FISHERIES SCIENCE
2008; 74: 573–578
Induced thermotolerance and expression
of heat shock protein 70 in sea cucumber
Apostichopus japonicus
Yunwei DONG* AND Shuanglin DONG
The Key Laboratory of Mariculture, Ministry of Education, Fisheries College, Ocean University of
China, Qingdao, 266003, China
ABSTRACT: Thermal limits, induced thermotolerance and the expression of heat shock protein 70
(Hsp70) in an echinoderm Apostichopus japonicus were studied. The sublethal and lethal temperatures for the juveniles were 30 and 34°C, respectively; a previous sublethal heat shock exposure
(30°C, 2 h) could increase the survival rates of the sea cucumbers when they were exposed to 34°C.
This induced thermotolerance could last for at least 2 days. Levels of Hsp70 increased substantially
after sublethal heat shock exposure and linearly decreased with time. This result indicated that a close
relationship existed between the induction of thermotolerance and the levels of Hsp70 in A. japonicus.
KEY WORDS: Holothurian, Hsp70, thermal limit, thermotolerance.
INTRODUCTION
Previous studies showed that animals which were
exposed to a previous ‘heat shock’ can acquire a
tolerance to otherwise lethal temperatures, know
as ‘induced thermotolerance’.1–3 Recent short-time
thermal history can influence the cellular stress
responses and induce the expression of heat shock
proteins.4–7 Acting as molecular chaperones, heat
shock proteins (Hsps), including members of the
Hsp70 family, assist in the refolding of stressdenatured cellular proteins and prevent these proteins from aggregating in the cell.8 Previous studies
indicate that Hsps are the primary mediators of
induced thermotolerance.9–13 Furthermore, expression patterns of Hsp are directly related to thermotolerance at the organismal level.5
The sea cucumber Apostichopus japonicus is a
common echinoderm species in the seas near
Japan, Korea and north of China, and has high
commercial value.14 In summer, the temperature in
a shallow pool for sea cucumber rearing (Shandong Province, China) can reach greater than 30°C
and cause large-scale mortality of sea cucumber
(Dong Y and Ji T, unpubl. data, 2006). Therefore, it
is important to know the thermal limits and induction of thermal tolerance of A. japonicus at higher
temperatures. To our knowledge, only limited
*Corresponding author: Tel: 86-532-8203-2435.
Fax: 86-532-8203-2435. Email: [email protected]
Received 21 June 2007. Accepted 18 December 2007.
doi:10.1111/j.1444-2906.2008.01560.x
research has focused on the thermal limits of A.
japonicus,15 and there are no studies on induced
thermotolerance in this species. In this study, we
examined the thermal limits, induction of thermotolerance and the expression of Hsp70 in A. japonicus by measuring the survival rates at different
treatments and the temporal profile of Hsp70
expression after a heat shock exposure.
MATERIALS AND METHODS
Collection and maintenance of animals
Juvenile sea cucumbers were sampled at 15°C
from Zhenzhong Aquaculture Corporation, Weihai,
Shandong Province, China. In the laboratory,
temperatures of the rearing water were gradually
increased to 20°C and kept at 20°C for 2 months.
Sea water was filtered using a sand filter and the
salinity was 28–30. One-half or two thirds of the
rearing water was exchanged by fresh equaltemperature sea water daily. Juveniles were fed ad
libitum daily at 13:00 hours with a laboratory-made
formulated feed (22.9 ⫾ 0.2% crude protein,
2.1 ⫾ 0.2% fat, 34.7 ⫾ 0.6% ash and 9.0 ⫾ 0.0%
moisture, 10.6 ⫾ 0.0 kJ/g), which mainly contained
powders of Sargassum spp., fishmeal, sea mud,
wheat, and vitamin and mineral premixes. Aeration
was provided continuously and the light was
natural light.
© 2008 Japanese Society of Fisheries Science
574
FISHERIES SCIENCE
Thermal limits
In total, 720 specimens were used in these studies.
The initial body weight of these animals was
0.5 ⫾ 0.1 g and individuals of essentially identical
weight were assigned at random to 72 aquaria
(10 inds/aquarium). There were nine tanks
(450 mm ¥ 250 mm ¥ 350 mm) at each of eight
temperatures (27, 28, 29, 30, 31, 32, 33 and 34°C)
with three tanks for each of the three exposure
duration times (1, 1.5 and 2 h). For each trial, the
sea cucumbers were put into a 2-L glass beaker.
