CHARACTERIZATION OF NOVEL NAKED AMOEBA ASSOCIATED WITH COASTAL CTENOPHORES (Mnemiopsis sp.)

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CHARACTERIZATION OF NOVEL NAKED AMOEBA ASSOCIATED
WITH COASTAL CTENOPHORES (Mnemiopsis
sp.) Margaret Wacera Mbugua, Andrew
Rogerson Biological Sciences, Marshall
University, Huntington, WV
ABSTRACT Coastal ctenophores (Mnemiopsis spp.)
also known as comb jellies harbor an undescribed
naked amoeba on their surface. Almost all the
ctenophore specimens examined from coastal
Florida had this associated amoeba (Versteeg,
pers. comm.). The nature of this symbiotic
association is unknown although there is
preliminary evidence from electron microscopy
suggesting that amoebae may be degrading the comb
plate surface of these planktonic grazers (Moss
et al., 2001). Since coastal ctenophores are
tolerant of a wide range of salinities, the
tolerance of the associated amoeba was also
studied. This provided useful information on the
nature of the association and information to aid
the identification of the amoeba species. Amoebae
were cultured in sea salt media (simulating their
natural environment) of varying salinities
ranging from 10g/l to 50g/l. Cell counts over
time were used to calculate generation times at
each salinity. Cell size (length and breadth
dimensions) and speed of locomotion was
determined by phase contrast microscopy. The
morphology of active cells (an important
diagnostic tool in identification) was determined
by light and scanning electron microscopy.
Amoebae, stained with the nucleic acid
fluorochrome acridine orange (stock solution 0.5
mg acridine orange in 10ml distilled water), were
imaged using confocal fluorescent microscopy to
determine nuclear number, shape and size. Results
showed that amoebae were unusual and probably new
to Science. Amoebae were also euryhaline,
surviving over the range 10 to 50 g/l salt (ocean
water is around 32 g/L). However, generation
times were highest at high sea salt
concentrations (reflecting slow growth) and the
velocity of amoebae was low at these high
extremes (40g/l and 50g/l) indicating that these
salinities were not optimal for the amoebae. Mean
length and breadth of the cells varied
inconsistently across the range of salinity
concentrations. To fully characterize this amoeba
and further explore the nature of the
association, molecular approaches and
ultrastructural studies (using TEM) will be
undertaken.
Introduction Ctenophores, also known as comb
jellies, are macroinverterbrates of the phylum
Ctenophora in the class Cnidaria (Fig. 1).
Ctenophores are important gelatinous grazers that
feed on plankton in the marine environment (Moss
et at, 2001). Common coastal ctenophores of the
genus Mnemiopsis are known to harbor protists on
their comb plates. One of these is an undescribed
naked amoeba that was isolated from the comb
plate surface. Transmission electron microscopy
(Fig. 2) has revealed apparent degradation of the
comb plate surface suggesting that this amoeba is
capable of destroying comb plate cilia (important
structures for ctenophore locomotion).
Understanding the nature of the symbiotic
association between the amoeba and the ctenophore
is inherently interesting since it appears to be
a widespread phenomenon. The association has
ecological implications for ctenophore
populations if the swimming and feeding
capabilities of animals with amoebal infestations
are reduced. The undescribed amoeba is also of
interest since this is new to Science.
These results are supported by the growth rate
determinations across the same range of
salinities. Counts over time gave growth curves
as shown in the one example (Fig. 8).
Fig. 3 Simultaneous transmitted DIC and
2-channel confocal images. At left green
fluorescence is presumed to be associated with ds
nucleic acid and small red inclusions as ss
precipitates (Darzynkiewicz, 1994). Areas of
green fluorescence in the confocal image
correspond with transparent material in the DIC
image while red inclusions appear as orange.
Scale is identical in both images.
Fig. 4 Amoeba cell fixed with 1 gluteraldehyde
and stained with DNA-specific fluorochrome (DAPI)
.
Fig. 8 Chart showing exponential growth curve at
40g/l.
Linear regression analysis was used to calculate
slopes of the exponential growth phase and the
growth rate constant (K) was calculated using
Stainers formula (1976) at the different
salinities. As shown in fig. 9, generation time
(1/K) was greater at higher salinity (implying
slow growth).
Scanning electron microscopy A 50 µl drop of
amoeba cell culture was placed onto a clean glass
cover slip (n 3) and left overnight in a moist
chamber (to prevent evaporation). This allowed
cells to adhere firmly to the glass surface. A
0.1M solution of sodium cacodylate buffer was
prepared in distilled water (pH 7.2). A drop of
2.5 gluteraldehyde in 0.05M cacodylate buffer
was added to amoebae on the coverslips (primary
fixation). After 30 mins the coverslips were
rinsed 2 times in 0.05M buffer by gently dipping
the coverslips into small staining jars (coplin
jars) containing the buffer. Each rinse lasted
about 30 secs. For postfixation, 2 osmium
tetroxide in 0.05M buffer was pipetted onto the
coverslips and left for 1 h. After fixation, the
coverslips were gently rinsed in distilled water
several times (30 secs in staining jars) and
dehydrated through an alcohol series 30, 50,
70, 85, 100,100 (15 min each solution).
