Effects of Rearing Temperature on Growth, Metabolism and Thermal

Aquaculture Research, 2013, 44, 1550–1559 doi:10.1111/j.1365-2109.2012.03162.x

Effects of rearing temperature on growth, metabolism and thermal tolerance of juvenile sea cucumber, Apostichopus japonicus Selenka: critical thermal maximum (CTmax) and hsps gene expression

Qing-Lin Wang1, Yun-Wei Dong2, Chuan-Xin Qin3, Shan-Shan Yu1, Shuang-Lin Dong1 & Fang Wang1 1The Key Laboratory of Mariculture, Ministry of Education, Fisheries College, Ocean University of China, Qingdao, China 2State Key Laboratory of Marine Environmental Science, College of Oceanography and Environmental Science, Xiamen University, Xiamen, China 3South China Sea Fisheries Research Institute, Chinese Academy of Fishery Science, Guangzhou, China

Correspondence: Y-W Dong, State Key Laboratory of Marine Environmental Science, College of Oceanography and Environmental Science, Xiamen University, Xiamen 361005, China. E-mail: [email protected]

Abstract Introduction

Effects of different rearing temperatures (16, 21 and Sea cucumber Apostichopus japonicus (Selenka) is 26°C) on growth, metabolic performance and ther- distributed along the Asian coast from 35°Ntoat mal tolerance of juvenile sea cucumber Apostichopus least 44°N including Russia, China, Japan and japonicus (initial body weight 7.72 ± 0.96 g, mean Korea (Sloan 1984; Liao 1997). Due to demand ±SD) were investigated in this study. During the 40- and high price, it has been exploited as an impor- day experiment, growth, metabolic performance, tant fishery resource for a long time. With the food intake and energy budget at different reared rapid increase in demand of beche-de-mer and sig- temperatures were determined. Sea cucumbers rear- nificantly declining wild caught stocks form overf- ing at 16°C obtained better growth (final body ishing, aquaculture of sea cucumber has become weight 11.96 ± 0.35 g) than those reared at 21 more economically viable. (10.33 ± 0.41 g) and 26°C (8.31 ± 0.19 g) Water temperature plays an important role in (P < 0.05), and more energy was allocated for growth and physiological performance of the sea growth at 16°C (162.73 ±11.85 J g1 d1) than cucumber (Li, Yang, Zhang, Zhou & Zhang 2002; those at 21(79.61 ± 6.76 J g1 d1) and 26°C Dong & Dong 2006; Dong, Dong, Tian, Wang & (27.07 ± 4.30 J g1 d1)(P < 0.05). Critical ther- Zhang 2006), and the low thermotolerance of A. mal maxima (CTmax) values of juvenile sea cucum- japonicus limits the national wide aquaculture bers reared at 16, 21 and 26°C were 33.1, 34.1 (Wang, Dong, Dong & Wang 2011). According to and 36.6°C, respectively, and the upregulation of our record, the water temperature in the field hsps in sea cucumbers reared at 26°C was higher aquaculture pond was 0.4 to 31.8°C in Jiaonan, than those acclimated at lower temperatures (16 Qingdao (35°53′N, 120°00′E), and frequently and 21°C), indicating that temperature acclimation exceeded 30°C in summer (from June to Septem- could change the thermal tolerance of the sea ber) (Dong Y. W. & Meng X. L, unpublished data). cucumber, and CTmax and hsps were sensitive indi- An, Dong and Dong (2007) found that the assimi- cators of the sea cucumber’s thermal tolerance. lation efficiency of sea cucumber decreased with temperature increasing, and adult A. japonicus Keywords: rearing temperature, sea cucumber, becomes inactive when water temperature exceeds growth, metabolic performance, thermal toler- 18°C and will aestivate at water temperatures ance, critical thermal maxima about 20–24.5°C (Liu, Li, Song, Sun, Zhang and Gu

