Introduction

The sea cucumber Apostichopus japonicus Selenka, which is believed to have aphrodisiac and curative properties, has been a prevailing aquaculture species in China (Fu et al. 2005). The total area of sea cucumber farming reached 153,626 hectare in China, and the total output was 137,754 tons in 2011 (DOF 2012). Cultivation in field ponds is one of the main culture modes in northern China. Because these ponds are usually shallow (2–3 m in depth), environmental factors are relatively sensitive to changing weather and tidal cycles. Based on our observations, the water temperature in these ponds can rapidly increase from 20 to 30 °C, and frequently exceeds 30 °C during the low tidal period in summer (Dong et al. 2008a). Furthermore, the salinity can also decrease to ~20 ppt after a summer rainstorm within several hours, which can cause large-scale motality of sea cucumber (Meng et al. 2011).

Temperature and salinity are critical factors affecting the behavior, metabolism, growth, life cycle and intra- and interspecific relationships of aquatic organisms (Kinne 1971). As previous studies have described, temperature (Dong et al. 2010; Wang et al. 2011, 2012) and salinity (Yuan et al. 2006; Meng et al. 2011) can influence growth, survival and physiological performance of the sea cucumber A. japonicus. Because of the dramatic changes of temperature and salinity in the aquaculture ponds after rainstorms, it is crucial to elucidate the combined effects of temperature and salinity on the physiological performance of sea cucumbers.

The osmolality of coelomic fluid in the sea cucumber changes with changes in salinity of the ambient water (Binyon 1972; Diehl 1986). Vidolin et al. (2002) reported that the gray sea cucumber (Holothuria grisea) could temporally regulate the osmolality of its coelomic fluids by possibly reducing the permeability of its body wall. In A. japonicus, the osmolality of the coelomic fluid changed rapidly and stabilized at 6 h after the osmotic shock when salinity changed from 32 ppt to different salinities (20, 25, 30 and 40 ppt) (Dong et al. 2008b). The osmoregulatory capacity of aquatic animals can be affected by temperature (Williams 1960; Charmantier 1998). For example, the osmoregulation of the shrimp Penaeus stylirostris was diminished at low temperature (Lemaire et al. 2002). In A. japonicus, an earlier heat shock can affect the activities of Na+/K+ ATPase and this then affects osmolality of coelomic fluids of this species (Dong et al. 2008b).

When animals suffer either heat stress (Tomanek and Somero 1999; Dutton and Hofmann 2009) or osmotic stress (Pan et al. 2000; Niu et al. 2008), heat shock proteins (HSPs) are induced. Acting as molecular chaperones, HSPs assist in the refolding of stress-denatured proteins and prevent those proteins from aggregating in the cell, thus helping the cell to cope with potential damage (Feige et al. 1996; Hartl 1996; Frydman and Höhfeld 1997; Morimoto 1998). Some studies have shown that HSPs play an important role in the sea cucumber A. japonicus, resisting both thermal stress (Dong and Dong 2008; Ji et al. 2008; Meng et al. 2009; Wang et al. 2011, 2012) and osmotic stress (Dong et al. 2008b; Meng et al. 2011). Superoxide dismutase (SOD), a primary antioxidase that is directly involved in eliminating reactive oxygen species (ROS), has been used as a main index of antioxidant defense (Wilhelm-Filho et al. 1993, 2001; Leiniö and Lehtonen 2005). In A. japonicus, the activity of SOD increased significantly during large fluctuations of temperature (Dong et al. 2008b) and in the early stage of estivation (Ji et al. 2008). However, there is a lack of knowledge about the combined effects of temperature and salinity on the expression of hsps mRNA in the sea cucumber.

In the present study, the osmolality of the coelomic fluid and the expression of hsps and sod mRNA levels in juveniles of the sea cucumber A. japonicus were investigated at different temperatures (16, 20, 24 and 28 °C) and salinities (22, 27 and 32 ppt) to elucidate the physiological response of sea cucumbers to acute changes of temperature and salinity. These results should be valuable in improving the management of sea cucumber aquaculture during rainstorms in summer.

