Difference in Thermotolerance Between Green and Red Color Variants of the Japanese Sea Cucumber
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Reference: Biol. Bull. 218: 87–94. (February 2010)
© 2010 Marine Biological Laboratory
Difference in Thermotolerance Between Green and
Red Color Variants of the Japanese Sea Cucumber,
Apostichopus japonicus Selenka: Hsp70
and Heat-Hardening Effect
YUN-WEI DONG1,*, TING-TING JI1, XIAN-LIANG MENG1, SHUANG-LIN DONG1, AND
WEI-MING SUN2
1
The Key Laboratory of Mariculture, Ministry of Education, Fisheries College, Ocean University of
China, Qingdao, People’s Republic of China, 266003; and 2Ocean School, Yantai University,
People’s Republic of China, 264005
Abstract. We studied thermal tolerance limits, heat-hard- Introduction
ening, and Hsp70 to elucidate the difference in thermotol-
Heat shock proteins (Hsps) have been observed in all
erance between two color variants of the sea cucumber
major phylogenetic lineages (reviewed by Feder and Hof-
Apostichopus japonicus. Green and Red variants occupy
mann, 1999). They play essential roles in protecting cells
different habitats and have different aestivation responses to
from damage generated by a variety of stressors, and their
high temperature in summer. In the absence of heat-hard- expression is closely regulated in a stress-dependent manner
ening, the variants showed no difference in the temperature (Morimoto, 1998). A major physiological function associ-
at which 50% of the individuals died: Green, 31.49 °C; Red, ated with the Hsps is induced thermotolerance, which is
31.39 °C. However, Green specimens acquired higher ther- defined as the ability of a cell or organism to become
motolerance than Red after a prior sublethal heat exposure. resistant to heat stress after a prior sublethal heat exposure
After 72 h of recovery from a heat-hardening treatment (Kregel, 2002).
(30 °C for 2 h), the survival of Green variants was more than The expression of heat shock proteins exhibits variation
50% and that of Red was less than 5% when they were that reflects both the evolutionary histories of species and
treated at 33 °C for 2 h. Levels of mRNA and protein for the recent thermal conditions that individuals have faced
Hsp70 were significantly higher in Green than Red after the (Hochachka and Somero, 2002). Tomanek and Somero
heat shock of 30 °C, and the stability of hsp70 mRNA of (1999) found that the temperatures at which Hsp synthesis is
Green was significantly higher than that of Red. Our find- first induced (Ton) and the temperature at which maximal
ings suggest that within the same species, different variants Hsp synthesis occurs (Tpeak) were different among conge-
that have similar thermal limits in the absence of heat- neric marine snails (genus Tegula). These congeners en-
hardening can acquire different thermotolerances after a counter different degrees of thermal stress after emersion
prior sublethal heat shock. The difference in induced ther- due to their occurrence at different heights in the intertidal
motolerance between Green and Red is closely related to the zone and different latitudinal distributions. The heat-shock
expression pattern of Hsp70, which was partly governed by response of four congeners of Lottia limpets was also stud-
the stability of hsp70 mRNA. ied to elucidate the adaptation and evolution of Hsp70 to
temperature. The two high-intertidal species, Lottia scabra
and L. austrodigitalis, had higher constitutive levels of
Hsp70 than the two low-and mid-intertidal congeners, L.
Received 27 April 2009; accepted 21 October 2009. pelta and L. scutum. No induction of Hsp70 occurred in L.
* To whom correspondence should be addressed. E-mail: dongyw@ouc. scabra. These findings suggest that high-intertidal conge-
edu.cn ners of Lottia employ a “preparative defense” strategy in-
8788 Y.-W. DONG ET AL.
volving maintenance of high constitutive levels of Hsp70 in (or populations of conspecifics) for comparison (Hochachka
their cells as protection against periods of extreme and and Somero, 2002). The slightly different thermotolerance
unpredictable heat stress (Dong et al., 2008a). and minor genetic divergence between Green and Red vari-
The sea cucumber Apostichopus japonicas Selenka pre- ants of A. japonicus provide an especially suitable study
sents another excellent study system for examining the system for examining fine-scale temperature adaptation.