The temperature of the water in the beaker was
adjusted to the designed values by a HBS-1000
water bath (EYELA, Tokyo, Japan) before the sea
cucumbers were put into the beaker. The water
temperature was recorded every 5 min. After
high-temperature exposure, sea cucumbers were
returned to 20°C sea water for 7 days. Mortalities
were recorded over time. In the period of the
experiment, the rearing conditions were similar to
those used during the acclimation period.
Induced thermotolerance
Sublethal and lethal temperatures were selected as
30 and 34°C, respectively, based on the results of
the experiment on thermal limits.
In total, 120 individuals were exposed to sublethal heat shock (SHS, 30°C, 2 h) and returned to
20°C for up to 7 days. The temperature control
method was similar to that in the experiment of
thermal limits. At each selected time (24 h, SHS24;
48 h, SHS48; 72 h, SHS72), three groups, each comprising 10 individuals, were selected randomly and
exposed only to lethal heat shock at 34°C for 2 h
(LHS). Subsequently, these animals were returned
to 20°C for 7 days to assess mortalities. Thirty sea
cucumbers that only experienced SHS heat shock
without LHS (NO-LHS) were transferred to 20°C for
7 days to assess the mortality of juvenile A. japonicus which only encountered 30°C heat shock.
Thirty individuals without previous sublethal
heat shock (NO-SHS) were exposed to lethal heat
shock (34°C, 2 h) and returned to 20°C for 7 days to
assess survival rate.
Y Dong and S Dong
The whole body was used when estimating
Hsp70 expression because the test animals were
too small to permit sampling of intestines and
other organs. Sea cucumbers were rinsed in sterile
sea water, then in distilled H2O, and homogenized
on ice with homogenization buffer in a Cell Lysis
Kit (BBI, Ontario, Canada) containing a protease
inhibitor cocktail. Homogenates were centrifuged
(10 000 ¥g, 10 min), and the supernatant was collected and kept at -70°C until use.
Gel electrophoresis of protein extracts was
performed in 10% sodium dodecylsulfate–
polyacrylamide gel electrophoresis (SDS-PAGE)
according to Laemmli.16 Protein samples were subjected to gel electrophoresis in the presence of
2-mercaptoethanol. Semi-dry electrotransfer was
performed onto PVDF-Immobilon membranes.17
Membranes were blocked and incubated with
H5147 antibody (Sigma, St. Louis, MO, USA)
(diluted 1:1000) for 2 h at 37°C. Then the immune
complexes were visualized by incubation with
antimouse IgG (horseradish peroxidase conjugated, Boster, Wuhan, China) followed by staining
with diaminobenzidine (DAB). Band intensity was
quantified using GeneTools software (Syngene,
Cambridge, UK).
Protein was determined as described by Bradford18 with bovine serum albumin as standard.
Hsp70 levels of sea cucumbers that were subjected to an acute temperature exposure (from 10
to 30°C) for 1 h were also analyzed and used as a
standard sample. Hsp70 levels in this study were
shown as values relative to the level of the standard
sample (relative unit, RU).
Statistics
The data were analyzed by SPSS for Windows statistical package (SPSS, Chicago, IL, USA). Probit
analysis was used to detect possible associations of
mortalities and heat shock temperatures.19 Differences in survival rates at SHS24, SHS48, SHS72,
NO-LHS and NO-SHS and differences in Hsp70
levels of control and samples at 1, 24, 48 and 72 h
were analyzed by one-way analysis of variance
(anova) followed by post hoc Duncan’s multiple
range tests. Differences were considered significant if P < 0.05.
Expression of Hsp70
At the selected times (1, 24, 48 and 72 h) after the
sublethal heat shock (30°C, 2 h), 10 SHS individuals
were randomly sampled and immediately frozen in
liquid nitrogen for analyses of Hsp70 expression.
Ten sea cucumbers without SHS were also sampled
as controls.
© 2008 Japanese Society of Fisheries Science
RESULTS
Thermal limits
The response of A. japonicus to thermal stress is
shown in Figure 1. There were no mortalities in the
Induced thermotolerance of sea cucumber
FISHERIES SCIENCE
80
60
40
20
0
b
100
Survival rate (%)
Survival rate (%)
100
575
27
28
29
30
31
32
33
34
O
Temperature ( C)
Fig. 1 Measured survivorship of Apostichopus japonicus at exposure durations of 1 h (䊐), 1.5 h (䊉) and 2 h
(䉭). Sample number n = 10. The lower limit of lethality
was 30°C, and 34°C killed all individuals tested.