1996; Yang, Zhou, Zhang, Yuan, Li, Liu & Zhang Materials and methods 2006). Aestivation can last for 4 months in some regions in China (Liu et al. 1996). Therefore, it is Collection and acclimation of animals essential to study the thermal limits of this species. Previous studies used upper or lower incipient Six-month old sea cucumbers were collected at lethal temperature (ILT) as an index reflecting 12°C in April from Jimo Aquatic Seeding Breeding thermal tolerance of sea cucumber (Meng, Ji, Farm, Qingdao, China. In the laboratory, water Dong, Wang & Dong 2009; Dong, Ji, Meng, Dong temperature was gradually increased to 16°C and & Sun 2010). The measurement of ILT involves maintained at 16°C for 2 weeks, before placing the abrupt transfer of samples to baths maintained animals in acclimatization conditions. at a sequence of constant, lethally high temperatures. However, critical thermal maxima (CTmax) Experimental protocol refers to the thermal point at which the locomo- tory activity of an animal becomes disorganized To acclimatize the experimental animals, water and the animal loses its ability to escape from con- temperature gradually increased at a rate of ditions which will promptly lead to its death when 1°Cd1 heated by 300 W electric heaters until heated from a previous acclimation temperature at the designated temperature, and kept at the desig- a constant rate just fast enough to allow deep nated temperature for 1 week. After acclimatiza- body temperatures to follow environmental tem- tion, the sea cucumbers were starved for 48 h and peratures without a significant time lag (Cox weighed by a balance of 0.001 g sensitivity. Forty- 1974; Becker & Genoway 1979). In the method of five sea cucumbers (wet weight of 7.72 ± 0.96 g) CTmax, samples are placed in an environment of were randomly selected and allocated into nine steadily increasing temperature. The ILT method glass aquarium (450 9 250 9 350 mm, about measures only mortification and, regrettably per- 40 L water). This allowed three treatments to be haps, the trauma of handling and transfer. How- tested in triplicates with five animals in each ever, the CTmax measures mortification and aquarium. The three treatments were setup at partial acclimation (Kilgour & McCauley 1986). three temperatures: 16, 21 and 26°C. The experi- To evaluate the capability of A. japonicus facing an ment lasted for 40 days. increasing thermal stress, therefore CTmax value was used in this study. Rearing conditions Thermal history can affect the upper thermal limit of aquatic animals, such as sea cucumber (Meng During acclimatization and throughout the experi- et al. 2009), fish (Das, Pala, Chakrabortyb, Manu- ment, sea cucumbers were fed to excess with a sha, Sahua & Mukherjeea 2004) and shrimp (Sel- laboratory-made formulated diet (9.94 ± 0.17% vakumar & Geraldine 2005). Acting as molecular moisture, 18.58 ± 0.23% crude protein, 2.67 ± chaperones, heat shock proteins (hsps) are extremely 0.06% fat, 42.66 ± 0.54% ash and 8.16 ± 0.00 sensitive to temperature (Frydman & Ho¨held 1997; kJ g1 energy), containing powders of Sargassum Morimoto 1998; Feder & Hofmann 1999; Picard spp., fish meal, sea mud, wheat, vitamin and 2002), and are closely related to organisms’ thermal mineral premixes. Seawater was filtered using a history (Tomanek & Somero 1999; Hochachka & So- sand filter and the salinity was 30–32 psu. One- mero 2002). The responses of hsp70 in the sea half or two-thirds of the rearing water was cucumber to acute thermal and osmotic stress have exchanged by fresh equi-temperature seawater been determined (Dong, Dong & Ji 2007, 2008; daily. Aeration was provided continuously, and Dong & Dong 2008; Dong, Dong & Meng 2008; Ji, the photoperiod was 12:12 h light:dark. Seawater Dong & Dong 2008). However, the responses of pH was about 7.8–8.2 and the concentration of hsp70 and hsp90 of A. japonicus to a long-term ammonia was less than 0.24 mg L1. Water tem- chronic thermal stress have not been assessed. perature, salinity, pH and ammonia concentration In this study, we want to test the hypothesis were determined with mercury thermometer that thermal history can change the thermal limits (accuracy ±0.2°C), salinity refractometer (AIAGO, of the sea cucumber A. japonicus, and the modifica- Kyoto, Japan), pH metre (PH3150i; WTW, tions of thermal tolerance are closely relate to the Munich, Germany) and hypobromite oxidation changes in expression of hsps. methods (Wu 2007) respectively.

8.23 ± 0.87 g were measured every 8 days. Prior Sample collection and data calculation to the test of oxygen consumption, sea cucumbers Five sea cucumbers were simultaneously sampled were starved for 24 h to reduce associated meta- from the acclimatized animals to determine the ini- bolic responses. The tested animal was put into a tial body composition. During the experiment, sea 330 mL conical ﬂask with a rubber plug individu- cucumbers were fed once a day at about ally, which was immersed into a water bath (Shuniu, 17:00 hour, faeces and uneaten feed were col- Chengdu, China) for temperature control. There lected by syphon 23–24 h later and then dried at were three replicates and two blank controls to 65°C to constant weight. The sea cucumbers were correct for the respiration of bacteria in the water. weighed every 20 days and the daily food supply Oxygen content of water samples was determined was precisely weighed and recorded. At the end of using the dissolved oxygen analyser (YSI 5000; experiment, the sea cucumbers were starved for YSI, Yellow Springs, OH, USA), and the OCR of 48 h, weighed and then dried at 65°C until con- the sea cucumber was calculated from the follow- stant weight achieved. ing equation: The energy content of the diets, faeces and ani- 1 1 mal samples were measured by a calorimeter OCRðlgO2h g Þ¼ðDtVt D0V0Þ=WT (PARR Instrument Company, Moline, IL, USA).