Following dehydration, the coverslips were added
to a 50 HMDS 50 ethanol solution for 10 min.
After two 10 min treatments of 100 HMDS the
sample was air dried. The coverslips containing
the fixed and dried amoebae were sputter coated
with 10nm thick gold/palladium and viewed on the
SEM JEOL 5310 scanning electron microscope at an
acceleration voltage of 20KV.
Fig. 2 Electron micrograph showing amoeba
crawling on ctenophore comb plate (cp) (adapted
from Moss et.al, 2001) Scale bar 1µm.
Fig. 1 Coastal Ctenophore Mnemiopsis sp.
isolated from coastal Florida. Arrow shows comb
plate surface.
MATERIALS AND METHODS   Salinity Tolerance
Sterile artificial seawater media of varying
concentrations was prepared by dissolving sea
salts (Sigma Scientific) in 1 litre of glass
distilled water (10g/l, 20g/l, 30g/l, 40g/l,
50g/l). Aliquots (8ml) of media at each salinity
were transferred into plastic Petri dishes (5.5
cm in diameter). For each salinity, 3 replicate
plates were prepared. Amoeba cells were harvested
from a dense exponentially growing culture by
dislodging cells from the bottom of the source
dish using a cell scrapper. Suspended cells were
agitated using a transfer pipette to evenly
suspend the amoebae. Five drops of amoebal
suspension were transferred to the experimental
Petri dishes. The bacterial prey suspension was
prepared by adding a loopful of E. coli to 10ml
distilled water. The suspension was shaken
vigorously and a dense drop of the bacterial
suspension was added to each plate. This ensured
that there was an abundance of bacterial prey in
all experimental dishes. Plates were incubated at
24C. Generation Time Generation time (h) was
estimated by averaging cell counts obtained from
three fields of view on each of three plates per
concentration using Leica DMI 4000B phase
contrast inverted light microscope with 63x long
working distance objective. The first count was
taken after the cells had settled (i.e. after 1h)
to determine the initial starting concentration
of cells (No). Subsequent counts were made every
12 h for 4 days on viable active cells (viable
active cells were attached to the substratum and
moved noticeably by pseudopodia). From the growth
curves generated over time, the division time
(generation time) of exponentially growing cells
was calculated. Velocity Rate of locomotion was
determined by computing the average distance
moved per second by ten randomly selected amoeba
observed in cultures at different salinities.
Size Length and breadth measurements (microns)
of 10 randomly selected individual amoebae
growing in different salinities were measured
from micrographs obtained using a Leica inverted
phase contrast microscope at 630x
magnification.  Fluorescence and Confocal
Microscopy In an attempt to elucidate the
three-dimensional morphology of the nucleus,
amoebae were stained with the DNA-specific
fluororchrome acridine orange (AO). A stock
solution of AO was prepared by adding 0.5 mg AO
to 10 ml distilled water. Before use, this stock
was further diluted 15 with distilled water. A
24µl aliquot of the diluted stock AO was added to
8ml amoeba cell culture (modified from Rogerson,
2005). These cells were grown in artificial
seawater medium at a salinity of 32g/l sea salt
to mimic the amoebas natural environment.
Fluorescence was detected on the confocal
microscope using PMT1 (585LP) and PMT2 (522/32).
PMT1 and PMT2 were used to collect emitted red
and green light respectively when excited with
blue light (488nm). Transmitted DIC images of
stained amoeba were also obtained. Acridine
orange is a vital nucleic acid stain that emits
green fluorescence when bound to dsDNA and red
fluorescence when bound to ssDNA or ssRNA.
Exclusive staining of dsDNA is dependant on
concentration of the acridine orange
(Darzynkiewicz et al., 1994). Acridine orange
can also bind non-specifically to other
components of the cell such as the outer covering
(glycocalyx) and intracellular inclusions such as
lipid bodies. For additional resolution of the
nucleus of this amoeba, DAPI staining was used
with conventional florescence microscopy. DAPI is
a DNA-specific fluorochrome that binds to A-T
base pairs. A stock solution of 10 mg DAPI in 10
ml distilled water was prepared. Cells in 5 ml
suspension were fixed with 1 glutaraldehyde and
stained with DAPI (5 drops) for 30 min in the
dark. After staining, cells were captured on a
0.2 µm pore size black membrane (Nuclepore) and
viewed under UV light by epifluorescence
microscopy at 900 x magnification.
Fig. 9 Chart comparing generation time at
different salinities.
Velocity Consistent with the slow growth rates,
least cellular activity was observed at higher
salinities (40g/l and 50g/l). On the other hand,
locomotion rates were highest at the lowest
salinity (10g/l) where cells grew fastest.
Distance traveled was measured every second.
However, since amoebae at 40g/l and 50g/l moved
very slowly, cell velocities were not calculated.
