optical density at
663 nm was taken of the supernatant, and the chlorophyll
content determined from the equation ODg3 = 9.83X
[chlorophyll] (Dawson,et. al.,1986). The pellet was
resuspended in 1 ml .5 M NaoH with 58 Triton X and allowed
to dissolve for about an hour. The methanol soluble and
insoluble fractions of the animal were both acidified by
addition of trichloroacetic acid to 108 final concentration,
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in a scintillatio counte








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RESULTS
Observations During Starvation
Upon arrival the E. hedgepethi were dark green
with glittery silver speckles. Under a dissection microscope
the green digestive gland could be seen to permeate nearly
every corner of their transparent bodies. After 15 days
without food, the sacoglossans had faded from their original
deep color but were still a dull lime green. After three
days of illumination, many of the slugs were distinctly
yellow in color. The slugs appeared to lose weight on
their strict sea water diet. Both green (fed) and yellow
(starved) slugs were observed to be positively phototaxic.
In both natural and artificial light, most slugs could be
found near the water line on the brightest wall of the glass
tank. When the flourescent lights, normally illuminating one
entire wall, were changed to illuminate only the bottom half
of the wall, most of the slugs migrated down into the
intense light.
Time Course of Chlorophyll Levels
Optical densities of the methanol extracts at 663 nm
were converted to ug chlorophyll per mg body weight and
plotted versus time (figure 1). Chlorophyll content
increased with feeding time. After 5 days it was near
C
the equilibrium value.
Time Course of Carbon Fixation
Counts per minute (cpm) of the methanol soluble and
insoluble fractions and their sum were normalized to slug
body weight and plotted versus time (figure 2). Carbon
fixation per mg slug increased with feeding time, peaking
at 5 days and then tapering off to the equilibrium value,
Carbon Fixed Per Unit Chlorophyll
The average fixation was 91527 cpm per ug chlorophyll,
with a standard deviation of 40763 cpm per ug chlorophyll.
There was no discernable trend with time; the variations
appeared random.
Intact Codium Versus Isolated Chloroplast Diets
Assays of slugs fed for 7 days on the different diets
are compared in Table 1, with data again normalized to slug
body weight. Slugs fed isolated chloroplasts contained only
1.68 as much chlorophyll per mg as slugs fed intact plants
for the same period of time. Carbon fixation in chloroplast-fed
slugs was just under 108 that of plant-fed slugs.
DISCUSSION
Owing to their availability and the rapid turnover
rate of their chloroplasts, E. hedgepethi were chosen
to follow the time course of re-establishing photosynthetic
capacity in starved animals. Intense light apparently
accelerates the removal of chloroplasts from animal tissue.
This is consistent with Hawes and Cobb's 1980 report that
chloroplasts in E. viridis undergo photodestruction,
and that damaged chloroplasts are removed from the animal
cytoplasm (Trench,1979).Thirty minutes feeding, the first
time point, was essentially equivalent to no feeding, for
the animals had not enough time to get settled and sink
their radulae into the algae. Two hours forty minutes
apparently was also insufficient, for chlorophyll content
and fixation both dropped, presumably due to variance in
the degree of starvation in the E. hedgepethi population.
After one day of feeding, however, chlorophyll content
and fixation increased significantly. The constant amount
of carbon fixed per ug chlorophyll in the animals throughout
the experiment suggests that the initial chloroplasts are
taken up and maintained intact; they are not degraded to
pay for any metabolic debt caused by starvation. Both
chlorophyll content and fixation could be reasonably
construed as logarithmically rising curves. Chloroplasts
reappear in the animals several times faster than they
disappear.
The ratio of fixed carbon that was methanol soluble
to that which was methanol insoluble was fairly constant
10
slugs
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ing
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ontinued to starve during this period.
101
content was 10 times lower than
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solated chloroplasts.
TERATURE CI
Dawson, R.M.C., Elliott, D.C., Elliott, W.H., Kenneth, M.J.
1986. Data for Biochemical Research, p.232.
Greene, R.W. 1970a. Malacologia, 10(2): 369-80.
Greene, R.W. 1970b. Marine Biol., 7: 138.
Hawes, C.R., and Cobb, A.H. 1980. The New Phytologist, 84: 375-
79.
McLean, N. 1976. Exp. Zool., 197: 321-30.
Shepard, D.C., Levin, Wendy B., and Bidwell, R.G.S. 1968.
Biochemical and Biophysical Research Communications, 32: 413-20.
Trench, R.K. 1975. Symposia for the Society of experiment
Biology, 29: 229-37.
Trench, R.K., and Ohlhorst, S. 1976. The New Phytologist, 76:
99-109.
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FIGURE LEGEND
Figure 1: Chlorophyll content mass * versus feeding
time. 2 slugs per data point. Dashed horizontal line
represents equilibrium value (i.e., assay of slugs which had not
been starved).
Figure 2: Photosynthesis mass  versus feeding time.
2 slugs per data point. Squares = methanol-soluble
photosynthate. Diamonds - methanol-insoluble photosynthate.
Triangles - total photosynthate. Dashed horizontal lines
represent equilibrium values (i.e., assay of slugs which had not
been starved).
FIG. 1.
ug chlorophyll /mg elug

9 8
3

9 2
Fig. 2.
cpm /mg alug
(Thousands)
ONOONOOSSS

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