Nitrogen content was determined by a Vario ELIII where Dt represents the oxygen content (lgO2 1 Elemental Analyzer (Elementar, Hanau, Germany). L ) in the test bottles; D0 is the oxygen content 1 Proximate compositions of diets were analysed (lgO2 L ) in the blank bottles; Vt and V0 are according to standard methods (AOAC 1990). volumes of the test bottles and blank bottles (L); Energy budget was constructed according to the W is the wet weight of the sea cucumber (g); T is equation: C = G + F + U + R, where, C is energy time duration (h). consumed; G is energy for growth; F represents energy of faeces produced; U is energy loss as Critical thermal maximum (CTmax) determination ammonia excretion and R stands for energy lost as respiration. The estimation of U was based on the After rearing of A. japonicus at each test tempera- nitrogen budget equation: U = (CN GN FN) 9 ture regime for 40 days, water temperature was 24 830 (Wang, Dong, Huang, Wu, Tian & Ma adjusted to 16°C at a rate of 1°Cd1. Then sea

2003), where CN is the nitrogen consumed from cucumbers were maintained at 16°C for 2 weeks. feed; GN, the nitrogen deposited in animal body; FN, After that, critical thermal maximum (CTmax) val- the nitrogen lost in faeces and 24 830, the energy ues were estimated using Critical Thermal Method- content in excreted ammonia (J g1). The value of ology (Cox 1974; Becker & Genoway 1979; R was calculated as the following energy budget Paladino, Spotila, Schubauer & Kowalski 1980; equation: R = C – G – F U. Beitinger, Bennett & McCauley 2000). For each Specific growth rate in terms of weight (SGRw) CTmax trial, sea cucumbers were placed, ten each, and energy (SGRe) were calculated as: into 10 L glass aquaria filled with fresh seawater of 16°C. Then one 300W heater was placed in the 1 SGRwð%day Þ¼100ðlnW2 lnW1Þ=D aquaria and aeration was provided to prevent thermal stratification. Water temperature was 1 SGReð%day Þ¼100ðlnE2 lnE1Þ=D measured with mercury thermometer (accuracy ±0.2°C). Temperatures increased at a rate of 1 where W2 and W1 are the final and initial body 0.30 ± 0.05°C min , which was slow enough to weight of the sea cucumbers (g) respectively; E2 track body temperature, but fast enough to pre- and E1 are the final and initial energy content of vent thermal acclimation (Becker & Genoway the sea cucumbers (J g1) respectively; D is the 1979). Temperature increase continued until sea duration of the experiment (d). cucumbers could not re-attach to the walls of the aquarium after being touched with a glass rod (Chen & Chen 2000). As half of the sea cucumbers Oxygen consumption reached loss of equilibrium, the water temperature Oxygen consumption rates (OCR) of the sea was recorded. Sea cucumbers were immediately cucumbers with a mean wet weight of removed from the CTmax chamber and returned

