Kamel, L.C.
ABSTRACT
Foettingeria,aciliated protozoan, is an endosymbiote
which occurs in the gastrovascular cavity of two species
of Anthopleura, a sea anemone native to the California
coast. Its population size is limited by the size of
its host.
Foettingeria sp. has two locomotion modes, swimming
and crawling. Crawling appears to be part of the foraging
mode. Feeding is done using a cytostome. The ciliate
appears to feed on tiny algal particles as well as
zooxanthellae released from the host. While it may de-
rive nourishment from mucous secretions as well as
from gut fluids, there is no evidence that Foettingeria
sp. harms its host even at high concentrations.
INTRODUCTION
The cited cases of symbiotic relationships in¬
volving protozoa are well-documented (Sleight, 1973).
One association on which there is little information
is on the endosymbiosis of the ciliated protozoan
Foettingeria sp. and Anthopleura. The apostome
Foettingeria actinarium has previously been described
from various European anthozoans (Chatton and Lwoff,
1950). Based on morphological features, an undes-
cribed species of Foettingeria has been reported to
occur commonly in the coelenteron of the sea anemone
A. xanthogrammica in southern California (Ball and
Moebius, 1955).
Kamel, L.C.
This investigation will focus on four aspects of
Foettingeria commonly found in A. xanthogrammica and
A. elegantissima in the area around Monterey, California.
This study focused on an examination of the morphology
of the ciliated protozoan as well as certain aspects
of its biology. Locomotion and feeding were studied,
Its incidence within its host and along various
heights and exposure gradients were examined. Its
temperature tolerance range was described. Finally.
an attempt was made to describe the nature of the
observed symbiosis.
MATERIALS AND METHODS
Collection of anemones for field studies
Sea anemones were collected by hand using a hammer
and chisel to remove the substrate the anemone was on.
A. elegantissima and A. xanthogrammica were obtained
at low tide from points around Mussel Point and under-
neath Monterey Wharf #2 where sunlight did not penetrate.
More than one animal from a clone of A. elegantissima
was collected. Metridium senile was collected from floats
in Monterey harbor and Corynactis californica subtidally
at a depth of 55 feet. For the field studies, clonal
A. elegantissima was collected at heights of -O.6m,
Om, O.6m, 1.2m and 1.8m. In the same way, A.
elegantissima clones were collected from Agassiz and West
Beach.
Collection and culturing of ciliates
Ciliates were removed from anemones by dissection
Kamel, L.C.
of the relaxed anemones in an isotonic solution of mag¬
nesium chloride under a dissecting microscope. Ciliates
found in the anaesthetizing solution were removed by
eye pipette dropper and placed in sea water. The sea
water provided an adequaté culture medium for up to
four days after which ciliates tended to slow down and
die.
Structural identification studies
Examination of morphological features of Foettingeria
was done using phase microscopy of a Zeiss microscope.
Internal structures viewed using various staining
procedures were identified.
Experiments on stress tolerance to temperature and starvation
Populations of fifty ciliates each were placed in
each of 14 plastic vials containing millipore-filtered
sea water. These samples wereplaced in a bath of running
sea water at approximately 14°C to allow for temperature
equilibration. Two vials each were then placed on ice
and in water baths of 3, 9, 15, 21, 24, 30°0 for one
hour. After one hour passed, survival of the population
was estimated. Ciliates which were non-motile and showed
signs of gross structural abnormalties were judged dead.
Starvation studies were conducted with populations
of fifty ciliates each. These were placed in 4 vials
containing millipore-filtered sea water and into a water
bath maintained within + 0.2°0 of 14°0 for seven and
fourteen days. Survival was estimated and internal and
external structural changes noted.
Kamel, L.C.
Feeding experiments
Populations of fifty ciliates were placed in each
of 10 vials, half containing the particulate dye India
ink and half containing carmine red. After a one day
incubation, the ciliates were removed, washed in
millipore-filtered sea water, and placed under a com¬
pound microscope to look for incorporation of the dye.
I
UE
Examination of morphology and internal structure
Using phase microscopy, the ciliate appears as a
tear-drop shaped protozoan ranging in size from
approximately 50 u to 100 u. This size range is the
same as the Foettingeria sp. noted by Ball and Moebius
(1955).
The ciliate is concave in shape with its ventral
side invaginated. Morphologically, the most obvious
structure is a spiral band which wraps around the
protozoan and meets at the posterior and anterior ends
(Figure 2). This band is filamentous in form. On
these microfilaments are located tufts of cilia
uniformly arranged around the organism. At the pos-
terior end wherethe band of cilia meet is a cytostome
approximately 10 u in diameter. Aside from these