sea cucumbers that were only exposed to the heat
shock of 30°C. However, all sea cucumbers were
killed when they were put into 34°C water for 1, 1.5
and 2 h. Therefore, 30 and 34°C were selected as
sublethal and lethal temperatures for this batch of
juvenile sea cucumbers. The temperature lethal to
50% of the sample (TL50) and 95% confidence
limits at 1, 1.5 and 2 h exposure were 32.74°C
[32.32, 33.22°C], 32.37°C [32.08, 32.68°C] and
30.94°C [30.71, 31.15°C], respectively. TL50 values
progressively decreased with the duration of exposure for juvenile A. japonicus.
b
b
80
60
c
40
20
a
all dead
0
NO-SHS
24h
48h
72h
NO-LHS
Hours after sublethal
heat shock
Fig. 2 Induced thermotolerance following exposure to
sublethal heat shock (SHS, 30°C) for 2 h. SHS sea cucumbers were incubated at 20°C for 24, 48 and 72 h, and then
were given a lethal heat shock (LHS, 34°C for 1 h). Survival rates were estimated after 1 week at 20°C. NO-SHS,
sea cucumbers without sublethal heat shock; NO-LHS,
sea cucumbers given SHS without LHS. Values are
mean ⫾ standard deviation (n = 3). Means with different
letters are significantly different (P < 0.05).
Induced thermotolerance
Exposed to lethal heat shock, the survival rates of
the animals with a previous sublethal heat shock
were higher than those without the sublethal heat
shock (F(4,14) = 84.25, P = 0.00). All sea cucumbers
without the previous sublethal heat shock (NOSHS) were killed at the lethal temperature. On
the other hand, all sea cucumbers given a sublethal heat shock without the following lethal heat
shock (NO-LHS) survived. The survival rates
(mean ⫾ standard deviation) in the SHS24, SHS48
and SHS72 groups were 90.0 ⫾ 10.0, 90.0 ⫾ 10.0 and
36.7 ⫾ 11.5%, respectively (Fig. 2).
Expression of Hsp70
The Hsp70 antibody used for Western blot analysis
only detected one band in all specimens (Fig. 3).
Fig. 3 Western blot image of Hsp70 in Apostichopus
japonicus after 30°C sublethal heat shock. Captions
above each lane correspond to exposure duration (h);
Std, standard. Equivalent amounts of protein (20 mg)
were loaded in 10% SDS-PAGE gel, followed by semidry
electrotransfer and immunostaining using diaminobenzidine (DAB).
Exposed to 30°C, an immediate onset of Hsp70
expression occurred. The maximum and minimum
levels of Hsp70 appeared at 1 and 72 h after the
SHS exposure, respectively. The Hsp70 levels after
1, 24 and 48 h heat shock exposure were significantly higher than that in the control. However,
there was no significant difference between the
Hsp70 level after 72 h heat shock exposure and the
control (F(4,49) = 32.34, P = 0.00) (Fig. 4).
© 2008 Japanese Society of Fisheries Science
FISHERIES SCIENCE
Relative levels of Hsp70 (RU)
576
60
b
50
c
40
d
30
20
10
a
a
0
01
24
48
72
Time after heat shock (h)
Fig. 4 Temporal expression pattern of Hsp70 of sea
cucumber Apostichopus japonicus after sublethal heat
shock exposure. The control level of Hsp70 (relative
units, RU) is plotted at 0 h after heat shock. Values are
⫾standard error (n = 10). Means with different letters are
significantly different (P < 0.05).
DISCUSSION
As an aquatic ectotherm, A. japonicus is sensitive
to changes of ambient temperature. Previous
studies showed that the appropriate temperature
for growth was between 12 and 21°C, and the
optimum was at approximately 15–18°C.20 Temperature fluctuations had significant effects on
growth of A. japonicus.21,22 In the present study,
most juvenile A. japonicus moved quickly at first,
and then aggregated when they were exposed to
high temperatures. This result indicated that
behavioral thermoregulation might occur when
sea cucumbers were exposed to high temperature.
At the relative lower temperatures of exposure (27,
28, 29 and 30°C), most sea cucumbers showed no
abnormal behaviors during the whole exposure.
However, at the relative higher exposure temperatures (31, 32, 33 and 34°C) sea cucumbers twisted
irregularly at first, and then were less active and
became almost quiescent. Some of them could
move and eat again, and others gradually became
white pellets of tissue heavily infested by microorganisms several days after the heat shock. Therefore, the sea cucumbers experiencing heat shocks
in the present study were returned to 20°C for
7 days to determine whether they were dead. All
sea cucumbers that experienced 30°C heat shock
survived and took food again. On the other hand,
all sea cucumbers that experienced 34°C heat
shock died. Therefore, 30 and 34°C were defined as
© 2008 Japanese Society of Fisheries Science
Y Dong and S Dong
the sublethal and lethal temperatures, respectively,
for juvenile sea cucumbers of the sizes used in this
experiment.