1552 © 2012 John Wiley & Sons Ltd, Aquaculture Research, 44, 1550–1559 Aquaculture Research, 2013, 44, 1550–1559 Effects of rearing temperature on sea cucumber Q -L Wang et al. to their acclimation temperature. There were four (10 pmol mL1), 15.875 lL of PCR-grade water, replicates at each test temperature regime. 0.125 lL(5UlL1) of Taq polymerase (Promega, Madison, WI, USA) and 1 lL of cDNA reaction mix. The PCR programmes was preceded by initial Semi-quantitative RT-PCR analysis of hsp70 and denaturation for 5 min at 94°C, followed by 30 hsp90 cycles (for hsp70, hsp90a and hsp90b) or 27 cycles After the determination of CTmax, sea cucumbers (fo b-actin) of 94°C for 45 s, 51°C (for hsp70, were returned to 16°C for a period of poststress hsp90a)or49°C (for hsp90b)or54°C (for b-actin) recovery and at selected time points (2, 6 and for 45 s, 72°C for 1 min and a final extension step 24 h), five specimens in each test temperature at 72°C for 10 min. The PCR products were elec- regime were randomly sampled and frozen in the trophoresed in 1.2% agarose gel stained with ethi- liquid nitrogen. Control animals were maintained dium bromide, after which the products were at 16°C without heat shock. purified from the gel and sequenced to confirm the Total RNA was isolated from approximately specificity of RT-PCR amplification. Electropheretic 80 mg of body wall tissues by Trizol Reagent images and the optical densities of amplified bands (Invitrogen, Carlsbad, CA, USA). A sample of 1 lg were analysed using GeneTools software (Syngene, of total RNA was used as the template for synthesis Frederick, MD, USA).The abundance of hsps mRNA of the first strand of cDNA. Partial b-actin gene was normalized to the corresponding b-actin (312 bp) was selected as reference housekeeping to abundance in all samples, and expressed as the normalize the level of expression between the sam- ratio optical densities of hsps and b-actin (Chsp70/ ples amplified using the primers from Meng et al. Cb-actin,Chsp90a/Cb-actin or Chsp90b/Cb-actin). (2009). Primers of the three genes Hsp70, Hsp90a and Hsp90b (Hsp70-F and Hsp70-R, Hsp90a-F and Statistics Hsp90a-R, Hsp90b-F and Hsp90b-R) were designed base on the sequences from Genbank (Ajhsp70, Data were analysed using SPSS version 16.0 (Chi- GH985449; Aj90a, JF907619; Aj90b, GH550976) cago, IL, USA). The homogeneity of variances in as shown in Table 1. data was tested using Mauchly’s Test of Sphericity. Semi-quantitative PCR conditions and compo- Oxygen consumption rate was analysed by nents for hsps and b-actin were optimized, espe- repeated measures analysis of variance. The differ- cially for the amplification cycles and annealing ence of SGR, body weight, energy parameters and temperatures. The optimized PCR was carried out CTmax among different test temperature regimes in 25 lL reactions containing 2.5 lLof109 PCR was analysed using one-way analyses of variance 1 buffer, 1.6 lL of MgCl2 (25 mmol L ), 2.0 lLof (ANOVA), followed by post-hoc Duncan Multiple dNTP (2.5 mmol L1), 1 lL of each primer Range Tests. For the semi-quantitative RT-PCR analysis of hsp70 and hsp90 experiment, two-way ANOVA was applied to discern significant differences Table 1 Primer sets designed for semi-quantitative RT- in hsps mRNA by the rearing temperature effect PCR analysis of hsps mRNA in sea cucumber Apostich- (16, 21 and 26°C) and by the time effect (within a opus japonicus rearing treatment at different time points). Differ- ences were considered significant at P < 0.05. Primers Primer sequences

Hsp70-F 5′-ATGCCTAGAACCAGTAGAGAAAG-3′ Results Hsp70-R 5′-TGTCGTTCGTGATGGTGATT-3′ Hsp90a-F 5′-TTGTTGAAAGGGAGGAGG-3′ Hsp90a-R 5′-GGCATCAGAGGCGTTAGA-3′ Growth Hsp90b-F 5′-TCTTTCTTAGGGAACTCATCTC-3′ Hsp90b-R 5′-CCTGTAGCATTCGTCATCG-3′ The temporal changes of body weight were differ- b-actin-F 5′-ACACGGTATCGTCACAAACTGG-3′ ent in the three treatments during the 40-day trial b-actin-R 5′-AGGATAGCGTGAGGAAGAGCAT-3′ (Fig. 1). For all the treatments, the body weight of the sea cucumber showed increasing trend, but it Hsp70-F and Hsp70-R are used for amplifying hsp70, Hsp90a- F and Hsp90a-R are used for amplifying hsp90a, Hsp90b-F and varied with different temperatures in a declining Hsp90b-R are used for amplifying hsp90b and b-actin-F and b- gradient of 16 > 21 > 26°C treatment. Sea actin-R are used for amplifying b-actin. cucumbers reared at 16°C had the highest body

Figure 1 Mean of body weight in the sea cucumbers Apostichopus japonicus at Day 0, Day 20 and Day 40 at Figure 3 The daily food consumption of the sea different rearing temperatures. Values (mean ± 1 SE; cucumbers Apostichopus japonicus in the three treat- n = 3) with different letters on the same day indicate a ments in a 40-day trial. During the whole experiment significant difference from each other (P < 0.05). period, the daily food supplied was precisely weighed and recorded. The uneaten feed was collected by syphon, and was dried at 65°C to the constant weight. weight (11.96 ± 0.35 g), whereas the ones reared Values are mean, n = 3. at 26°C had the lowest body weight (8.31 ± 0.19 g). On Day 40, body weights among different and 21°C kept eating actively during the whole temperatures were significantly different (F = 2,6 experimental period, and sea cucumbers at 16°C 31.112, P = 0.001). ingested more food than that at 21°C. The daily After the 40-day experiment, Specific growth rate food intake fluctuated in treatment 26°C, and an of sea cucumbers in terms of energy and body obvious decrease in food consumption occurred weight (% d1) tended to decrease with increasing from Day 13 to the end of the experiment. Overall, temperature (Fig. 2). SGRw in 26°C sea cucumber the food consumption varied with different temper- was significantly lower than that in treatment of 16 atures in a declining gradient of 16 > 21 > 26°C and 21°C(F = 19.186, P = 0.002), and SGRe in 2,6 treatment. 16°C sea cucumber was significantly higher than the other treatments (F2,6 = 20.359, P = 0.002). Oxygen consumption