Materials and methods

Collection and acclimation of animals

About 700 juvenile sea cucumbers, with a mean body wet weight of 0.92 ± 0.20 g (mean ± SE), were collected from Jimo Aquatic Seeding Breeding Farm, Qingdao, P. R. China. The animals were acclimated in several aquariums (1,700 × 750 × 350 mm) at 16 °C for 4 weeks. During acclimation, sea cucumbers were fed ad libitum with a laboratory-made formulated diet (9.94 ± 0.17 % moisture, 18.58 ± 0.23 % crude protein, 2.67 ± 0.06 % fat, 42.66 ± 0.54 % ash and 8.16 ± 0.00 kJ g−1 energy), containing powders of Sargassum spp., fishmeal, sea mud, wheat, vitamin and mineral premixes.

Aeration was provided continuously, and lighting was provided by incandescent lamps at a photoperiod of 12 h light: 12 h dark. Seawater was filtered using a sand filter, and the salinity was ~32 ppt. One-half or two-thirds of the rearing water was exchanged with fresh seawater daily. Water pH was about 7.8, and the concentration of ammonia was less than 0.24 mg l−1. Water temperature, salinity, pH and ammonia concentration were determined with a mercury thermometer (accuracy ± 0.2 °C), a salinity refractometer (AIAGO, Japan), a pH meter (PH3150i, WTW, Germany), and a hypobromite oxidation method (Wu 2007), respectively.

Experimental protocol

Based on water temperature and salinity values in June–August along the coast of Shandong Province, 12 environmental regimes were designated as combination of temperatures of 16, 20, 24 and 28 °C (T16, T20, T24 and T28) and salinities of 22, 27 and 32 (S22, S27 and S32). Each regime had three replications. After acclimation at 16 °C and 32 ppt (control) for 1 month, 360 sea cucumbers (ten individuals in each group) were allocated into 36 50-L plastic aquariums and reared in filtered aerated seawater maintained in the conditions as mentioned above. These aquariums were fully randomized and not blocked. Target temperatures above ambient were maintained by 300-W heaters connected to a temperature regulator provided with a thermocouple. The salinities of seawater were adjusted by adding aerated tap water. The rearing conditions were similar to those used in the acclimation period.

Osmolality, hsps and sod mRNA expressions

After being transferred to the preprepared seawater at different temperatures and salinities, one specimen was sampled at 2, 6, 12, 24, 36 and 48 h from each glass aquarium for measuring the osmolality of the coelomic fluid (n = 3). Their body wall and intestine were quickly removed and frozen in liquid nitrogen (~−200 °C) for analysis of hsps and sod mRNA expressions (n = 3). About 30 μl, coelomic fluid was extracted using 1.0 ml disposable syringes and No. 21 gauge 1.5′′ needles. To make sure that all the fluid was from the coelom, the body wall was pierced carefully at a small angle until the tip of the needle reached the coelomic cavity, without touching internal organs. The osmolality was measured using a Fiske 210 Micro-Sample Osmometer (Advanced Instruments, USA).

Total RNA was isolated from approximately 80 mg of body wall tissues by Trizol Reagent (Invitrogen, USA). A sample of 1 μg of total RNA was used as the template for synthesis of the first strand of cDNA. Partial β-actin gene (312 bp) was selected as reference housekeeping to normalize the level of expression between the samples amplified using the primers from Meng et al. (2009). Primers of the four genes hsp70, hsp90a, hsp90b and sod (Hsp70-F and Hsp70-R, Hsp90a-F and Hsp90a-R, Hsp90b-F and Hsp90b-R, Sod-F and Sod-R) were designed based on the sequences from GenBank (Ajhsp70, accession no. GH985449; Aj90a, accession no. JF907619; Aj90b, accession no. GH550976; Ajsod, accession no. X64060.1) as shown in Table 1.