heat-shock response. This species commonly occurs in shal- Previous studies indicated that the expression of heat shock
low coastal waters from the intertidal zone to depths more protein was very important for the sea cucumber in dealing
than 100 m along the North Pacific coast (Liao, 1997). with thermal and osmotic stress (Dong et al., 2008b, c). A
Temperature is a critical factor affecting the survival, previous exposure to sublethal heat shock (30 °C, 2 h) could
growth, and reproduction of the species (Yang et al., 2005; increase the survival rates of the sea cucumbers when they
Dong et al., 2006). According to records for 2005–2006, the were later exposed to 34 °C. The induced thermotolerance
water temperature in wild ponds used for the culture of sea could last for at least 2 days, and a close relationship existed
cucumbers had dramatic seasonal variations. The water between the induction of thermotolerance and the levels of
temperature ranged from ⫺0.4 to 31.8 °C in Jiaonan, Qing- Hsp70 in the sea cucumber (Dong and Dong, 2008). In the
dao, People’s Republic of China (35°53⬘N, 120°00⬘E), and present study, we investigated the difference in induced
it often exceeded 30 °C in summer (from June to Septem- thermotolerance between Green and Red variants, and the
ber) (Dong, Y.-W., unpubl. data). When the water temper- constitutive and inducible expression of Hsp70 in transcrip-
ature is above 25 °C, adults of A. japonicas usually enter a tional and post-transcriptional levels. These results help to
state of aestivation to cope with the unfavorable conditions elucidate further the difference in thermotolerance between
(Choe and Ohshima, 1961; Yang et al., 2006). Characteris- these two variants of Japanese sea cucumber, and provide
tics of aestivation in A. japonicus include fasting, gut tract insight into the evolutionary differences in the heat-shock
degeneration, weight loss, and metabolic rate depression (Ji responses between two variants within the same species.
et al., 2008). Large-scale mortality frequently takes place in
summer when the temperature is too high or the duration of Materials and Methods
high temperature is too long.
According to variation in body color, A. japonicus can be Animal collection and acclimation
divided into Red, Green, and Black variants, as first reported One-year-old Green (wet weight 0.55 ⫾ 0.03 g) and Red
by Mitsukuri (1912). Choe and Ohshima (1961) found that (wet weight 0.45 ⫾ 0.03 g) variants were collected from
there were distinct differences in morphological character- Jiaonan Aquatic Seeding Breeding Farm, Qingdao, and
istics, ecology, and distribution between Green and Red Haiyang Aquatic Seeding Breeding Farm, Yantai, respec-
variants. The colors of the Green and Red variants are dark tively (both in Shandong province, People’s Republic of
bluish green and brownish red, respectively. The shapes of China), and both of them were acclimated at 16 °C for 3
Polian vesicles are different in Green and Red variants; the weeks. There was no significant difference in initial body
former is stumpy with a round tip, while the latter is slender weight between the two variants (P ⬎ 0.05). The Green
with a projected tip. The Green variant has a preference for variant is native to Qingdao, and the Red variant was
somewhat lower salinity and often inhabits sand and introduced from Kyushu, Japan, in 2006. The one-year old
muddy-sand areas. In contrast, the Red variant is often Red variant individuals used in the present study were
found in rock pebble and gravel. Recently, using isozyme acquired from artificial breeding in Yantai.
(Kanno and Kijima, 2003) and microsatellite analysis Seawater was filtered using a sand filter, and the salinity
(Kanno et al., 2006), researchers found genetic differences was 28 –30 psu. One-half or two-thirds of the rearing water
between the Red and the other two variants. Tolerances of was exchanged using fresh equi-temperature seawater daily.
the Red variant to salinity (Yamamoto et al., 2003) and high Aeration was provided continuously, and the photoperiod
temperature (Yamamoto et al., 2005) were different from was 12 h light:12 h dark. The sea cucumbers were fed ad
those of the Green and Black variants. In summer when the libitum daily at 1300 h on a commercial formulated feed
water temperature was high, almost all Red variants were in (22.9% ⫾ 0.2% crude protein, 2.1% ⫾ 0.0% fat, 34.7% ⫾
aestivation compared to only part of the Green variant 0.6% ash, and 9.0% ⫾ 0.0% moisture; 10.6 ⫾ 0.0 kJ g⫺1
population (Choe and Ohshima, 1961). This divergence in energy), which mainly contained powders of Sargassum
behavior might be due to different tolerances to high tem- spp., fish meal, sea mud, wheat, and vitamin and mineral
perature between the variants (Yamamoto et al., 2005). premixes.