Daily food consumption During the whole experimental period, OCR in 16°C sea cucumber was stable (from 14.63 ± 0.84 lg The daily food intake among the three treatments 1 1 1 1 O2 g h to 16.37 ± 1.06 lgO2 g h ) (Fig. 4). was different (Fig. 3). Individuals in treatments 16 The OCR in 21 and 26°C sea cucumber decreased 1 1 from 22.05 ± 1.41 lgO2 g h and 23.12 ± 1 1 0.75 lgO2 g h at Day 0 to 16.85 ± 1.36 lg

Figure 2 Effect of different temperatures on SGRw and SGRe of the sea cucumber Apostichopus japonicus after Figure 4 Effects of different temperatures on the oxy- 40-day rearing. Values (mean ± 1 SE; n = 3) with dif- gen consumption rate (OCR) of the sea cucumber Apos- ferent letters in the same column indicate a signiﬁcant tichopus japonicus at different temperatures. Values are difference from each other (P < 0.05). mean ± 1 SE, n = 3.

Table 2 Effect of temperature treatments on the oxygen cantly higher than the other treatments (F2,6 = consumption rate (OCR) of the sea cucumber (Apostich- 18.056, P = 0.003). opus japonicus) at different time points (Day 0, 8, 16, 24, 32, 40) Effects of different rearing temperatures on the Type III hsp70 and hsp90 expression sum of Mean Source squares d.f. square F P Two-way ANOVA analysis showed that there were signiﬁcant differences in hsp70, hsp90a and hsp90b Time 99.54 5 19.908 15.691 <0.001 expression among different rearing temperatures Temperature 184.231 2 92.116 147.344 <0.001 Time 9 72.61 10 7.261 5.723 <0.001 and time points after thermal shock (Table 4). Temperature The expression of hsp70, hsp90a and hsp90b after heat shock increased rapidly (Fig. 6). The peak Data were analysed by repeated measure analysis of variance, values of hsp70 occurred at 6 h in all treatments n = 3. (16°C, Chsp70/Cb-actin = 1.083 ± 0.089; 21°C, Chsp70/

Cb-actin = 1.319 ± 0.299; 26°C, Chsp70/Cb-actin = 1 1 1 1 O2 g h and 17.25 ± 1.64 lgO2 g h at 1.618 ± 0.105). The peak values increased with Day 40 respectively. Repeated measure analysis of the rising of temperature, although there was no variance showed that the OCRs of sea cucumbers signiﬁcant difference among different acclimation among different treatments were signiﬁcantly dif- temperatures (F2,6 = 1.992, P = 0.217) (Fig. 6a). ferent: 26°C > 21°C > 16°C (Table 2). At 24 h, the expression of hsp70 decreased to the initial level. The expression patterns of hsp90s were similar Energy budget to those of hsp70 (Fig. 6b and c). The peak values

Energy budgets for the sea cucumbers rearing at of hsp90s occurred at 6 h (16°C, Chsp90a/Cb-actin = different temperatures are presented in Table 3. 1.070 ± 0.065, Chsp90b/Cb-actin = 0.571 ± 0.048;

Animals reared at 16°C spent more energy out of 21°C, Chsp90a/Cb-actin = 1.495 ± 0.031, Chsp90b/Cb-actin consumed food on growth (G, 9.70%), but less = 0.615 ± 0.036; 26°C, Chsp90a/Cb-actin = 1.576 ± energy on respiration (R, 32.76%) than those of 0.213, Chsp90b/Cb-actin = 0.642 ± 0.017), and they the other two temperature treatments varied with different temperatures in an increasing (P<0.05).The energy deposited as growth and gradient of 26 > 21 > 16°C treatment. faeces decreased with rearing temperature increase (P<0.05). Conversely, the consumption of energy Discussion for respiration increased with temperature increase (P<0.05). The growth rates (SGRw and SGRe) of sea cucumbers were temperature-dependent, and animals reared at 16°C had higher growth rate than those Critical thermal maximum (CTmax) determination reared at the other two temperatures. The differ- Critical thermal maximum of the sea cucumber ence in growth should relate to the following rea- increased with the rising of rearing temperature sons. Firstly, the difference in growth was due to (Fig. 5). The CTmaxs of the sea cucumber rearing the decreased food consumption with increasing at 16, 21 and 26°C were 33.1, 34.1 and 36.6°C, rearing temperature (Fig. 3). As Forseth and Jons- respectively, and the CTmax in 26°C was signiﬁ- son (1994) described, theoretically the optimal

Table 3 Energy allocation in sea T (°C) G (% C1) R (% C1) F (% C1) U (% C1) cucumber Apostichopus japonicus rearing at different temperatures 16 9.70 ± 0.25c 32.76 ± 0.35a 53.66 ± 0.25c 3.88 ± 0.07b 21 4.89 ± 0.38b 41.98 ± 0.60b 48.87 ± 0.57b 4.26 ± 0.04b 26 2.85 ± 0.31a 56.95 ± 0.74c 37.20 ± 0.83a 3.01 ± 0.30a

