Abstract
The symbiotic sea anemone Anthopleura elegantissima has been shown
to exhibit a salient physiological response to elevated thermal stress that entails
the expulsion of its endosymbiotic algae, Symbiodinium californium. A.
elegantissima clones were subjected to a 48 h thermal stress at 24 °C and 31 °C.
The kinetics of the bleaching event was quantified in the latter treatment such
that more than 50% (2.32 x 10° / 5.51 x 10°; units in number of alga cells/mg
animal protein) of the algae were expelled at the end of the 48 h, and complete
loss of algae was noted 13 days after the stress. Two anti-murine Hsp 70
monoclonal antibodies, 3A3 and N27F3-4 (N27), were utilized to probe for
constitutive and inducible Hsp 70 family proteins by immunoblotting using
anemone samples taken from both elevated temperature groups at six time
points during and after the thermal stress. The 3A3 mAb recognized a
constitutive 70kD protein in stressed and unstressed anemones at both the 24 °C
and 31 °C treatments. In the 31 °C treatment, the N27 mAb revealed rapid
induction (seen at six hours) of a 70kD protein in all thermally stressed
anemones, but not in the unstressed anemone. Also, the N27 mAb revealed a
low molecular weight 30kD cross-reactive protein present in both stressed and
unstressed anemones. In the 24 °C elevated temperature group, the N27 mAb
revealed no sign of the 70kD protein at all sample times, nor was it present in
the unstressed anemone. However, the constitutive 30kD protein was present
in all samples including the unstressed anemone. Qualitative changes in
soluble proteins in response to both treatments were visualized by
398 methionine labeling during and after thermal stress. An aposymbiotic
anemone subjected to a 48 h, 31 °C heat stress showed induction of the 70kD
protein; constitutive 70kD protein was also seen with the 3A3 mAb.
Identification and temporal correlation of physiological and biochemical
responses to elevated temperature stress in cnidarians is novel and instructive
for further study of the stress response in invertebrates.
Introduction
Cnidarian-alga symbioses have been the subject of numerous studies of
the impact of environmental stressors such as high UV light levels, salinity
fluctuations, heavy metals, and variations in water temperature. In response to
extreme environmental insults, a breakdown of the symbiotic relationship can
occur in which the host expels the symbiont in large quantities (Hoegh¬
Guldberg, 1989; Glynn, 1990; Lesser et al., 1990; Muscatine et al. 1991). This
response, termed ’bleaching,“ has been studied extensively in tropical coral and
anemone symbioses, however, little work has been done on animals that
inhabit temperate environments.
The temperate sea anemone Anthopleura elegantissima (Brandt)
maintains an endosymbiotic association with the photosynthetic dinoflagellate
Symbiodinium californium (McNally et al., 1994). It should be noted, however,
that aposymbiotic A. elegantissima, anemones lacking algal symbionts, are
known to occur naturally and to inhabit locations with low or no solar radiation
(Buchsbaum, 1968). A. elegantissima, which is the most abundant sea anemone
found along the North American Pacific coast (Hand, 1955), is an intertidal
animal that is subjected to largely unpredictable variations in temperature, pH,
salinity, and water loss (Fitt et al., 1982; Taylor and Littler, 1982). The organismal
stress response of A. elegantissima and its congener A. xanthogrammica have
been elucidated by Buchsbaum (1968), Engebretson and Martin (1994), and
O'Brien and Wyttenbach (1980). The present investigation attempts to draw
conclusions regarding not only the physiological stress response of A.
elegantissima to elevated temperature, as discussed in Buchsbaum (1968), and
O'Brien and Wyttenbach (1980), but also the underlying biochemical changes.