Comparative study of temperature responses in different
color variants is useful for elucidating the evolution of an
Thermal tolerance limit
organism’s physiological adaptation to temperature. To ad-
dress question about thresholds of thermal perturbation and The upper thermal limits of Green and Red variants were
adaptation, a critical first step is to select appropriate species assessed according to the method described in Dong andHEAT HARDENING OF SEA CUCUMBER 89
Dong (2008). Ten specimens of each variant were used at Table 1
each of the four temperatures (30, 31, 32, and 33 °C). For Primer sets designed for semi-quantitative RT-PCR analysis of inducible
each trial, individuals were placed into a 2-l glass beaker. hsp70 mRNA in the sea cucumber Apostichopus japonicus1
Water temperature in the beaker was adjusted to the desired
values in an HBS-1000 water bath (EYELA, Tokyo, Japan) Primers Primer sequences
before the sea cucumbers were put into the beakers. The Hsp-F 5⬘-ATGCCTAGAACCAGTAGAGAAAG-3⬘
water temperature was recorded every 5 min. After 2 h of Hsp-R 5⬘-TGTCGTTCGTGATGGTGATT-3⬘
exposure at test temperatures, sea cucumbers were returned -actin-F 5⬘-ACACGGTATCGTCACAAACTGG-3⬘
to 16 °C for 7 days. Mortalities were recorded over time. -actin-R 5⬘-AGGATAGCGTGAGGAAGAGCAT-3⬘
During the experiment, the rearing conditions were similar 1
Hsp-F and Hsp-R are used for amplifying hsp70, and -actin-F and
to those used during the acclimation period. -actin-R are used for -actin.
Induced thermotolerance
On the basis of the experiment on thermal limits, tem- cycles (for -actin) at 95 °C for 30 s, 55 °C for 30 s, 72 °C
peratures of 30 and 33 °C were selected as the sublethal and for 1 min, and a final extension step at 72 °C for 10 min. The
lethal temperatures, respectively. The temperature control PCR products were separated in 1.2% agarose gel stained
method was similar to that in the experiment on thermal with ethidium bromide. To confirm the specificity of RT-
tolerance limits. A total of 150 individuals of each variant PCR amplification, the products were purified from the gel
were exposed to sublethal heat shock (30 °C, 2 h) and and sequenced. The optical densities of amplified bands
returned to 16 °C for up to 7 days. At each selected time (2, were analyzed using GeneTools software, ver. 3.04 (Syn-
24, 48, and 72 h) during this period, three groups, each gene, USA), and the abundance of hsp70 was normalized to
comprising 10 individuals, were selected randomly and the corresponding -actin abundance in all samples and
exposed to lethal heat shock (33 °C, 2 h). Subsequently, expressed as the ratio of the optical densities of hsp70 and
these animals were returned to 16 °C for 7 days to assess -actin (Chsp70/C-actin).
mortalities.
Western blotting
Expression of Hsp70 after heat shock Expression of Hsp70 at the protein level was quantified
At the selected time points (2, 24, 48, and 72 h) after the following the methods of Dong et al. (2008b). Specimens
sublethal heat shock (30 °C, 2 h), 10 individuals of each were treated with homogenization buffer in the Cell Lysis
variant were randomly sampled and immediately frozen in kit (BBI, Canada) containing a protease inhibitor cocktail.
liquid nitrogen for subsequent analysis of the levels of There were five replicates per treatment. Specimens were
Hsp70. As controls, 10 of each variant without heat shock homogenized on ice and centrifuged (10 000 ⫻ g, 15 min).
were sampled. The supernatant fraction was collected and kept at ⫺70 °C
until use. Protein was determined as described by Lowry et
al. (1951) with bovine serum albumin as standard.
