Fryefield
ABSTRAC
Different areas of anemone were found to have differen
relative amounts of chlorophyll-a, and differential apparent
division rates of zooxanthellae.
Pheophytin-a was used as an
assay of compounds released to anemone tissue due to zooxan-
thellae degeneration. Pheophytin appears to be periodical
accumulated in the anemone tissue, indicating a possible use
chloroplastic material from lysed cells as a nutritiona
source
Pes.
CTIOE
The investigation of the benefits received by the host
anemone in its symbiotic relationship with the zooxanthellae,
intracellular and intercellular dinoflagellates, has been
limited to the transfer of organic compounds between the
anemone and viable populations of the algae. Trench (1971
demonstrated that zooxanthellae in vitro release photosyn-
thetically fixed 'C to the host tissue in the form of glycerol
alanine, glucose, and organic acids, without lysis of the alga
1968) and ammonia (Kawaguti, 195
cells. Phosphate (von Holt,
ransfer in corals have also been studied.
It is known (Huscatine, 1974) that anemones sustain their
zooxanthellae in a steady-state, with new cells being produced
and degenerate cells being extruded in the mucus (Taylor, 1968)
These degenerate cells have enormous potential for the anemone
a source of organic and inorganic nutrients. Tonge (1975)
reports that the giant clam Tridacna gigas transports its
symbiotic algae from the sunlight to the inner tissues wher
they become senescent. The senile algae then enter the blood
stream where they are digested, releasing their nutritional
benefit to the host.
This paper will investigate the possibilit
f a similar behavior between the sea anemone Anthopleur
xanthogrammica and its zooxanthellae.
According to Yonge (1930), coelenterates do not diges
their algal symbionts, so the investigation for farming
behavior must consider possible nutrients derived from degen-
Fryefield Pag
senescen
Further evidence
ooanthellae.
ained from a description of the movement of the zooxanthe
ithin the anemone.
ATERIALS AED METIC
1 anemones were collected intertidally from Hopkins
larine Station. In the field,
were oriented such that
they
each received approximately the same amount oi
sunlight, and
all except anemone fi were collected at about 9:00 A.M. of the
day
they were to be assayed. Anemone
remained in an aquar
ium with less than normal light for one week befor-
bein
assayed
Assays for chlorophyll-a and pheophytin-a were
conduc
anemones i through 45 by homogenizing separately
the
oral disc, uppe
tissue
tentacles, mesenteries,
lovin
extracted with ace
vere
column, and middle columm.
Pigments
readings were made at the appropriate wave
density
optical
lengths
on a Unicam (model SP600) spectrophotometer. Amoun
gment were determined by the formulas stated in Stricklan
Parsons (1968).
the variot
ivision rates were determined
macerating
ue areas in M acetic acid and counting
the number
algal cells per 100 cells in a random field. M
lividir
distinction was made between senescent or degenerate cel
and viable cells in the
16 through 710 wer
count.
Anemone
these observations
Pa
field
Cultured zooxanthellae were used to determine the protein
content of a single algal cell. The zooxanthellae were
collected by centrifugation of homogenized anemone tissue
and cultured in enriched seawater medium. Protein determin-
ation was performed by the Lowry method (1951). Population
counts of the cultures were made with a hemocytometer.
Chlorophyll determination of a zooxanthellae population
was performed by acetone extraction, after collecting the alga-
centrifugation of anemone homogenate.
They were washed
in sterile seawater and recentrifuged five times.
Populati
counts were again made in a hemocytometer.
The amounts of chlorophyll-a vary greatly between different
areas of the anemone. The results of the chlorophyll assay
are summarized in Table 1 and Figure 1. Tentacles, mesenteries
and middle column have statistically distinct mean relative
values (student's T test, p C.05), while the oral disc an
apper column are nearly equal.
Pheophytin-a is magnesium-free chlorophyll-a. It is the
form in which chlorophyll is released from algal cells. The
pheophytin-a/ chlorophyll-a ratios are in Table 2 and Figure
can be seen to exhibit a wide range of values, even when
converted to relative terms. The oral disc shows a rather
tight distribution, while all other tissue parts have larg
efield
andard deviations, close to the mean values
The apparent rates of division for each tissue are shown
in Table 3 and Figure 3. Statistically, the mean tentacle
value is distinctly lowest and the mean upper
column value
is distinctly highest (student's T test, p K.05). The mese
teries, oral disc, and middle column have very similar mear
division rates.