Table 1 Primer sets designed for semi-quantitative RT-PCR analysis of hsps mRNA in the sea cucumber Apostichopus japonicus
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Semi-quantitative PCR conditions and components for hsps and β-actin were optimized, especially for the amplification cycles and annealing temperatures. The optimized PCR was carried out in 25 μl reactions containing 2.5 μl of 10 × PCR buffer, 1.6 μl of MgCl2 (25 mmol l−1), 2.0 μl of dNTP (2.5 mmol l−1), 1 μl of each primer (10 pmol ml−1), 15.875 μl of PCR-grade water, 0.125 μl (5 U μl−1) of Taq polymerase (Promega, USA) and 1 μl of cDNA reaction mix. The PCR program was preceded by initial denaturation for 5 min at 94 °C, followed by 30 cycles (for hsp70, hsp90a, hsp90b and sod) or 27 cycles (for β-actin) of 94 °C for 45 s, 51 °C (for hsp70, hsp90a) or 49 °C (for hsp90b and sod) or 54 °C (for β-actin) for 45 s, 72 °C for 1 min and a final extension step at 72 °C for 10 min. The PCR products were electrophoresed in 1.2 % agarose gel stained with ethidium bromide (EB), after which the products were purified from the gel and sequenced to confirm the specificity of RT-PCR amplification. Electropheretic images and the optical densities of amplified bands were analyzed using GeneTools software (Syngene, USA). The abundance of hsps and sod mRNA was normalized to the corresponding β-actin abundance in all samples and expressed as the ratio of optical densities of genes and β-actin (C hsp70/Cβ-actin, C hsp90a/Cβ-actin, C hsp90b/Cβ-actin or C sod /Cβ-actin).

Statistical analyses

The data were analyzed using SPSS for Windows (version 13.0; SPSS, Chicago, IL, USA). Data were tested for normal distribution using the Shapiro–Wilk test and for homogeneity of variances using the Levene test. Two-way ANOVA followed by post hoc Tukey multiple range test was applied to analyze the differences in osmolality of coelomic fluids in different temperature and salinity treatments. One-way ANOVA followed by post hoc Tukey multiple range test was applied to test the effect of salinity at each level of temperature and the effect of temperature at each level of salinity. The differences in gene expressions in different treatments were analyzed using one-way ANOVA followed by post hoc Tukey multiple range test or Kruskal–Wallis H test followed by Siegel–Tukey test depending on heterogeneity variances. Differences were considered significant at P < 0.05.

Results

Osmolality

The osmolality of the coelomic fluid changed immediately when the animals were transferred to low salinity treatments and reached osmotic balance within 6 h (Fig. 1, dotted lines represent the osmolality of ambient water in each experimental regime). Two-way ANOVA analysis showed that the interaction between temperature and salinity was significant (Table 2). The osmolality of coelomic fluid after 2 h of hypo-osmotic exposure (ΔOP2h) differed statistically between different temperatures within the same salinity (Fig. 2). Values of ΔOP2h at high temperature (28 °C) were significantly higher than those at low temperatures (16 and 20 °C) in low salinities (27 and 22 ppt).

Fig. 1
figure 1

The osmolality of the coelomic fluid in the sea cucumber Apostichopus japonicus at selected time points in 32, 27 and 22 ppt at A 16 °C, B 20 °C, C 24 °C and D 28 °C (mean ± S.E., n = 3). Dotted lines indicate the osmolarity of surrounding water in each treatment

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Table 2 Effects of temperature and salinity on the differences in osmotic pressure of coelomic fluid of sea cucumbers Apostichopus japonicas between 0 and 2 h (n = 3)
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Fig. 2
figure 2

The differences in osmolality of coelomic fluid of sea cucumbers Apostichopus japonicus from different treatments between 0 and 2 h. Differences between these groups were assessed by two-way ANOVA followed by post hoc Duncan multiple range test. Values with different lower case letters are significantly different (P < 0.05) among different temperature levels at the same salinity, and values with different capital letters are significantly different (P < 0.05) among different salinity levels at the same temperature (mean ± S.E., n = 3)

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Expression of hsps and sod mRNA

The expressions of hsp70, hsp90 s and sod mRNA showed similar temporal patterns in different treatments (Fig. 3, 28 °C as examples). Levels of hsps mRNA increased immediately after thermal and osmotic stresses, and the maximum values of hsps and sod mRNA in all treatments occurred at 6 h after the stresses and then decreased to the initial levels.