Semi-quantitative RT-PCR analysis of hsp70
Gel electrophoresis of protein extracts was performed in
Total RNA was isolated from about 80 mg of body wall 10% polyacrylamide gels according to Laemmli (1970).
tissues by Trizol reagent (Invitrogen, USA). A sample of 1 Protein samples (each of 30 g total protein) were subjected
g of total RNA was used as the template for synthesis of to gel electrophoresis in the presence of dithiothreitol. Semi-
the first strand of cDNA. We selected the -actin gene as an dry electrotransfer was performed according to Kyhse-
internal control. Two pairs of primers, Hsp-F and Hsp-R, Andersen (1984) onto PVDF-Immobilon membranes (0.45
-actin-F and -actin-R, were designed based on the se- m, Millipore). Membranes were blocked and incubated
quences from our previous work (Table 1). with monoclonal anti-Hsp70 antibody (H5147, Sigma,
Semi-quantitative PCR conditions and components for USA) (diluted 1:3000) for 2 h at 37 °C. Then the immune
hsp70 and -actin were optimized, especially for the am- complexes were visualized by incubation with anti-mouse
plification cycles and annealing temperatures. The opti- IgG (horseradish peroxidase conjugated) followed by ECL
mized PCR was carried out in a 25-l reaction volume reagents (Amersham, USA) and exposure of blots to X-ray
containing 2.5 l of 10⫻ PCR buffer, 1.6 l of MgCl2 (25 film (Kodak, USA). Band intensity was quantified using
mmol l-1), 2.0 l of dNTP (2.5 mmol l-1), 1 l of each GeneTools software (Syngene, USA). An equal quantity of
primer (10 pmol ml-1), 15.875 l of PCR-grade water, 0.125 protein (30 g) of a sea cucumber that had been subjected
l (5 U/l) of Taq polymerase (Promega, USA), and 1 l of to an acute temperature flux (from 10 °C to 30 °C) for 2 h
cDNA reaction mix. The PCR program was performed at was also added to each gel. The Hsp70 level of this sample
95 °C for 5 min, followed by 28 cycles (for hsp70) or 26 was designated as a standard for normalization. Hsp7090 Y.-W. DONG ET AL.
levels in this study were expressed as values relative to the
level of the standard sample (relative unit, RU) (%).
Statistics
Data were analyzed using an SPSS 13.0 for Windows
statistical package (SPSS Inc., Chicago, IL). The homoge-
neity of variances of data was tested using Mauchly’s test of
sphericity. The temperatures at which 50% of the sea cu-
cumbers died (TL50) and the 95% confidence limits were
calculated using Probit analysis. For the heat-shock exper-
iment, differences in hsp70 mRNA and Hsp70 levels among
different variants and time points after heat shock were
determined using two-way ANOVA. At the same time
point, difference in survival, hsp70 mRNA, and Hsp70
levels between Green and Red variants were analyzed using
independent samples t-tests. The temporal patterns of hsp70 Figure 1. Induced thermotolerance in sea cucumber Apostichopus
mRNA levels at 2, 24, and 48 h after sublethal heat shock of japonicus following exposure to a sublethal heat shock (30 °C) for 2 h.
30 °C were fitted to the data using linear regression, and the After the sublethal exposure, sea cucumbers were incubated at 16 °C for 2,
difference in the slopes of the regressions were analyzed 24, 48, and 72 h, and then were given another heat shock (33 °C, 2 h).
Survivals were estimated after one week at 16 °C. Values are mean ⫾ S.D.
using an analysis of covariance (ANCOVA) (␣ ⫽ 0.05).
(n ⫽ 3). Asterisk indicates significant difference in survival between Green
and Red variants (P ⬍ 0.05).