C, energy consumed in food; G, energy deposited for growth; F, energy lost in faeces; U, energy lost in excretion; R, energy lost in respiration. Values (percentage in mean ± 1 SE; n = 3) with different letters in the same column indicate a signiﬁcant difference from each other (P < 0.05).

described (Beitinger & Bennett 2000; Beitinger et al. 2000; Hernańdez, Bu¨ckle, Guisado, Baroń& Estavillo 2004), the CTmax method represents an efficient way for comparing the relative thermal tolerance of stressed aquatic poikilotherms, and CTmax is valuable for assessing the relative temperature tolerance of organisms acclimated to different temperatures. Therefore, sea cucumbers reared at high temperature acquired high thermotolerance, and the CTmax values were good indi- cator of thermal tolerance of the sea cucumber. Figure 5 Effects of different rearing temperatures on These results coincided with the study of Beitinger critical thermal maximum (CTmax) of sea cucumbers. et al. (2000), who suggested that acclimation tem- ± = Values (mean 1 SE; n 3) with different letters indi- perature exerted a major effect on the temperature cate a significant difference from each other tolerance of tested animals and it was strongly (P < 0.05). linearly related to CTmax value. temperature for growth should be close to the opti- Sea cucumbers rearing at 26°C had higher mum temperature for maximum food consump- hsp70 mRNA level than the other treatments, and tion, if there is enough oxygen to metabolize the this was consistent with the previous studies that food consumed. Secondly, the OCR of tested ani- acclimation at higher temperatures could increase mals did increase with rising temperature in this the ability of synthesizing proteins under thermal study, and the decrease in sea cucumber growth stress (Tomanek & Somero 1999). The high levels should relate to the increase in respiration at high of hsp70 mRNA indicated high level of protein temperature. Finally, the difference in energy allo- damage and high capability to cope with the envi- cation was another important reason resulting in ronment stressors (Lindquist 1986; Feder & Hof- the variation in growth at different temperatures. mann 1999; Tomanek & Somero 1999; Po¨rtner The energy budget provides a framework for the 2002; Dong, Dong & Ji 2008). In this study, the evaluation of various ways in which nutrients are expression pattern of hsp90 mRNA was similar to utilized and for the comparison of energy utiliza- that of hsp70 (Fig. 6b and c). This result indicated tion strategies between different species or the that hsp90s could serve as sensitive biomarkers in same species for different ecological factors (Law- monitoring the impact of temperature stress on rence & Lane 1982). The energy out of consumed the sea cucumber A. japonicus. food that animals spend on respiration was In this study, hsps expressions of sea cucumbers 32.76%, 41.98% and 56.95% in treatment of 16, increased with the rising of rearing temperatures 21 and 26°C, respectively (Table 3), indicating the (Fig. 6). The expression of hsps is a process of energy allocated into respiration increased with energy consumption. When the hsps are up-regu- rising of rearing temperature. lated, cellular energy demands may increase dra- In this study, CTmax was positively correlated matically owing to the energy requirements with the rearing temperature. As previous studies associated with activating transcription, synthesing

Table 4 Effects of rearing temperatures (16, 21 and 26°C) on the hsps expression of the sea cucumber at different time points (0, 2, 6, 24 h)

hsp70 hsp90a hsp90b

d.f.1 d.f.2 F P d.f.1 d.f.2 F P d.f.1 d.f.2 F P

16°C 3 11 17.350 0.001 3 11 8.827 0.006 3 11 4.355 0.043 21°C 3 11 1.402 0.311 3 11 45.107 0.000 3 11 10.298 0.004 26°C 3 11 14.193 0.001 3 11 4.431 0.041 3 11 5.326 0.026 d.f.1, degrees of freedom within groups; d.f.2, total degrees of freedom. The temporal changes of hsps expression were analysed using one-way analyses of variance (ANOVA) followed by post-hoc Duncan Multiple Range Tests. n = 3 in all treatments.

Acknowledgments

This study was supported by the Chinese National Science Foundation (30400333), National Key Technologies R&D Program of China (2006BAD09A01), Reward Research Foundation for Talented Young and Middle Aged Scientists of Shandong, China (BS2009NY019).