Papers on another temperate anemone, Anemonia viridis, also document
bleaching and biochemical changes in response to elevated temperature stress
(Miller et al., 1992; Sharp et al., 1994). In particular, changes in soluble proteins
have been discussed within the context of heat shock proteins (Hsp) and the
heat shock response (for reviews see Lindquist, 1986; Craig and Lindquist, 1988;
Sanders, 1993)
In the present study, the response of A. elegantissima to thermal stress is
characterized on the level of the organism in terms of algal expulsion and at the
biochemical level in terms of qualitative and quantitative changes in soluble
protein expression. The constitutive and inducible isoforms of a 70kD Hsp 70
protein as well as a low molecular weight 30kD Hsp 70 cross-reactive protein are
the focus of the quantitative assays. The identification of constitutive and
inducible Hsp 70 isoforms at two different elevated temperatures, 31 °C and 24
raises interesting questions regarding the stress response and upper thermal
limits of organisms in light of extensive discussion in the literature of the role
of Hsps in the phenomenon of thermotolerance and environmental adaptation
(Lindquist, 1986; Bosch et al., 1988; Sanders et al., 1991; Sanders, 1993; Sharp et al.
1994)
Materials and Methods
Collection and maintenance of A. elegantissima
Symbiotic specimens of the experimental organism were collected from a
single clonal mat in the intertidal zone at Hopkins Marine Station, Pacific
Grove, CA. Symbiotic animals were collected from rocks in full sunlight,
whereas naturally-occurring aposymbiotic animals were collected from
underneath the adjacent Monterey Bay Aquarium. Animals were maintained
in outdoor, running seawater tanks (referred to as control tanks, subsequently)
set to 13 °C, beneath screened sunlight for at least one week, but not more than
one month, prior to experimentation.
Heat stress experimental procedure
Specimens, which had been settling into glass dishes during the
acclimatory period, were transported within their dishes from the control tanks
directly to a twelve liter aquarium containing seawater preheated to 24 °C or 31
C. The tanks designated for thermal shock were situated indoors, beneath low
light levels, and were equipped with air stones and a cover to prevent
hypersaline conditions (periodically during the heat shocks, salinity
measurements were taken with a American Optics portable refractometer to
insure that normal salinity levels (32 %) were maintained). Directly after
exposure to 48 h of thermal stress, animals were returned to the control tanks
for recovery.
Determination of algal densities
During the experimental regime described above, clips were taken from
the anemone’s columns at intervals. Animals were removed from the tank,
and a small piece of column was excised with a razor blade whilst the anemone
remained attached to the glass bowl. Also, anemone extracts used for
immunoblots were prepared from animals distinct from those that were clipped
for algal numbers. The clips were then homogenized in filtered seawater (FSW)
using a plastic pestle and a siliconized microcentrifuge tube. The homogenates
were centrifuged in a microfuge at 4 °C for 12 minutes at 9,000 X g to pellet the
algae. The animal supernatant containing the soluble protein was decanted, and
its protein concentration was determined by the method of Bradford (1976). The
algae were resuspended in FSW and quantified using a hemacytometer and
algal cell numbers were related to animal protein.
Preparation of A. elegantissima homogenates
Animals sampled both during and after the heat stress were frozen in
liquid N2 and stored at -20°C until further processing. Anemones were
removed from the freezer and finely minced with a razor blade. Protein
samples from each anemone were obtained by grinding whole animals in a 2m.
Teflon-glass homogenizer (Wheaton Instruments, Millville, NJ) with -1.5ml
homogenization buffer: 40 mM Tris, 10 mM EDTA, 1 mM PMSF, ImM
pepstatin, 1mM leupeptin and 10mM chymostatin, pH 7.4. Homogenates were
centrifuged for 12 minutes at 17,500 X g in a microfuge at 4 °C to pellet algae and
animal debris. Supernatants were removed and centrifuged as above a second
time to pellet remaining algae; also, pellets were inspected microscopically for
evidence of lysed and damaged algae, but none was detected. Supernatants were
then kept on ice until resolution by 1-D SDS-PAGE. Protein concentrations were
determined as described above (Bradford, 1976).
(898]-methionine labeling of A. elegantissima
Symbiotic A. elegantissima in 10ml beakers containing 7ml of FSW were
placed in a recirculating water bath at 15 °C and allowed to adjust to the
temperature for 45 min prior to addition of the isotope. Anemones were
exposed to 250 umoles quanta : m’2 . s+ of light, which is above the saturation
point for photosynthesis in isolated algae from A. elegantissima (Muller-Parker,
pers. comm.), for the duration of the experiment. Fifty uCi of L-828lmethionine
(Amersham, Arlington, III.) were added to each vessel after the 45 min pre¬
incubation, and the animals were labeled for 4 h. Each animal was then rinsed
in three successive 10 ml volumes of 0.2 M L-methionine in 40 mM Tris, pH 7.4.