Results
Thermal tolerance limits
individuals (F1, 24⫽ 6.781, P ⫽ 0.019). There was no
The mortalities of sea cucumbers increased with temper- significant effect of the interaction between color variants
ature between 30 and 33 °C. The survival of Green variants and time points after heat shock. After 72 h of recovery, the
at 30, 31, 32, and 33 °C were 100%, 70%, 30%, and 0%, survival of Red individuals was significantly lower than that
respectively. The survival of Red variants at those temper- of Green individuals (P ⫽ 0.044).
atures was 100%, 60%, 20%, and 0%, respectively. There
were no mortalities in either Green or Red individuals that Temporal change of Hsp70 expression
were exposed to a heat shock of 30 °C. However, all sea
The levels of hsp70 mRNA were determined using semi-
cucumbers were killed when they were put into 33 °C water
quantitative RT-PCR and normalized to the corresponding
for 2 h. Probit analysis showed that TL50 values and 95%
-actin abundance in all samples. The bands of hsp70 were
confidence limits of Green and Red variants were 31.49 °C
very clear, and there were obvious differences in the optical
(30.10, 32.14 °C) and 31.39 °C (30.01, 32.18 °C), respec-
intensities of different bands (Fig. 2). After exposure to
tively.
30 °C, an immediate onset of hsp70 transcription occurred
in both Green and Red individuals (Fig. 3). Two-way
Induced thermotolerance
ANOVA analysis showed that the hsp70 mRNA levels after
After a previous heat shock of 30 °C, sea cucumbers were 2 h of heat-shock exposure were significantly higher than
returned to water of 16 °C for recovery (2, 24, 48, or 72 h) those in the unheated controls (F4, 50 ⫽ 30.875, P ⬍ 0.001),
and then were put into water of 33 °C to measure their and decreased with the recovery duration at 16 °C. Two-
survival. There was no mortality in either Green or Red way ANOVA analysis showed that the levels of hsp70
variants exposed to only 30 °C. Post-recovery survival after mRNA in Green individuals were significantly higher than
a heat shock of 33 °C showed the occurrence of heat- those of Red individuals (F1, 50 ⫽ 19.604, P ⬍ 0.001), and
hardening. After heat-hardening, the survival of Green vari- the hsp70 mRNA levels in Green individuals after 24, 48,
ants was respectively 80%, 83.33%, 66.67%, and 50% after and 72 h recovery were significantly higher than hsp70
2, 24, 48, and 72 h of recovery from the heat shock of 30 °C mRNA in Red individuals at corresponding time points.
(Fig. 1). The survival of Red individuals was 62.5%, There was no significant effect of the interaction between
66.67%, 45.83%, and 4.167%, respectively, after the same color variants and time after heat shock. The temporal
intervals of recovery from the heat shock of 30 °C. The pattern of hsp70 mRNA after the sublethal heat shock of 30
survival after the heat shock of 33 °C decreased with the °C could be fitted to the data using linear regression: HspG
duration of recovery (F3, 24⫽ 5.058, P ⫽ 0.012), and the ⫽ 1.205 ⫺ 0.017T (Green variant; R2 ⫽ 0. 831); HspR⫽
survival of Red individuals was lower than that of Green 0.989 ⫺ 0.020T (Red variant; R2 ⫽ 0.627). HspG and HspRHEAT HARDENING OF SEA CUCUMBER 91
Figure 2. Increases of hsp70 mRNA induced by heat shock in Green and Red variants of sea cucumber
Apostichopus japonicus. C, control samples without heat shock; 2 h, 24 h, 48 h, and 72 h respectively represent
hsp70 mRNA levels after 2, 24, 48, and 72 h of recovery from the heat shock.
are hsp70 mRNA levels in Green and Red variants, respec- When exposed to 30 °C, the expression of Hsp70 in both
tively; T is time of recovery after the heat shock of 30 °C Green and Red individuals increased immediately, and then
(from 2 h to 48 h). The results of ANCOVA showed that the showed a trend of decrease until the end of the experiment
slope of hsp70 mRNA versus time (2, 24, and 48 h) in the (F4, 40 ⫽ 22.093, P ⬍ 0.001) (Fig. 4). The maximum and
Red variant was significantly higher than the slope for the minimum levels of Hsp70 appeared at 2 h and 72 h after the
Green variant (F2, 30 ⫽ 32.950, P ⬍ 0.001). heat shock of 30 °C. The Hsp70 levels of Green individuals
The expressions of Hsp70 in sea cucumbers that were were significantly higher than those of Red individuals at
treated with a heat shock of 30 °C were determined at time points of 2 h and 24 h (F1, 40 ⫽ 18.494, P ⬍ 0.001).