References

An Z.H., Dong Y.W. & Dong S.L. (2007) Temperature effects on growth-ration relationships of juvenile sea cucumber Apostichopus japonicus (Selenka). Aquaculture 272, 644–648. AOAC (1990). Official methods of analysis. In: Association of official analytical chemists, (15th edn) (ed. by K. Hel- drich), pp. 1298. AOAC, Arlington, VA, USA. Becker D.C. & Genoway R.G. (1979) Evaluation of the critical thermal maximum for determining thermal tolerance of freshwater fish. Environmental Biology of Fishes 4, 245–256. Beitinger T.L. & Bennett W.A. (2000) Quantification of the role of acclimation temperature in temperature tolerance of fishes. Environmental Biology of Fishes 58, 277–288. Beitinger T.L., Bennett W.A. & McCauley R.W. (2000) Temperature tolerances of North American freshwater fishes exposed to dynamic changes in temperature. Environmental Biology of Fishes 58, 237–275. Chen J.C. & Chen W.H. (2000) Salinity tolerance of Hali- otis diversicolor supertexta at different salinity and temperature levels. Aquaculture 181, 191–203. Cox D.K. (1974). Effects of three heating rates on the Figure 6 The expression pattern of hsp70 (a), hsp90a critical thermal maximum of bluegill. In: Thermal Ecol- (b) and hsp90b (c) in the sea cucumbers after the ogy (ed. by J.W. Gibbons & R.R. Sharitz), pp. 158–163. CTmax determination. At selected time points, five sea Nat. Tech. Inf. Ser., Springfield, Virginia, USA. cucumbers were sampled randomly in each treatment. Das T., Pala A.K., Chakrabortyb S.K., Manusha S.M., Values are mean ± 1SE(n = 3 for all data points). Sahua N.P. & Mukherjeea S.C. (2004) Thermal tolerance and oxygen consumption of Indian major carps acclimated to four temperatures. Journal of Thermal of Hsps and replacing damaged proteins, and Biology 29, 157–163. energy costs induced by thermal stress would nega- Dong Y.W. & Dong S.L. (2006) Growth and oxygen con- tively reduced the energy available for growth (So- sumption of the juvenile sea cucumber Apostichopus mero 2002; Tadashi, Takao, Hiroshi, Shin, japonicus (Selenka) at constant and fluctuating water Masaaki, Hideaki, Tadashi, Seiji & Ryuichi 2004). temperatures. Aquaculture Research 37, 1327–1333. Therefore, the high consumption of energy owing Dong Y.W. & Dong S.L. (2008) Induced thermotolerance to hsps synthesis and function should partly lead to and the expression of heat shock protein 70 in sea the decrease in growth rate of sea cucumbers at cucumber Apostichopus japonicus Selenka. Fisheries Sci- high temperature. ence 74, 573–578. In conclusion, rearing at higher temperatures Dong Y.W., Dong S.L., Tian X.L., Wang F. & Zhang M.Z. could improve the thermal tolerance of sea cucum- (2006) Effects of diel temperature fluctuations on growth, oxygen consumption and proximate body bers indicated by the higher CTmax value, and the composition in the sea cucumber Apostichopus japonicus increased thermal tolerance at high temperature Selenka. Aquaculture 255, 514–521. was closely related to the expression of hsps.