Zooxanthellae were found to have 2.5x10 mg of chlo
yll-a per algal cell. In addition, 2.15mg of pheophytin-a
was found in the same population which contained approximately
5210
zooxanthellae. Cultured zooxanthellae were determined
37.9x10 mg of protein per algal cell.
have
Observations were made during the course of this inves
ation regarding the extrusion of zooxanthellae from the
anemone. Muscatine (1974) reports periodic extrusion of
ucus containing zooxanthellae from the mouth. Possible
urces for these zooxanthellae are the mesenteries, as rep
Taylor (1968), and the large mass of zooxanthellae
bserved in the lumen of the tentacles.
JUSSI
The results which were derived from this research lend
to a variety of interpretations regarding algae
themselves
xanthogrammica. Most of this discrepancy is
farming in A.
due to the wide range of values for the pheophytin-a/ chlorc
e
treat each of these
y in the following discussion.
First, consider the chlorophyll-a assay for the various
tissue areas. From the reports of Bogorad (1962), these
relative amounts give a reasonable indication of the number
ooxanthellae in the various tissues.
These amounts remain
relatively stable throughout the five anemones.
This stabil.
s probably not due to the time of day at which the anemones
ere collected and assayed, because anemone Fi was kept ir
han normal light for a week and still compare
wel
mean values.
The division rates give some idea as to the movemen
zooxanthellae within the anemone. These are apparent
division rates, and are not meant to suggest that the alga
ells are necessarily dividing any more rapidly in any portion
Rather, these division rates give a measur-
of the anemone.
the viabilit
of the population of zooxanthellae'in tha

tissue with a high percentage of senescent
ea, since an
ls will show a low percentage of dividing cells.
gal
This suggests that the mass of zooxanthellae in the lumen
the tentacles may actually be senescent cells in the
rocess
are the area of loe
being extruded, as the tentacles
ate of division.
There are two possibilities for these differential divisi
First is that the zooxanthellae in each tissue
part
ates.
transported to any other area within the anemone
The
ryef
d Pag
divide in the tissue and are accumulated there until
sin
xtrusion takes place directly from that tissue. The differen
ial division rates are thereby caused by differential accum-
ulation rates. Alternatively, the senescent zooxanthellae
may be constantly transported from the upper column, middle
column, and oral disc to the tentacles and mesenteries for
legeneration and extrusion.
In either case, the lack of variation
suggests that the movement and extrusion is a continual proces
the most part, and any periodic extrusion involves an
amount of zooxanthellae which does not significantly affec
rom which periodie
the division rates. The most likely area
extrusion could take place without affecting the divisi
rate is the tentacles.
The mean pheophytin-a/ chlorophyll-a ratios show
negatite correlation with the division rates, as shown in
This is to be expected, as a population with
igure 4.
igh amount of senescent zooxanthellae would show a low
division rate and a high amount of degenerate cells, hence
a high pheophytin concentration. Furthermore, the negative
correlation indicates that the pheophytin-a present in the
Issues is not caused by a continual leakage from the algal
cells, which would occur in the case of a turnover of chlord
phyll in those cells.
There is an apparent contradiction, though, because th
pheophytin-a/ chlorophyll-a ratios show wide variances which
e not reflected in the division rates.
This sugge
Pag
fie.
iodic, rather than continual, extrusion. If the ratios ar
determined instead by an accumulation of the pheophytin-a
itself which has been released from
degenerating zooxanthellae,
the fluctuations would have no effect upon the apparent division
rates. With the small number of trials, it is possible that
luctuations in the apparent division rates do occur, but
were not observed in my five anemones. It does seem likely
though, that if the fluctuations are present, they would hav
become visible in at least one tissue part of one anemone,
simply on the basis of the wide occurrence of variance in th
pheophytin-a/ chlorophyll-a ratios.
The results indicate
therefore, that a high number of senescent cells creates:
latively high amount of pheophytin. But whereas the senes
entand degenerate zooxanthellae are continually
extruded,
causing the division rates to remain constant, the pheophytir
accumulated in the tissue and extruded periodically.