Animals were then frozen in liquid N2 and placed at -20 °C until further
processing. Äfter homogenization and protein isolation were carried out as
described above, scintillation counts were determined by a Packard 2200CA
liquid scintillation counter and 150,000 cpm of animal proteins were set aside for
gel electrophoresis.
Gel electrophoresis
A. elegantissima proteins (15ug) were separated by one-dimensional
sodium dodecyl sulphate polyacrylamide gel electrophoresis (1-D SDS-PAGE) on
12.5% minigels (8x7 cm) of Imm thickness (methods modified from Laemmli,
1970). Pre-stained molecular weight standards (Bio-rad Laboratories, Hercules,
CA) were loaded onto gels for the determination of the apparent molecular
weights of the proteins. Gels were subsequently stained with Coomassie blue
Gels of 3581-labeled proteins were left unstained, fixed in 40% methanol, 10%
acetic acid for 30 minutes and then enhanced with Resolution
chemiluminescence autoradiography enhancer (Electron Microscopy Sciences)
according to the manufacturer’s instructions. Following enhancement, gels
were dried and exposed to Kodak XAR-5 film at -70 °C.
Immunoblotting
Protein transfer from unstained gels to nitrocellulose membranes was
performed according to the procedure described by Burnette (1981) with slight
modifications. Proteins were transferred in a Hoefer TE22 transfer apparatus
overnight at 12 ?C at a constant current (25mA) in 25 mM Tris, 192 mM glycine,
20% methanol, 0.01% SDS buffer, pH 8.3 (modified from Towbin et al., 1979).
After transfer, nitrocellulose membranes were removed from the gel and
blocked in TBS/Milk/Tween (60ml 5X TBS, 15g Carnation nonfat dry milk,
0.3ml Tween20) for at least on hour at 37 °C. Blots were then probed with either
anti-murine Hsp70 343 monoclonal antibody (received from Dr. Sean Murphy,
Northwestern Univ., Illinois) in a 1:10000 dilution of TBS/Milk/Tween or anti-
murine Hsp70 N27F3-4 (N27) monoclonal antibody (StressGen, Victoria, B.C.,
Canada) in a 1:1000 dilution of TBS/Milk/Tween. Primary antibodies were
Incubated overnight in seal-a-meal bags on a slow rotary shaker at 12 %C. Blots
were then washed successively with 1X TBS, TBS/Tween (1X TBS, 0.5% Tween).
and IX TBS for 10 minutes each (30 minutes total). Secondary antibody probing
occured with goat anti-murine IgG (whole molecule) horseradish peroxidase
conjugate (Sigma, St. Louis, MO) diluted 1:1000 in 1X TBS for 1 h with rapid
shaking. Blots were rinsed as above for 30 min and subsequently incubated in
Renaissance western blot Enhanced Chemiluminescense (ECL) reagents
(DuPont NEN, Boston, MA) for 1 minute and imaged on Kodak XAR-5 film.
Densitometric analysis
X-ray films of immunoblots were scanned by transmittance using a
Pharmacia ImageMaster densitometer and analyzed with Pharmacia
Image Master Software (Version 1.0) according to prescribed methods.
Sampling times and notation used
The following notation for sampling times will be used in this paper: S
denotes time during the 48 hour stress period and R denotes time after the 48 h
stress, in the recovery period. For example, the first sampling point for 1-D SDS
PAGE is S6, which signifies a time point six hours into the stress period; the
final time point is R15d, or 15 days into the recovery period, which is actually 17
days after initiation of the experiment. As seen in this example, if the notation
contains a "d," the number indicates days; in all other notation, hours are
implied.