different time points (no-heat control, 2, 24, 48, and 72 h). There was no significant effect of the interaction between
Only one band could be detected using the monoclonal color variants and time after heat shock.
anti-heat shock protein 70 antibody (H5147, Sigma). As we
found in previous studies (Dong and Dong, 2008; Dong et Discussion
al., 2008b), this band should include both constitutive and
inducible isoforms of Hsp70 in the sea cucumber. Temperature is a critical factor for growth, survival, and
distribution of the sea cucumber Apostichopus japonicus in
its natural habitat. As previous studies described, A. japoni-
cus can survive in water of 0 °C (Liao, 1997), and the
Figure 3. Temporal patterns of hsp70 mRNA in Green and Red
variants of sea cucumber Apostichopus japonicus after a sublethal heat
shock of 30 °C. C, control samples without the heat shock. After 2, 24, 48,
and 72 h of recovery from the heat shock, hsp70 mRNA was measured Figure 4. Temporal patterns of Hsp70 in Green and Red variants of
using semi-quantitative RT-PCR. Chsp70/C-actin was the ratio of optical sea cucumber Apostichopus japonicus after a sublethal heat shock of 30 °C.
densities of hsp70 and -actin. Values are mean ⫾ S.D. (n ⫽ 5). Asterisks C, control samples without the heat shock. After 2, 24, 48, and 72 h of
indicate significant differences in hsp70 mRNA between Green and Red recovery from the heat shock, Hsp70 levels were measured using western
individuals. Inset: The linear regressions of hsp70 mRNA versus time blotting. Values are mean ⫾ S.D. (n ⫽ 5). Asterisks indicate significant
(from 2 h to 48 h) after the sublethal heat shock of 30 °C. differences in Hsp70 between Green and Red individuals.92 Y.-W. DONG ET AL. optimal temperature for growth is between 12 and 21 °C Thus, variation of Hsp70 level in protein between unheated (Dong et al., 2005). In the present study, all individual Green and Red individuals was not a consequence of dif- survived a heat shock of 30 °C for 2 h. When heat-shock ferences in the corresponding mRNA of the inducible iso- temperature kept increasing, mortality was high; all indi- form, and might be due to the different levels of constitutive viduals died when they were exposed to heat shock of 33 °C isoform. for 2 h, which agrees with our previous observation (Dong In the present study, unheated Green and Red individuals and Dong, 2008). The survival rate of Red individuals was that were acclimated at 16 °C for 3 weeks had small slightly lower than that of Green individuals at temperatures amounts of inducible hsp70 mRNA. As reported in previous of 31 and 32 °C, but there was no difference in the temper- studies (Dong et al., 2006; Yang et al., 2006), a temperature atures at which 50% of the sea cucumbers died (the TL50 of 16 °C is optimal for the growth of the sea cucumber and values) between Green (30.10 –32.14 °C) and Red (30.01– cannot induce upregulation of inducible hsp70. The half life 32.18 °C) individuals overall. These results indicated that of Hsp70 of the sea cucumber is rather short (⬍3 days; there was no difference in thermotolerance between Green Dong and Dong, 2008), so the 3-week acclimation at 16 °C and Red when they encountered acute thermal stress. How- in the present study is long enough for the decay of induc- ever, Yamamoto et al. (2005) found that the tolerance of ible hsp70. Buckley et al. (2001) reported that mussels Red specimens to high temperature was lower than that of (Mytilus trossulus) that were acclimated at 10 –11 °C with- Green specimens. The differences between these two stud- out heat shock also had a baseline expression of inducible ies might be due to differences in the heat-shock protocols. hsp70. Thus, a baseline expression of inducible hsp70 Water temperature in Yamamoto’s experiment increased should exist in unheated sea cucumbers to protect against slowly, and it was more of a chronic stress than an acute other unknown stresses besides temperature. After 72 h of stress. In the present study, individuals were put into pre- recovery from the sublethal heat shock, hsp70 mRNA levels heated water and the heat shock was an acute stress. of the Red variant declined below the initial level in un- In the study of induced thermotolerance, we found that heated individuals. The low levels of hsp70 mRNA at 72 h Green individuals could acquire higher