structures, any other feature is obscured by a yellow¬
tan cytoplasm which is uniformly shaped in the ciliate
(Figure 1). This, in turn, is surrounded by a clear
layer of ectoplasm ususally 10 u in thickness.
Kamel, L.C.
The use of organelle-specific dyes has uncovered
a few internal traits of the ciliate although the
amorphous brown pigment obscures many observations.
Among the internal characteristics is the observation of
similar nuclei ranging 23-32 in number. With congo
red dye these are arranged uniformly in the ciliate.
Also present in significant numbers are mitochondria.
These appear to be 1 u across and 1 to several u in
length. Upon lysis of the plasma membrane and analysis
of internal contents, one notices the presence of food
vacuoles. In the feeding mode these vacuoles contain
partially-dissolved algal particles. In addition to
the food vacuoles, there are present in numbers too
large to quantify vessicles approximately 1 u in diameter.
It is these vessicles which contain the yellow-tan
pigment observed in the intact organism.
Swimming and feeding behavior observations
Observations of Foettingeria in millipore-filtered
sea water indicates that the protozoan tends to ex-
hibit two modes of movement, crawling and swimming.
Crawling is defined here as movement on a particular
substrate. When the protozoan is placed in a solution
of Protoslo, a viscous solution which retards movement,
the action of the cilia can be observed. The cilia move
in close waves which can be seen sweeping back over the
surface of the body as the animal swims or crawls.
The direction of movement is changed by alteration in
the direction of the beat of the cilia, which coincides
Kamel, L.C.
with the direction of the movement of the wayes.
Feeding behavior was observed with two particulate
dyes. Using India ink, it is observed that there is in-
corporation of the dye into specific parts of the organism,
Upon cell lysis, these dye particles are observed incor¬
porated in food vacuoles.
Distribution studies
Distribution studies of Foettingeria sp. was
conducted on four different sea anemones: Anthopleura
elegantissima, both of the clonal and solitary types,
A. xanthogrammica, Metridium senile and Corynactis
californica (Figure 3). In the clonal A. elegantissima,
the rate of infection was particularly high with more than
eight out of ten animals infected with the protozoan.
The solitary A. elegantissima and A. xanthogrammica
did not show as high an infection rate as A.
elegantissima clones, but were infected in 58% of
the cases. In contrast, no animals of M. senile
or C. californica contained the ciliate.
In collections from the protected Agassiz Beach
and exposed West Beach of Mussel Point, a larger
number of ciliates were consistently found in A.
elegantissima and A. xanthogrammica from the more
protected area of Mussel Point (Figure 5). Foettingeria
were found throughout the gut of the anemone including
the tentacular gut extensions. These was a correlation
of the number of ciliates found in a particular host and
the size of its host (Figure 6). As host size increased.
Kamel, L.C.
the number of ciliates increased but leveled off at
an oral disc diameter of around 7 cm.
Stress tolerance studies
As seen in Figure 7, Foettingeria from the inter-
tidal rocks shows a sharply peaked temperature tolerance
rnage. The optimum range, a survival of 80% or higher.
occurred from 3 - 24°0. The survival dropped off quickly
on both sides of this range, however. Furthermore,
dark pigmented ciliates showed a wider temperature
optimum range than ciliates collected from aposymbiotic
anemones collected from under the Monterey Wharf.
Ciliates placed in starvation conditions showed
noticeable shanges after a one week period. After this
time there was still approximately 75% survival in the
population. Survivors, however showed bleaching of the
yellow-tan pigment they had begun with. After two weeks
of starvation, survival rate had decreased to 7%. Those
that did survive underwent encystment. Encystment was
characterized by a thickening of the clear plasma layer
surrounding the organism. Agitation of the cells in-
terrupts the encystment.
DISCUSSION
Movement and feeding studies
Movement by Foettingeria sp. appears to be one of
two types. Both of these types may have specific func¬
tions. The swimming mode may be used solely for loco¬
motion from one place to another. Crawling, on the
other hand, may be specific to foraging behavior. When
Kamel, L.C.
the ciliate is crawling along a particular substrate,
it may well be taking up particles for digestion.
The particulate dye experiments showing incorporation
of dye particles in food vacuoles is consistent with the
microscopic observation of a cytostome structure.
While pinocytosis does occur in ciliates, such as
Opalina ranarum, an opalinid protozoa found in frog
faeces (Corliss, 1955), feeding in Foettingeria
sp. appears to occur via the cytostome.
Distribution studies
From the experiments involving percent infection
of Foettingeria sp. in A. elegantissima and A. xan-
thogrammica and two other sea anemones, M. senile