Results
Kinetics of algae loss in response to elevated temperature
Visual inspection of the 31 °C heat stressed anemones did not reveal
remarkable discoloration during the 48 h trial, but two and three days into the
recovery period the oral disks of anemones appeared markedly lighter. Time
points at which the anemones were sampled were as follows: prior to heat
stress (S0), at the end of heat stress (S48), 48 h into the recovery period (R48),
four days into the recovery period (R4d) and 13 days into the recovery period
(R13d). The 48 h thermal stress at 31 °C resulted in the loss of more than 50 % of
algae during the thermal stress (Fig. 1). The number of algal cells per mg
animal protein at SO was 5.51 x 10°, however, at S48, when the animals were
being replaced to control conditions, that number dropped to 2.32 x 10°. The
subsequent two sample points revealed a less precipitous decline in the number
of algae, however, at 13 days post-shock no algae were seen on slides prepared
from the column clips. Anemones appeared completely bleached by one week
post-stress, although, no samples were taken between R4d and R13d time points.
No algal density samples was taken on the 24 °C heat stressed anemones.
Although the anemones appeared mildly lighter in color, no marked bleaching
effect was noted after two weeks of observation.
Immunoblots of 24 °C and 31 °C heat stressed A. elegantissima utilizing the 3A3
anti-Hsp 70 mAb
In order to determine the effect of hyperthermal stress on the presence of
Hsp 70 proteins, protein samples from heat stressed A. elegantissima were
probed with the 3A3 mAb that cross reacts with the Hsp 70 family of proteins.
Samples of heat stressed anemones were taken at six time points: S6, S24, S48
R24, R5d, and RlOd. What is referred to as the control anemone in all western
blots (Figs. 2-7) is an unstressed A. clegantissima that was maintained in the
control tanks. In the far left lane(s) of all blots (Figs. 2-7) is what is referred to as
the positive control: purified Hsp 70 from bovine brain (Sigma, St. Louis, MO)
with a molecular weight of 70kD. A single protein appearing at 70kD that cross-
reacts with the 343 mAb occurs with equal intensity at all time points, including
control anemone, and at both elevated temperatures (Figs. 2 and 3; 31 % and 24
C respectively). The apparent molecular weight of this protein is exactly equal
to that of the positive control Hsp 70 protein described above. Densitometric
scans of the immunoblots shown in Figs. 2 and 3 confirm that the protein is of
70kD in apparent molecular weight and that there is no significant variation in
intensity over the time course (data not included)
Immunoblots of 24 °C and 31 °C heat stressed A. elegantissima utilizing the N27
anti-Hsp 70 mAb
10
The second mAb used, N27, is also specific for the Hsp 70 family of
proteins; it was employed to complement the 3A3 mAb as it recognizes à distinct
epitope. The six time points, plus control anemone, that were sampled and
subsequently probed with the 3A3 mAb were also employed in the N27
immunoblots. Immunoblots of 31 °C heat stressed anemones probed with the
N27 anti-Hsp 70 mAb showed two proteins, one at 70kD, the other at 30kD, in
animals from all time points during and after heat stress (Fig. 4), but not in the
control anemone lane where only the 30kD protein appeared (Fig. 4, lane 4).
Densitometric analysis did not offer robust conclusions regarding the changes in
the 70kD protein intensity over the time course (data not shown). In contrast to
the induction of the 70kD protein in response to heat stress at 31 °C, anemones
subjected to 24 °C heat stress showed no induction of a 70kD protein (Fig. 5).
However, a protein at 30kD was present in all animals including the control.
Immunoblots of 31 °C heat stressed aposymbiotic A. elegantissima probed with
3A3 and N27 anti-Hsp 70 mAb
To determine the effect of the presence of algae on the occurrence of the
Hsp 70 family of proteins in response to elevated temperature, proteins from à
heat stressed aposymbiotic A. elegantissima were probed with the 343 and N27
mAbs. The aposymbiotic anemone was exposed to a 31 °C heat stress for 48h.
and its protein was isolated immediately. Lanes 2 and 3, in both Figs. 6 and 7,
are the control aposymbiotic anemone and the heat stressed aposymbiotic
anemone, respectively. Results shown in Figs. 6 and 7 coincide with the results
of the 31 °C heat stressed symbiotic anemones (Figs. 2 and 4). A 70KD protein
that cross-reacts with the N27 anti-Hsp 70 mAb in the 48 h heat stressed
aposymbiotic A. elegantissima (Fig. 7, lane 3) is at the same molecular weight as
the positive control, however, no 70kD protein is apparent in the control
anemone, lane 2. The 3A3 mAb recognizes a consistent amount of protein in
the heat stressed and control anemones (Fig. 6). An interesting observation is
the lack of a protein at 30kD in both control and heat stressed aposymbiotic
anemones probed with the N27 anti-Hsp 70 mAb (Fig. 7, lanes 2 and 3).
