| 1 The effects of acute temperature change and diel fluctuating temperature on isozyme patterns of LDH, EST and SOD of sea cucumber Apostichopus japonicus were studied. In the experiment of acute temperature change, young A. japonicus were subjected to heat exposure (from 10 to 20℃) and cold exposure (from 20 to 10℃), respectively. Samples were taken at 0 h, 1 h, 3 h, 12 h, 24 h and 72 h after heat or cold exposure, respectively. In the experiment of diel fluctuating temperatures, young A. japonicus were subjected to four temperature treatments (18℃, 18±2℃, 18±4℃and 18±6℃). Results showed that there was no expression of LDH isozyme. In the experiment of acute temperature changes, within 12 h heat exposure, the EST and SOD bands were induced. After 1 h cold exposure, a new EST-2 band was detected. After 3 h cold exposure there were new SOD-2 and SOD-3 bands induced. In the experiment of diel fluctuating temperature, no change was detected in the EST and SOD isozyme pattern. It indicated that there were immediately adaptive responses in A. japonicus exposed to acute temperature changes, which were significant to adapt in the intertidal zone.2 Sea cucumbers A. japonicus were collected monthly from a field cultured pond in Jiaonan from July 2005 to June 2006 (except September, January and February). Changes of activities of SOD, CAT, GST and expression of Hsp70 in body walls and intestines were studied. Results showed that the activities of CAT (in the two tissues), SOD and GST (only in body walls) increased gradually from summer to autumn, and peaked in October or November. In spring, all the activities maintained the low levels. Enhanced antioxidant responses occurred in autumn after aestivation of sea cucumber, which were significantly different with those in spring. The high level of Hsp70 in body wall was present in November and March, but that in intestine appeared in August. Hsp70 level in intestine correlated positively with the water temperature. It indicated that Hsp70 expression was tissue-specific. Exposed to thermal stress, intestine was more sensitive than body wall. Because body walls participate in respiration, acitvities of antioxidant enzymes in body walls were connected with oxidate stress. CAT and GST, SOD and Hsp70 have positive correlations, respectively.3 Aestivation is an adaptation of the sea cucumber A. japonicus to high temperature, however, the causations and physiological responses of aestivation are not well understood. This study deals with the relationship between temperature and aestivation. Sea cucumbers were allocated into four treatments. In two treatments of temperature elevation, the ambient temperature gradually was increased from 16°C to 26°C linearly (treatment FA) or by a fluctuating temperature profile (treatment FB). Two control treatments maintained constant temperatures of 16°C and 26°C, and were designated as optimum temperature of growth and threshold of aestivation, respectively. During the 40-day experiment, body weight, oxygen consumption, daily food intake, catalase (CAT) and superoxide dimutase (SOD) activities and heat shock protein 70 (Hsp70) levels were determined periodically. When the temperature gradually increased from 16°C to 26°C, the body weight of the tested sea cucumbers decreased gradually. After the ambient temperature reached 26°C, the tested sea cucumbers in treatments of FA and FB were reared at 26°C for an additional twenty days. During this period, symptoms of aestivation appeared in the tested sea cucumbers. Activities of antioxidases and Hsp70 levels increased when the ambient temperature increased from 16°C to 26°C, and decreased when the temperature was kept at 26°C. These results indicate that aestivation in A. japonicus is an adaptive strategy to reduce the production of reactive oxygen species (ROS) and denatured proteins which were induced at high temperature.4 Many farmers transported juvenile sea cucumbers to the south of China in late autumn and maintained them there until next spring. The difference of thermal resistance will be determined in sea cucumbers A. japonicus with the different thermal histories. Sea cucumbers were acclimated at 22?C (WA) or 12?C (CA) for 30 days and then maintained at 17?C for a week. After that, the lethal temperature of WA and CA sea cucumbers was 31?C , and the temperature lethal to 50% of the sample (TL50) in WA and CA sea cucumbers were 32.06?C (31.41-32.72?C) and 31.5?C, respectively. To determine the temporal profile of Hsp70 expression, juveniles were exposed to 30?C for 2h and returned to 17?C. Hsp70 expressions in the visceral mass were slightly different between the two groups of juveniles. After 2h recovery from heat shock (25, 27, 29, 31, 32?C), the temperature of maximal responses was measured. The maximum induction temperatures for body wall in WA and CA sea cucumbers were 31?C and 29?C, respectively. Hsp70 level in visceral mass in CA sea cucumbers was lower than that in WA sea cucumbers at heat shock (29?C and 31?C). These results showed that the thermal history could change the thermal resistance, which was related to the changed expression pattern of Hsp70 in the sea cucumber. Exposed to thermal stress, visceral mass might be more sensitive than body wall in sea cucumbers.5 According to body color variation, sea cucumber A. japonicus can be divided into red, green and black three variants, and there are distinct differences in thermotolerance and aestivation at summer between red and other two variants. In the present study, thermal limit, induced thermotolerance and heat shock protein 70 (Hsp70) levels after heat shock were determined to elucidate the difference in thermal adaptation between red and green variants. Our results indicated that there was no significant difference in the temperature lethal to 50% of the sample (TL50) between red (30.01-32.18℃) and green (30.10-32.14℃), but green could acquire higher thermotolerance than red after a prior sublethal heat exposure (30℃for 2h). After 72-hour recovery from heat shock of 30℃for 2h, the survival rate of green was over 50% and the survival rate of red was less than 5% when they were treated in a heat shock of 33℃for 2h. Two-way ANOVA analysis showed that Hsp70 levels of green in both transcriptional and posttranscriptional levels were significantly higher than those of red at different time points after the heat shock of 30℃, and the stability of hsp70 mRNA of red was significantly lower than that of green. Our findings suggest that different vatiants within same species, which have similar thermal limit, can acquire different thermotolerance after a prior sublethal heat shock and the difference of induced thermotolerance between green and red is closely related to the different expression pattern of Hsp70, especially the stability of hsp70 mRNA.
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