After exposure to a 30°C heat shock, some sea
cucumbers could survive a subsequent 34°C heat
shock exposure. This result indicated that a previous sublethal heat shock could induce thermotolerance in A. japonicus, as described in previous
studies of other species.1,3,9,13,23,24 In the present
study, the duration of the induced thermotolerance in the sea cucumber was shorter than that of
Pacific oyster Crassostrea gigas.3 After a sublethal
heat shock, the induced thermotolerance of
C. gigas remained for approximately 14 days. The
survival rates of sea cucumbers in both SHS24 and
SHS48 groups were 90 ⫾ 10% after LHS heat shock
exposure. However, the survival rate decreased
rapidly for SHS72 (Fig. 2). This result indicated that
the induced thermotolerance decreased significantly from the third day after the sublethal heat
shock in A. japonicus. This difference of the duration of the induced thermotolerance between
A. japonicas and C. gigas might be caused by the
difference in the temporal pattern of heat shock
proteins.
In the present study, only one band could be
detected using H5147 antibody (Sigma). There may
be more bands that could not be separated by onedimensional gel used in the present study. Therefore, it was difficult to tell whether the band was
the constitutive or inducible isoform of Hsp70.
Because the intensity of the band increased
approximately 10 times after the heat shock, and
other researchers found a dose-dependent
increase in the level of Hsp70 using the same antibody in the sea urchin embryos,25 this band could
represent the inducible level of Hsp70 in the sea
cucumber.
The expression of Hsp70 was induced 1 h after
the SHS exposure in A. japonicus (Fig. 4). The
enhancement of Hsp70 was consistent with the
cellular need for protein repair and stabilization.
Heat shock proteins can protect other proteins
from unfolding, refold denatured protein, or target
denatured protein for degradation.10,24,26 This high
Hsp70 level after the sublethal heat shock in A.
japonicus indicated a high level of protein damage
and a high ability of A. japonicus to respond
adaptively to heat shock, as described in previous
studies.5,11,27,28
The temporal profile of Hsp70 expression
showed that the levels of Hsp70 increased initially,
and then linearly decreased with time after the
sublethal heat shock exposure (Fig. 4). This result
suggested that the half life of Hsp70 in A. japonicus
was rather short and was similar to that of some
other animals (6–9 h in Drosophila, 2 days in
Induced thermotolerance of sea cucumber
FISHERIES SCIENCE
Morris hepatoma 7777 cells, ⱕ6 h in Tegula funebralis).27,29,30 However, the high level of Hsp70 in
C. gigas after heat shock could maintained for at
least 14 days.3 The difference in the temporal patterns of Hsp70 in different species might be attributed to the length and severity of the thermal stress
and to the stability of hsp70 mRNA, which may vary
as a function of temperature.7
The appearance and decay of Hsp70 shared a
close temporal relationship with the induction and
disappearance of thermotolerance in A. japonicus.
The relationship might occur in the translational
level. At high temperature, the synthesis of Hsps
during heat shock blocks the synthesis of nonHsps in some organisms because of the preferential translation of hsp70 mRNA.9,23,31–34 The
enhancement of Hsp could prevent the inhibition
of non-Hsp synthesis during subsequent exposures to heat.35,36 Therefore, acclimation to higher
temperatures could lead to an increased ability
to synthesize proteins at higher temperatures.5
Therefore, the higher survival rate of the SHS sea
cucumbers at the lethal temperature exposure
might be attributed to their increased ability to
synthesize proteins at 34°C.
CONCLUSION
The sublethal and lethal temperatures for juvenile
A. japonicus of the sizes used in this experiment
were 30 and 34°C, respectively. A previous sublethal heat shock could increase the animal’s thermotolerance, and this induced theromotolerance
was maintained for at least 2 days; there was a
close relationship between the induction of thermotolerance and the expression of Hsp70 in the
sea cucumber A. japonicus.
ACKNOWLEDGMENTS
We are grateful to Professor GN Somero of the
Hopkins Marine Station, Stanford University, for
useful comments on this study and careful revision
of the writing. We thank XL Shan and MJ Liu for
raising the animals. This work was supported by
the Chinese National Science Foundation (Grant
no. 30400333) and National Key Technologies R&D
Program of China (grant nos 2006BAD09A01,
2006BAD09A06).
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