Comparison of newly-synthesized protein profiles in the 24 °C and 31 °C heat
stressed A. elegantissima
In order to determine the effect of temperature stress on protein profiles
of anemones, sample anemones at different time points were incubated in
LI°9S]-methionine to allow visualization of de novo protein synthesis. The
three time points shown, S24, R24, and R5d show qualitative changes in soluble
proteins in comparison with controls. Figures 8 and 9 are autoradiographs of 31
C and 24 °C heat stressed anemones respectively, incubated for four hours.
Dramatic intensity changes occur with at least four proteins over the
experimental time course in the 31 °C heat stressed anemones (see arrows, Fig.
8). One interesting point in the autoradiograph of the 24 °C heat stressed
anemones is the enhancement of a 29kD protein during the time course (Fig. 9,
see arrow).
Discussion
This study demonstrates the temporal correlation of bleaching and its
associated biochemical changes in a cnidarian-algal symbiosis subjected to
thermal stress; more specifically, it qualitatively and quantitatively describes the
breakdown of the symbiotic relationship and the modifications in heat shock
protein expression. Low intertidal A. elegantissima subjected to a 48 h, 31 %C
thermal stress, as described by Buchsbaum (1968), resulted in expulsion of algae
within two weeks (see also, O’Brien and Wyttenbach, 1980). Monoclonal
antibodies raised against the highly conserved Hsp 70 family recognized an
induced 70kD protein in the 31 °C heat stressed anemones, however, heat stress
at 24 °C did not show induction of this protein, nor did the animals bleach. This
is of possible interest because 24 °C may not be a physiologically relevant
temperature. Present in both groups of heat stressed anemones were
constitutive Hsp 70 proteins with molecular weights of 30kD and 70kD.
Induction of a 70kD Hsp 70 in symbiotic cnidarians has not been
extensively studied (Bosch et al., 1991; Gellner, K. et al., 1992; Miller et al., 1992;
Hayes and King, 1995), however, its presence in a wider range of marine species
has been more thoroughly described (Ha, 1993, Matorin, 1994; Sanders, 1994).
The N27 anti-murine Hsp 70 mAb was raised against a 70kD heat shock protein
from HeLa cells and is thought to be specific for constitutive and inducible
forms of Hsp 70 (StressGen, Victoria, B.C., Canada; Sanders, 1994). Sanders
(1994) has shown N27 to cross-react with inducible 70kD proteins in 4 species of
fish, 2 species of molluscs, and one species each of crustacean, ground squirrel,
and alga. In the majority of these experiments, Sanders (1994) subjected animals
to hyperthermal stress near their lethal limits. Therefore, it is not surprising
that N27 would recognize a heat-inducible 70kD protein in the 31 °C thermally
stressed anemones (death occurs at 33 °C, Buchsbaum, 1968). However, the lack
of the protein in the 24 °C heat stressed anemones is remarkable in that 24 °C is
a temperature that is beyond the range of physiological relevance as well. Sea
water temperatures at Hopkins Marine Station during the last 75 years varied
from 8 to 19 °C (Barry et al., 1995). A. elegantissima is a low-to-mid intertidal
organism that may encounter temperatures slightly above those at the sea
13
surface because of the slightly elevated temperatures in tide pools. However, it
seems likely that the heat shock response in A. elegantissima could only be
elicited by thermal stress in concert with additional environmental stressors
such as high UV light levels, salinity fluctuation and dessication.
The anti-murine monoclonal antibody 3A3 has been used more
extensively in experimental investigations than the N27 mAb. It recognizes an
epitope located between amino acids 504-617 of human Hsp 70, a region shown
to be involved in stress-induced nucleolar localization (Sanders, 1994; Sharp et
al., 1994; Hayes and King, 1995; Affinity Bioreagents data sheets). The antibody
reacts with several antigens of the Hsp 70 family, including the inducible form,
Hsp 72, and the constitutive form, Hsp 73. Sanders (1994) found the 3A3 mAb to
be the most broadly cross-reactive, across species, among four anti-Hsp 70 mAbs
tested-N27 included. The Ab recognized distinct constitutive and inducible
forms of Hsp 70 proteins at 70kD in equal numbers of species. In the present
study the Ab recognized the constitutive form of the 70kD protein in all
animals, stressed and unstressed, at both thermal stress temperatures. The
epitope of the protein that the mAb recognizes must be responsible for a
consequential function in thermally stressed animals as it is highly conserved
across phyla.