thermotolerance in the Red variant might be partly due to the downregulation than Red specimens after a previous sublethal heat shock of the baseline expression of inducible hsp70 during recov- (30 °C for 2 h). Though the aestivation of the sea cucumber ery from sublethal heat shock. After a 72-h recovery from is a complex physical phenomenon, the higher plasticity of heat shock, post-transcriptional levels of Hsp70 in both thermotolerance in Green individuals provides an explana- Green and Red variants were also lower than in the corre- tion of the difference in aestivation between Green and Red sponding initial unheated individuals. The downregulation variants. When water temperature increases continuously in should include decreases of both constitutive and inducible early summer, the higher thermotolerance induced by the isoforms of Hsp70. Further study is needed to clarify the previous high temperature could offer more protection cause of the downregulation of Hsp70 during recovery after against thermal stress in the Green variant. Therefore, it the sublethal heat shock in the sea cucumber. may not be necessary for some Green individuals, especially The appearance and decay of Hsp70 shared a close tem- young sea cucumbers, to enter a state of aestivation. How- poral relationship with the induction and disappearance of ever, the acquired thermotolerance is only a temporary induced thermotolerance in A. japonicus (Dong and Dong, protection against thermal stress. The mechanism by which 2008). In the present study, this relationship in both Green the plasticity of thermotolerance affects aestivation is still and Red individuals could also be detected. Hsp70 levels unclear. Therefore, additional investigation of the relation- increased immediately after the heat shock, and then ship between induced thermotolerance and entry into aesti- showed a trend of decrease until the end of the experiment. vation is necessary. The temporal pattern of hsp70 mRNA was similar to that of In the present study, the band detected using the current Hsp70. The close relationship between Hsp70 and thermo- antibody (H5147, Sigma) should include both constitutive tolerance indicated that heat shock could enhance the levels and inducible isoforms, as described in a previous study of Hsp70, which consequently enhanced the tolerance of the (Dong and Dong, 2008). Because of the difficulty in distin- sea cucumbers against subsequent thermal stress. guishing the constitutive and inducible isoforms of Hsp70, There were obviously differences in expression patterns it was hard to identify the inducible isoform of Hsp70 using of both transcriptional and post-transcriptional levels Hsp70 Western blotting analysis with the H5147 antibody. The after heat-shock exposure between the two color variants. RT-PCR analysis with specific primers was more effective. Two-way ANOVA analysis showed Hsp70 in both levels of Using specific primers, only the inducible isoform of hsp70 Green were significantly higher than those of Red individ- could be amplified. The Hsp70 levels in unheated Green uals. The adaptive response of the inducible isoform of heat individuals were higher than those of unheated Red indi- shock protein 70 to thermal stress could be described using viduals. However, there was no significant difference in the levels of hsp70 mRNA. As shown in Figure 3, there was hsp70 mRNA between unheated Green and Red individuals. no significant difference in hsp70 mRNA between unheated
HEAT HARDENING OF SEA CUCUMBER 93
Green and Red individuals. After exposure to a heat shock comments on this study and careful revision of the writing.
of 30 °C, hsp70 mRNA levels increased immediately in This work was supported by the Chinese National Science
both variants, and there was no significant difference in Foundation (Grant No. 30400333, 30771661), National Key
these levels between Green and Red specimens after a 2-h Project of Scientific and Technical Supporting Programs
recovery. However, the levels of hsp70 in Red individuals Funded by Ministry of Science & Technology of China
after 24 h and 48 h of recovery were significantly lower than (Grant No. 2006BAD09A01), and the 111 Project
those of Green individuals. After a 48-h recovery, no ex- (B08049).
pression of hsp70 could be detected in Red individuals. The
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