Dong Y.W., Dong S.L. & Ji T.T. (2007) Stress responses Liu Y.H., Li F.X., Song B.X., Sun H.L., Zhang X.L. & Gu to rapid temperature changes of the juvenile sea B.X. (1996) Study on aestivating habit of sea cucum- cucumber (Apostichopus japonicus Selenka). Journal of ber (Apostichopus japonicus): ecological characteristic of Ocean University of China (English Edition) 6, 275– aestivation. Journal of Fishery Sciences of China 3,41– 280. 48 (In Chinese with English abstract). Dong Y.W., Dong S.L. & Ji T.T. (2008) Effect of different Meng X.L., Ji T.T., Dong Y.W., Wang Q.L. & Dong S.L. thermal regimes on growth and physiological perfor- (2009) Thermal resistance in sea cucumbers (Apostich- mance of the sea cucumber Apostichopus japonicus Sele- opus japonicus) with differing thermal history: the role nka. Aquaculture 275, 329–334. of Hsp70. Aquaculture 294, 314–318. Dong Y.W., Dong S.L. & Meng X.L. (2008) Effects of Morimoto R.I. (1998) Regulation of the heat shock tran- thermal and osmotic stress on growth, osmoregulation scriptional response: cross talk between a family of and Hsp70 in sea cucumber (Apostichopus japonicus Sel- heat shock factors, molecular chaperones, and nega- enka). Aquaculture 276, 179–186. tive regulators. Genes & development 12, 3788–3796. Dong Y.W., Ji T.T., Meng X.L., Dong S.L. & Sun W.M. Paladino F.V., Spotila J.R., Schubauer J.P. & Kowalski (2010) Difference in thermotolerance between red and K.T. (1980) The critical thermal maximum: a tech- green color variants of the Japanese sea cucumber, nique used to elucidate physiological stress and adap- Apostichopus japonicus Selenka: Hsp70 and heat hard- tation in fishes. Revue Canadienne de Biologie 39, 115– ening effect. Biological Bulletin 218,87–94. 122. Feder M.E. & Hofmann G.E. (1999) Heat shock proteins, Picard D. (2002) Heat-shock protein 90, a chaperone for molecular chaperones, and the stress response: evolu- folding and regulation. Cellular and Molecular Life Sci- tionary and ecological physiology. Annual Review of ences 59, 1640–1648. Physiology 61, 243–282. Po¨rtner H.O. (2002) Climate variations and the physio- Forseth T. & Jonsson B. (1994) The growth and food logical basis of temperature dependent biogeography: ration of piscivorous brown trout (Salmon trutta). Func- systemic to molecular hierarchy of thermal tolerance tional Ecology 8, 171–177. in animals. Comparative Biochemistry and Physiology Frydman J. & Ho¨held J. (1997) Chaperones get in touch: 132, 739–761. the hip-hop connection. Trends in biochemical sciences Selvakumar S. & Geraldine P. (2005) Heat shock protein 22,87–92. induction in the freshwater prawn Macrobrachium mal- Hernańdez M., Bu¨ckle F., Guisado C., Baroń B. & Estav- colmsonii: acclimation-influenced variations in the illo N. (2004) Critical thermal maximum and osmotic induction temperatures for Hsp70. Comparative Bio- pressure of the red sea urchin Strongylocentrotus fran- chemistry and Physiology 140, 209–215. ciscanus acclimated at different temperatures. Journal of Sloan N.A. (1984). Echinoderm fisheries of the world: a Thermal Biology 29, 231–236. review. In: Echinodermata (ed. by B.F. Keegan & B.D.S. Hochachka P.W. & Somero G.N. (2002) Biochemical O’Connor), pp. 109–124. A. A. Balkema Publishers, Adaptation. Oxford University Press, New York, USA. Rotterdam. Ji T.T., Dong Y.W. & Dong S.L. (2008) Growth and phys- Somero G.N. (2002) Thermal physiology and vertical iological responses in the sea cucumber, Apostichopus zonation in intertidal animals: optima, limits, and costs japonicus Selenka: aestivation and temperature. Aqua- of living. Integrative and Comparative Biology 42, 780– culture 283, 180–187. 789. Kilgour D.M. & McCauley R.W. (1986) Reconciling the Tadashi K., Takao K., Hiroshi A., Shin E., Masaaki N., two methods of measuring upper lethal temperatures Hideaki T., Tadashi Y., Seiji K. & Ryuichi T. (2004) Mild in fishes. Environmental Biology of Fishes 17, 281– heat shock induces autophagic growth arrest, but not 290. apoptosis in U251-MG and U87-MG human malignant Lawrence J.M. & Lane J.M. (1982). The utilization of glioma cells. Journal of Neuro-Oncology 68, 101–111. nutrients by post-metamorphic echinoderms. In: Echi- Tomanek L. & Somero G.N. (1999) Evolutionary and noderm Nutrition (ed. by M. Jangoux & J.M. Lawrence), acclimation-induced variation in the heat-shock pp. 368–371. A. A. Balkema Publishers, Rotterdam. responses of congeneric marine (genus Tegula) from dif- Li B.Q., Yang H.S., Zhang T., Zhou Y. & Zhang C.X. ferent thermal habitats: implications for limits of ther- (2002) Effect of temperature on respiration and excre- motolerance and biogeography. Journal of Experimental tion of sea cucumber, Apostichopus japonicus. Oceanolo- Biology 202, 2925–2936. gia et Limnologia Sinica 33, 182–187 (In Chinese with Wang F., Dong S.L., Huang G.Q., Wu L.X., Tian X.L. & English abstract). Ma S. (2003) The effect of light color on the growth of Liao Y. (1997) Fauna Sinica: Phylum Echinodermata Class Chinese shrimp Fenneropenaeus chinensis. Aquaculture Holothuroidea. Science Press, Beijing. 228, 351–360. Lindquist S. (1986) The heat-shock response. Annual Wang Q.L., Dong Y.W., Dong S.L. & Wang F. (2011) Review of Biochemistry 55, 1151–1191. Effects of heat-shock selection during pelagic stages on

thermal sensitivity of juvenile sea cucumber, Apostich- Yang H.S., Zhou Y., Zhang T., Yuan X.T., Li X.X., Liu Y. opus japonicus Selenka. Aquaculture International, doi: & Zhang F.S. (2006) Metabolic characteristics of sea 10.1007/s10499-011-9436-x. cucumber Apostichopus japonicus (Selenka) during aesti-

Wu Z.Z. (2007) Improved method of NH4–N determined vation. Journal of Experimental Marine Biology and Ecol- by hypobromite oxidation in water. Marine environmen- ogy 330, 505–510. tal science 26,84–87 (In Chinese with English abstract).