The low molecular weight (LMW) proteins are more species-specific than
the other major stress protein families and less highly conserved (Craig, 1985;
Lindquist, 1986; Sanders, 1993). This family of proteins is induced under adverse
environmental conditions and has long been implicated in thermotolerance
(for review see Sanders, 1993). In fact, LMW stress proteins have been shown to
accumulate in desert succulents acclimatized to high temperatures (Kee and
Noble, 1986). The induction of four LMW proteins, at 28, 29, 30 and 31kD, in
response to elevated temperature shock in temperate anemones has been
documented by Miller et al. (1992) and Sharp et al. (1994). Using 1-D SDS-PAGE,
Miller et al. (1992) showed induction of a 30 and 31kD protein in response to a
short term 32 °C heat stress. Utilizing 1-D SDS-PAGE and western blotting with
the 3A3 anti-Hsp 70 mAb, Sharp et al. (1994) demonstrated constitutive
expression of 28 and 29kD proteins which is enhanced after short term, thermal
stress at 32 °C. Sharp et al. (1994) performed numerous experiments to
demonstrate that the 28 and 29kD doublet of LMW proteins are not products of
proteolytic breakdown of a 70kD Hsp 70. This is also a concern with regard to
the present study
It is possible, but unlikely that the 30kD protein recognized by N27 at 24
and 31 °C is a breakdown product. Three enzyme inhibitors (chymostatin,
leupeptin, and pepstatin) as well as PMSF and the chelating agent ÉDTA are
present in the homogenization buffer. Hsps have characteristic and stable
breakdown products. The 70kD protein in the Hsp 70 family is proteolytically
reduced to two doublets ranging from 40-44kd and 18-22kd (Lindquist, 1986).
No second LMW Hsp 70 cross-reactive protein in the 30-40kd range was
observed in any thermal stress experiments reported on here. Although, it is
possible that only one of the two proteolytic products contains the epitope
recognized by the antibody. However, the varying amount of the 30kD protein
when compared to the 70kD protein (Figs. 4 and 6) is further evidence against
the 30kD protein being a breakdown product. Assuming proteolysis is
proportional to total amount of protein, neither the inducible nor the
constitutive 70kD protein is a likely source of the 30kD protein if it is the
product of proteolysis. Additional evidence that the LMW protein is not a
breakdown product is its absence in the aposymbiotic anemone experiment.
It was not surprising that the 31 °C thermally stressed aposymbiotic
anemone showed the same pattern of inducible and constitutive expression of
the 70kD Hsp 70 as the symbiotic anemone. Naturally occurring symbiotic and
aposymbiotic A. elegantissima may be genetically identical, i.e. from the same
clone (Buchsbaum, 1968), but differ phenotypically with regard to presence of the
symbiotic algae. In addition, phenotypic differences in terms of biochemical
expression between symbiotic and aposymbiotic A. elegantissima have been
ascribed to the presence of algae in experiments performed by Weis and Levine
(in print, 1995). Thus, the lack of the 30kD protein in the stressed and unstressed
aposymbiotic A. elegantissima is not inconceivable. However, labeling the
protein a symbiosis-specific, constitutive LMW Hsp 70 may be premature.
Undoubtedly, further experiments must be performed to understand the
differential expression of the 30kD and 70kD Hsp 70 within the species, and how
it relates to the proteins' functions in the two animals.