Fig. 3
figure 3

Temporal patterns of heat shock proteins mRNA (hsp70, hsp90a and hsp90b) and superoxide dismutase mRNA (sod) in A 32 ppt, B 27 ppt and C 22 ppt in the body wall of sea cucumbers Apostichopus japonicus at 28 °C (mean ± S.E., n = 3)

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The maximum values of mRNAs in the body wall at 6 h were variable among different salinity treatments (Fig. 4). For hsp70, the levels in S22 and S27 were significantly higher than that in S32, and there was no significant difference between S27 and S22. For hsp90a, there was no statistical difference among the three salinity treatments. For hsp90b, the expression pattern was similar to that of hsp70. Gene levels at S27 and S22 were significantly higher than that at S32. For sod, there was no statistical difference among the three salinity treatments. The expressions of mRNAs in the intestine were identical to those in the body wall (Tables 3, 4; Fig. 5).

Fig. 4
figure 4

Levels of A hsp70, B hsp90a, C hsp90b and D sod mRNA in the body wall of sea cucumbers Apostichopus japonicus at 6 h after the temperature and salinity stresses. Differences between these groups were assessed using one-way ANOVA followed by post hoc Duncan multiple range test or Kruskal–Wallis H test followed by post hoc S–N–K test. Data in the same column having different lower case letters are significantly different (P < 0.05) among different temperature levels, and data in the same row having different capital letters are significantly different (P < 0.05) among different salinity levels (n = 3)

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Table 3 One-way ANOVA table of gene expressions of hsp70, hsp90a, hsp90b and sod mRNA in the body wall and intestine of sea cucumbers Apostichopus japonicas at 6 h after the temperature and salinity stresses (n = 3)
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Table 4 Kruskal–Wallis H test table of gene expressions of hsp70, hsp90a, hsp90b and sod mRNA in the body wall and intestine of sea cucumbers Apostichopus japonicas at 6 h after the temperature and salinity stresses (n = 3)
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Fig. 5
figure 5

Levels of A hsp70, B hsp90a, C hsp90b and D sod mRNA in the intestine of sea cucumbers Apostichopus japonicus at 6 h after the temperature and salinity stresses. Differences between these groups were assessed using one-way ANOVA followed by post hoc Duncan multiple range test or Kruskal–Wallis H test followed by post hoc S–N–K test. Data in the same column having different lower case letters are significantly different (P < 0.05) among different temperature levels, and data in the same row having different capital letters are significantly different (P < 0.05) among different salinity levels (n = 3)

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The maximum levels of mRNAs in the body wall were also distinct in different temperature treatments (Fig. 4). The levels of hsps mRNA (hsp70, hsp90a and hsp90b) were up-regulated with temperature increase. The levels of hsps mRNA in T24 and T28 were significantly higher than that of T16, and there were significant differences between T28 and T20. There was no significant difference in sod mRNA among different temperature treatments. In the intestine, the levels of hsps mRNA in T24 and T28 were significantly higher than that of T16. The levels of sod mRNA in T28 was significantly higher than that of T16 (Tables 3, 4; Fig. 5).

Discussion

Juveniles of sea cucumber Apostichopus japonicas Selenka prefer to live in the low intertidal zone (+0.4 m above Chart Datum) on rocky substrates (Yusuke et al. 2006). In this area, sea cucumbers encounter extreme temperature and salinity variations during low spring tides, especially after a rainstorm in summer when the body temperature of the sea cucumber can increase rapidly from 20 °C to over 30 °C and the salinity can decrease from 32 to 20 ppt (Dong and Meng, unpublished data). So, we investigated combined effects of acute temperature and salinity stresses on related physiological responses of this species.