and C. californica, many conclusions can be drawn.
It is possible that the difference in incidence is due
to the difference in physical factors at the collection
sites. It seems probable, however, that Foettingeria
sp. is specific to Anthopleura. In the life cycle of
F. actinarium, the ciliate encysts on a crab where it
is later ingested by a sea anemone (Lwoff, 1950). It
may well be that the ciliate encysts on an intertidal
crab taken in the die to intertidal Anthopleura.
This would be an interesting aspect for further inves-
tigation.
In the experiments investigating ciliate number
as a function of host size, it seems logical that the
number of ciliates would increase as its host size
increases. It is interesting that the number of ciliates
Kamel, L.C.
level off in the larger anemones but no explanation
is apparent.
tress test studies
The eifference in the temperature tolerances of the
pigmented ciliate and the translucent ciliate could be
explained by the difference in the arease in which they
were collected, Mussel Point versus underneath Monterey
Wharf. This would argue that environmental history of
the organism might be important in stress tolerance.
Another possibility may be that the pigmented ciliate
may be more temperature-hardy due to its diet -- one
that includes zooxanthellae. A third possibility is that
these two ciliates represented are genetically different.
however the bleaching of intertidal ciliates with
starvation as well a s transplantion studies need to
be done.
The starvation studies showing bleaching of pigment
after one week and cyst formation in a small sample
after two weeks provides some interesting interpretations.
The bleaching observed may be a result of the ciliate
using up stored food sources. Encystment may be the
extreme case when these stores are depleted. Cyst
formation after two weeks indicates that Foettingeria
sp. has the capacity to form two types of cysts. The
first is a reproductive cyst already outlined in the
studies of Chatton and Lwoff (1950) involving F.
actinarium. The second described here is a protective
cyst brought on by starvation conditions. This is not
10
11
Kamel, L.C.
unique to Foettingeria sp. Many protozoa, such as soil
amoeba Acanthamoeba (Neff and Neff, 1969), encyst in
response to starvation, dessication, overcrowding.
accumulation o f metabolic wastes or raised temperatures.
Experiments to determine under what conditions encystment
in Foettingeria sp. is reversible has yet to be carried out.
LITERATURE CITED
Ball, G. and Moebius, R. 1955. A Foettingeriid (Apostomea)
from the Sea Anemone Anthopleura xanthogrammica.
J. Protozool. 2 (suppl.), 2.
Corliss, J.O. 1955. The Opalinid infusorians: flagellates
or ciliates? J. Protozool. 2, 107-11.
MoLaughlin, J., and Zahl, P. 1959. Axenic zooxanthellae
from various invertebrate hosts. Annals, New
York Academy of Science 77, 55-9.
Lwoff, A. 1950. Problems of Morphogenesis in Ciliates.
Wiley, New York.
Neff, R. and Neff, R.H. 1969. The biochemistry of ameobic
encystment. Symp. Soc. Exp. Biol. 23, 51-9.
FIGURE LEGEND
Figure 1.
Photomicrograph showing top view of Foettingeria
sp. Filamentous band of cilia and food particles
in vacuoles (anterior end of animal) can be
identified.
Illustration of external structure (left) and
Figure 2.
internal structure of ciliate. External struc¬
ture shows bands of cilia and cytoxtome at pos¬
terior end of animal. Internal structure shows
the number of nuclei located in the cytoplasm.
Figure 3. Percent incidence of Foettingeria sp. in five
different species of central California coast
anemones.
Number of ciliates per anemone collected at
Figure 4.
various tidal heights along a vertical transect.
Anemones collected were A. elegantissima.
Figure 5. Number of ciliates found per anemone at
protected area (Agassiz Beach) and exposed area
(West Beach) of Mussel Point. Anemones collected
were A. elegantissima.
Number of ciliates as a function of diameter of
Figure 6.
anemone oral disc. Anemones sampled were of the
type Anthopleura collected under similar conditions.
Bars represent standard deviation from mean value.
Percent survival as a function of temperature.
Figure 7.
Samples of 100 ciliates were collected under
similar conditions and placed at temperatures of
0, 3, 9, 15, 21, 24, and 30°0 for one hour.
Squares represent dark-pigmented ciliates collected
from protected area of Agassiz Beach on Mussel Point,
Circles represent translucent ciliates collected from
anemones underneath Monterey Wharf.
9.
L
5

3
4
ooc
4
OOO
o

Fig. 2
80
60
F 40
E
20

PERCENT INCIDENCE IN FIVE CENTRAL
COAST ANEMONES
82
N=17
63
N=19
58
N=17
N=18 N=13
Animal
—
Tidal Height
-0.6m
—
0.Om
O.6m
—
1.2m
1.8m
Exposure Point
Agassiz Beach
West Beach

Mean Number of Ciliates per Anemone

29 (N = 6)
—
13 (N = 5)

—
23 (N = 6)

16 (N = 5)
—
(N = 7)
Mean Number of Ciliates per Anemone
21
(N = 7)
(N = 5)
9
Std.
6.7
3.2
—
54
—
9.6
0.0
Fig.
Std. Dey.
64
2.7
Fig.S
30
- 20
10

N=10
N=12
N-11
6
2
Diameter of oral disc
cm
+N=12
N=15
—
10 12
Po.6
TEMPERATURE TOLERANCE
100

60
40
20
a2
12
18
Temperature °0
24
100
80
160
40
20
14.7