The [3°8lmethionine experiment did not corroborate the induction of the
70kD protein in the 31 °C heat stressed anemone experiments. The comparison
of newly synthesized (de novo) proteins in control and heat stressed anemones
reveals qualitative differences in proteins with high turnover rates, but low
relative abundance. It may be enlightening to try and infer ug quantities of the
70kD protein from the western blots to characterize its overall abundance. Ir
Figs. 2 and 3, lug of purified Hsp 70 was loaded in lane 3. All proteins probed
with the 3A3 mAb are of less intensity. Therefore, if we assume that all lanes
contain at most 0.75ug of the 70kD protein, and it is known that 15ug of total
protein was loaded in each lane, its abundance would be less than 5% of total
protein loaded. Assuming that the 3A3 mAb binds with equal affinity to the
bovine brain isolated Hsp 70 positive control and the constitutive and inducible
forms of the 70kD protein observed, the 70kD protein is not highly abundant in
proportion to the total ugs of protein loaded. This may be an explanation for the
lack of the appearance of the inducible 70kD Hsp 70 in the 328lmethionine
experiment.
This paper is a basis for further study of bleaching and its correlation to
associated biochemical changes in response to thermal stress in symbiotic
anemones. As stated before, the loss of endosymbionts in A. elegantissima and
its congener A. xanthogrammica in response to thermal stress has been studied
by Buchsbaum (1968) and Ö’Brien and Wyttenbach (1980). The generation of
aposymbiotic anemones within two weeks of elevated temperature stress at 31
C is interesting within the context of a threshold idea for bleaching. All algae
were expelled in response to the 31 °C heat shock, however, Buchsbaum (1968)
reports the arrival at this optimal temperature stress as the product of much
trial and error. Thermal shock at levels just below 30 °C does not result in
complete loss of algae; temperatures above 32 °C results in death. Therefore, the
complete bleaching effect is restricted to a narrow range of temperatures. The
idea of a range, or more likely a threshold, with regard to the induction of heat
shock proteins has also been made apparent in this study. It is a widely held
belief that heat shock proteins exist to protect organisms from the damaging
effects of short term environmental stress. There is abundant evidence in the
literature to support this notion (Sander, 1993). The temperature at which
induction occurs is usually near the upper portion of the organism’s natural
growth range (Lindquist, 1986; Sanders, 1993). Moreover, the acquisition of
tolerance has been shown to be correlated with the induction of Hsps (Bosch et
al., 1988; Sanders, 1991; Sharp et al 1994). There is evidence in conflict with this
idea, but the interpretation of results is complicated by the nature of Hsps in
terms of their constitutive and inducible isoforms which may or may not be
enhanced in response to different environmental stressors. The results of this
study support the idea that Hsps are not the only mediator of resistance to
thermal stress. A. elegantissima utilizes only constitutive Hsp 70 isoforms to
allow it to adapt to the range of environmental temperatures found in its
environment. In other words, induction of Hsps is not strongly correlated to
ecologically relevant, thermal stress. Therefore, in response to thermal stresses
near its upper, natural growth range, it is likely that the anemone employs other
sorts of physiological mechanisms for tolerance and protection.
The results of this study raise questions that must be answered before
further investigation of environmental stressors, their induction of heat shock
proteins, and the relevance of the stress protein response to in situ populations
of A. elegantissima can be directed and fruitful. The idea of a threshold for Hsp
induction introduced above is one that is instructive for this path of
investigation. First, the temperature threshold for the induction of various heat
shock proteins must be found. Second, the duration of the thermal stress
required to induce the Hsps must be described. Subsequently, once the
laboratory answers to these first two questions are provided, extensive fieldwork
must be done to determine the relevance of the acquired knowledge to what is
actually occurring in the intertidal zone.
Conclusion
Few studies have attempted to relate the physiological response of whole
organisms to underlying biochemical changes that result from environmental
insults such as elevated temperature. I have demonstrated a correlation
between such biochemical changes and the heat-induced loss of endosymbionts
in Anthopleura elegantissima. Algal expulsion due to a 48 h, 31 °C thermal
stress was complete within two weeks. Induction of a 70kD Hsp 70 was also seen
in anemones subjected to this heat shock. Thermal stress at a more
18
physiologically relevant temperature, 24 °C, did not result in induction of the
70kD Hsp 70, nor was marked bleaching observed. Also, a 30kD Hsp 70 was
expressed constitutively in both groups of thermally stressed anemones.
These findings lead to the conclusion that constitutive expression of a 70kD and
30kD Hsp 70, in concert with other physiological mechanisms, function to
confer tolerance to environmental extremes of temperature.