The osmolality of coelomic fluid in the sea cucumber changed rapidly with the change of ambient water salinity, and this change was temperature dependent. In the present study, osmolality of coelomic fluid reached osmotic balance with the environment within 6 h. The rapid change of osmolality in A. japonicus facing hypo-osmotic stress indicates that this species has poor ability for osmoregulation as previous studies described (Ruppert et al. 1994; Dong et al. 2008b; Meng et al. 2011). Furthermore, the change of osmolality in the first 2 h was variable among different temperature treatments, and the osmolality of coelomic fluids of the sea cucumber is temperature dependent. High temperatures (24 and 28 °C), above the optimal temperature for growth (16–20 °C), can aggravate the decrease in osmolality of the coelomic fluid in low salinity.

The rapid decrease in osmolality of coelomic fluid in the sea cucumber is related to the limited energy supply against salinity stress. The aerobic metabolism decreased at high temperatures, as described in our previous studies (Dong et al. 2008a; Ji et al. 2008). When water temperature was over 24 °C, the oxygen consumption decreased continually with the elongation of thermal stress. Therefore, the limited energy supply is an important reason for the rapid decrease in osmolality of the sea cucumber at high temperatures.

Besides the decrease in aerobic metabolism, the increase in energy consumption is another reason for the low cellular energy status at high temperatures. In the present study, significant up-regulation of hsp70 and hsp90 s mRNA occurred at high temperatures. Large amounts of energy are required at several events in the heat shock responses, such as the activation of transcription of heat shock genes, the synthesis of HSPs, the repair and replacement of damaged proteins and the ATP-requiring chaperoning by HSPs (Somero 2002). Hence, less energy is allocated into growth, reproduction, defenses against salinity stress (Na+/K+ ATPase synthesis, for example) and so on (Somero 2002; Tadashi et al. 2004).

Heat shock proteins (HSPs) and SOD are often regarded as defense mechanisms to environmental stresses (Feder and Hofmann 1999; Parihar et al. 1997). The expression of HSPs and SOD was tissue specific, and the internal organs were more sensitive than the body wall (Ji et al. 2008). This might be the reason for the lack of significant effects on sod expression in the body wall. The up-regulation of HSP at high temperature indicates an increase in protein damage. Previous studies showed that when suffered osmotic stress, the concentrations of intracellular ions of echinoderms was altered, so did enzymes of intermediary metabolism. The enzyme Na+/K+-ATPase (the ‘sodium pump’), which is responsible for the regulation of Na+ and K+ by transferring ions, is fundamental to osmotic regulation in most eukaryotic cells (Balshaw et al. 2001; Jorgensen et al. 2003). The activity of Na+/K+ ATPase of A. japonicus increased initially and reached a maximum value within 6 h after osmotic challenge (Dong et al. 2008b). As previous studies described, the activity of Na+/K+-ATPase in the estuarine crab, Chasmagnathus granulate Dana was the highest at 30 °C and a significant enzyme inhibition was observed at 40 °C (Castilho et al. 2001). The activity of Na+/K+-ATPase in the turbot Scophthalmus maximus was positively correlated with temperature from 10 to 22 °C, but when the temperature was increased further, the enzyme activity was inhibited or even lost (Imsland et al. 2003). Therefore, the stability of Na+/K+ ATPase is impaired at high temperatures.

The poor osmoregulatory capability at high temperature is also related to the change of biochemical pathways at high temperature. The enhancement of HSP during thermal stress blocks the synthesis of non-heat shock protein in some organisms because of the preferential translation of hsps mRNA (Storti et al. 1980; Morimoto et al. 1990). Therefore, the transcription and translation of proteins/enzymes involved in osmotic regulation are blocked at high temperatures, and then the osmoregulatory capability is decreased. Further, studies should be carried out to clarify the effect of thermal stress on osmotic regulation of the sea cucumber A. japonicus, especially the interaction between HSP and Na+/K+ ATPase.

In conclusion, the osmoregulation and hsps mRNA expression of the sea cucumber were affected by temperature. The decrease in osmolality of sea cucumber’s coelomic fluid in a hypo-osmotic environment is aggravated at high temperatures. Therefore, effective ways should be taken (e.g., draining off the upper freshwater, exchanging water and adding man-made sea water) to prevent dramatic changes of salinity in sea cucumber culture ponds induced by rainstorms in summer.