Size Measurements were exclusively done on
amoebae attached to the substratum for the 10g/l,
20g/l and 30g/l sea salt media experiments. This
was not possible for 40g/l and 50g/l since most
amoebae were floating forms. Here, measurements
included attached and rounded floating forms.
Results showed that length and breadth
measurements remained constant across all
salinities and that regardless of culture
conditions, amoebae averaged 5.5 um in length
(Table 1).
Fig 5 Preliminary SEM image showing typical
morphology of naked amoeba, definitive
identification will require further work .
Results and Discussion Morphology of cells The
DIC photomicrographs (Figs. 6, 7 10) clearly
show the morphology of these unidentified
amoebae. Cells are small (around 6 µm in length)
with a very faint (thin) anterior hyaline zone
(Fig. 7). In moving cells, this zone changes
shape rapidly. The thinness of the hyaline zone
and its markedly changing shape is unusual in
amoebae. Cells have occasional trailing filaments
from the posterior uroid. The confocal
microscopy with AO showed several inclusions in
this amoeba, perhaps lipid drops (Fig. 3). This
prevented clear characterization of the nucleus.
When DAPI was used, the nucleus was evident (Fig.
4). The single nucleus is characteristically
amoeboid with a prominent (unstained) central
nucleolus. The morphology of the cell was also
revealed by SEM (Fig. 5). Additional samples will
be examined to confirm that this micrograph is
typical, however, the cell does show a flattened
hyaline zone and a raised cell body. An unusual
feature seen in this micrograph is the appearance
of the surface undulations. The cell appears to
be covered by short projections that might, in
part, explain the rapidly changing appearance of
cells observed by light microscopy.
Table 1 Average breadth/length dimensions and
Average Locomotion Rate at Different Salinities
with Standard Error Values
Salinity (g/l)
Breadth (µm)
Length (µm)
Velocity (µm/sec)
10
2.4 /- 0.16
6.4/- 1.24
0.59/- 0.10
20
2.7/- 0.33
4.8/- 0.33
0.43/- 0.03
30
3.3/- 0.26
6.5/- 1.00
0.3/- 0.04
40
2.8/- 0.39
5.7/- 0.40
No data
50
3.8/- 0.20
4.2/- 0.20
No data
Conclusion Survival across a wide range of
salinities illustrates that this amoeba is
euryhaline. It is surprising that maximum growth
of this supposed marine amoeba was at 10 g/l
rather than the salinity of seawater (32 g/l).
This suggests that the source of the amoeba might
be in brackish water or even from freshwater
runoff. Although minimum activity was seen at
the highest salinities, cells continued to
reproduce. Salinity did not affect the size or
morphology of cells, however, amoebae were most
active at the lowest salinity. DAPI staining on
fixed cells was superior to acridine orange for
detailing the nucleus of this amoeba. The
nonspecific binding displayed by AO obscured
nuclear detail, even when confocal microscopy was
used. The most distinctive feature of this
amoeba at the light microscope level is the
extremely thin, and rapidly changing, hyaline
zone. At the SEM level, unusual surface
projections were observed. Future work to further
characterize this amoeba will include additional
SEM as well as Transmission Electron Microscopy
(TEM). Complementary molecular studies are being
undertaken by collaborators at Woods Hole
Oceanographic Institute.
Fig 6 7 Transmitted DIC images showing active
amoeba with visible hyaline zone (white arrow)
and trailing filament from posterior uroid (red
arrow).
Salinity Tolerance Amoebae survived at all the
salt concentrations tested over the range 10 to
50 g/l. At the highest salinities, more of the
population was observed as floating cells, rather
than as attached motile cells. This suggests that
these higher salinities (40g/l and 50g/l) were
close to the survival limit for these amoebae.
Surprisingly, fastest growth was found at 10
g/l salt. Here the generation time was around 8.5
h. This was shorter than the generation time at
30 g /l (the salinity of coastal water). Under
these conditions, amoebae divided every 12 h.
Additional experiments will be conducted to
determine whether this amoeba can grow rapidly at
0 ppt salt (i.e. freshwater conditions).
Acknowledgements I wish to acknowledge Dr.
Michael Norton and David Neff for maintenance of
the MIBC imaging facilities. I am grateful for
the support and invaluable advice given by Andrew
Rogerson during this study. Collaborators at
Woods Hole Oceanographic Institute and Auburn
University provided useful information integrated
in this study, made possible through the National
Science Foundation (NSF) grant.
References Moss G. A. et al. (2001) Protistan
Epibionts of the Ctenophore Mnemiopsis mccradyi
Mayer Hydrobiologia, Vol. 451 pp. 295-304,
Kluwer Academic Publishers Rogerson et al.
(1994) Estimation of Amoeba Cell Volume from
Nuclear Diameter and its Application to Studies
in Protozoan Ecology Hydrobiologia, Vol. 284 pp.
229-234 Kluwer Academic Publishers Darzynkiewicz
Z. (1994) Simultaneous Analysis of Cellular RNA
and DNA Content Methods in cell Biology, Vol. 41
pp. 401-420, Academic Press
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Fig. 10 Transmitted DIC time lapse photography
of active amoebae on glass cover slip.