Acknowledgments
I am grateful to Dr. R. Paul Levine and Dr. Virginia M. Weis for their
unwavering enthusiasm and dedication to this project. The offering of their
knowledge, insight, and support has been deeply appreciated. I would also like
to extend thanks and good feelings to my labmates, Archana Dhawan, Ajna
Pisani, Nicole Boudreaux, and Sunny Sanders and Davey. My experience at
Hopkins will remain with me, in my pleasant thoughts, for years to come.
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Figure Legend
Figure 1. Algal loss from A. clegantissima vs. time in response to 31 %
temperature stress. Samples taken from animals at the following times: SO, S48,
R48, R4d, R13d. Stress period is marked on abscissa with a dark band. Each point
= mean + SE, (n=3).
Figure 2. Immunoblot using 3A3 anti-Hsp 70 mAb of A. elegantissima soluble
protein extracts during and after 31 °C temperature stress. Lanes 1-3 are a
dilution series of purified bovine brain Hsp 70, containing 0.lug, O.3ug, and
0.9ug respectively. Fifteen (15) ug of sample protein derived from animals was
loaded in lanes 4-10. Lane 4 is the control anemone, no heat stress. Lanes 5-10
contain protein from thermally stressed anemones: lane 5 anemone removed
from stress at 6 hours; lane 6 anemone removed from stress at 24 hours; lane 7
anemone removed from stress at 48 hours; lane 8 anemone removed from
control tank after 24 hours of recovery from thermal stress; lane 9 anemone
removed after 5 days of recovery; lane 10 anemone removed after 10 days of
recovery.
Figure 3. Immunoblot using 343 anti-Hsp 70 mAb of A. elegantissima soluble
protein extracts during and after 24 °C temperature stress. Lane assignments are
same as Fig. 2. Same amounts of animal protein and purified Hsp 70 loaded as
Fig. 2. Sample time points for lanes 5-10 are same as Fig. 2.
Figure 4. Immunoblot using N27 anti-Hsp 70 mAb of A. elegantissima soluble
protein extracts during and after 31 °C temperature stress. Lane assignments are
24
same as Fig. 2. Same amounts of animal protein and purified Hsp 70 loaded as
Fig. 2. Sample time points for lanes 5-10 are same as Fig. 2.
Figure 5. Immunoblot using N27 anti-Hsp 70 mAb of A. elegantissima soluble
protein extracts during and after 24 °C temperature stress. Lane assignments are
same as Fig. 2. Same amounts of animal protein and purified Hsp 70 loaded as
Fig. 2. Sample time points for lanes 5-10 are same as Fig. 2.
Figure 6. Immunoblot using 343 anti-Hsp 70 mAb of aposymbiotic A.
elegantissima soluble protein extracts after 31 °C temperature stress. Fifteen (15)
ug of anemone protein was loaded in lanes 2 and 3. Purified Hsp 70 (0.25 ug)
loaded in lane 1. Control aposymbiotic anemone, lane 2. Thermally stressed
aposymbiotic anemone, S48, in lane 3.
Figure 7. Immunoblot using N27 anti-Hsp 70 mAb of aposymbiotic A.
elegantissima soluble protein extracts after 31 °C temperature stress. Lane
assignments are same as Fig. 6. Same amounts of anemone proteins and
purified Hsp 70 loaded as Fig. 6.
Figure 8. Autoradiograph of L[82S)-methionine-labeled soluble protein profiles
derived from 31 °C temperature stressed A. elegantissima. Lane 1 contains pre¬
stained molecular weight markers. Equal counts (150,000 cpm) were loaded in
lanes 2-7. Lanes 2, 4 and 6 contain protein derived from control anemones.
Thermally stressed anemones were incubated at the following times: lane 3 =
S24; lane 5 = R24; lane 7 = R5d. Arrows point to proteins that show marked
changes in intensity along the time course.
Figure 9. Autoradiogragh of L[328)-methionine-labeled soluble protein profiles
derived from 24 °C temperature stressed A. elegantissima. Same cpms loaded as
Fig. 8. Lane assignments and sampling times are same as Fig. 8. Arrow
connotes same as in Fig. 8.
6.00E405
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Fig. 1
means + SE: n =3
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