The pheophytin-a which is released by the degeneratin,
gal cells provides a good indication of other compounds
which are also released to the anemone tissue for possibl
Bonner (1950) reports that chlorophyll is stable in t
hloroplasts only when in association with other chloroplastie
materials, such as chloroplastic proteins. When the alga
legenerates, the chloroplast is the first organelle to lyse
ing its contents to the anemone. Thus, an accumulatior
pheophytin-a could indicate an accumulation of other
mpounds which would be of more use to the anemone.
Sine
yefield
chloroplast, a h
the zooxanthella is comprised mainl
of
ercentage of protein per cell is chloroplastic protein.
Th
zooxanthellae can be estimated to release as much as 15. me
f protein per milligram of pheophytin-a to the anemone.
he basis of the apparent accumulation of pheophytin-a, it
seems very possible that the anemone is receiving benefi
rom at least some of these algal products.
At the beginning of this investigation, I defined farm
behavior as involving the transportation of zooxanthellae
within the anemone plus some benefit to the anemone from
he digested or degenerate zooxanthellae. On the basis o
idence, the exploration of zooxanthellae transport coi
ident with the algal
life cycle is inconclusive, although the
lifferer
ial division rates suggest the possibility of some
such movement. On the subject of benefit to thé anemone;
eems fairly definite that pheophytin-a and some other
chloroplastic materials are being released to the anemone
Whether the anemone is using these products is unknown.
the pheophytin is being accumulated in the tissues as the
sults suggest, then it would seem likely that the anemone
in fact, using the products of degeneration. There i
ertainly a need for more work in this area to determine
onclusive.
y whether the anemone is able to make use
s degenerate zooxanthellae as a nutritional source
vefield
ACKVONLEDGNE
would like
appreciation to
eapres
r.
Phillips for
providing me with countless references and
gre
eal of his time and patience.
I would also like to thank
within the real.
Isabella A. Abbott for keeping my ideas
feasibility, for giving me more than a modicum of helpful
riticism on this paper, and especially
for encour
when I wanted
punt.
Fryefield
1962. Chlorophylls.
Pages 385-404 in R.
Sogorad
and biochemistry of algae.
win, ed., Physiology
Academic Press, N.Y.
nner, J. 1950. Plant biochemistry. Academic Press,
Y. 536 pages.
aguti, S. 1953. Anmonium metabolism of the reef eo
Okayama Univ. 1:171-176.
Biol.
O. H., et al. 1951. Protein measurements with f
Chem. 193:265-275.
phenol reagent.
J. Biol.
1974. Endosymbiosis of cnidarians and alga
tine,
359-395 in L. Muscatine and H. Lenhoff, eds.,
ages
Y: Reviews and
new perspectives
enterate biolog
mic
ess, N.Y.
D. H. and Parsons, T. R. 1968. A practi
ook of seawater analysis. Fish.
Res. Board Can
161.
311 pages.
1968. In situ studies on the cytoch-
ltrastructure of a symbiotic marine dinoflagelle
Biol. iss. U.K. 48:349-366
Mar.
The physiology and biochemistry o
nch, R. K. 1971.
zookanthellae symbiotic with marine coelenterates.
Liberation of fixed
C by zooxanthellae in vit
77.
237-25

Proc. Roy. Soc. Lond.
ong
age
Uptal
1968.
phosphates by zooxanthell
Comp.
Biochem.
26:1071-10
Studies on the physiol
corals.
1930.
With notes on the speed of
Digestive enzymes.
sient. Rep
Barrie
gestion by A. G. Nicholls.
Pages 59-81
Reef Exped.,
Vol.
1975.
Sci. Am
32(4):96
Giant clams.
C. M.
ryefield
CAP
IOr
TABI
Table One: Actual values
chlorophyll-a in milligrame
pigment per milliliter of tissue homogenate.
ble Two: Actual values of
the pheophytin-a/ chlorophyll
atio in milligrams of pheophytin per m.
ran
hlorophyll.
hree: Actual apparent division rates
ividing cells per 100 cell
Page
el
IGURE
IONS
Figure
Mean relative amounts oi
the
nemone tissues.
Standar
kets
Mean relative pheophytin-
chlor
ure
the anemone
Standard
tissues.
brackets.
gure 3: Mean relative division rates in
Standard deviation values are in bracket
ure 4: Relative
chlorophyll-a, pheophytin-a/ chlorophyl.
and division rate values in the anemone tissues.
Note
the negative correlation between the division rates ar
the pheophytin-a/ chlorophyll-a ratios.
one
enta
lese
idd
Columr
olum
lum
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yefie
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