Chinese Journal of Oceanology and Limnology Vol. 28 No. 2, P. 218-223, 2010 DOI: 10.1007/s00343-010-9016-3

Metabolic rates and biochemical compositions of Apostichopus japonicus (Selenka) tissue during periods of inactivity*

BAO Jie (包杰), DONG Shuanglin (董双林)**, TIAN Xiangli (田相利), WANG Fang (王芳), GAO Qinfeng (高勤峰), DONG Yunwei (董云伟) Key Laboratory of Mariculture, Ministry of Education, Ocean University of China, Qingdao 266003, China

Received Feb. 16, 2009; revision accepted May 4, 2009

© Chinese Society for Oceanology and Limnology, Science Press and Springer-Verlag Berlin Heidelberg 2010

Abstract Estivation, hibernation, and starvation are indispensable inactive states of sea cucumbers Apostichopus japonicus in nature and in culture ponds. Generally, temperature is the principal factor that induces estivation or hibernation in the sea cucumber. The present study provided insight into the physiological adaptations of A. japonicus during the three types of inactivity (hibernation, estivation, and starvation) by measuring the oxygen consumption rates (Vo2) and biochemical compositions under laboratory conditions of low (3°C), normal (17°C) and high (24°C) temperature. The results show that the characteristics of A. japonicus in dormancy (hibernation and estivation) states were quite different from higher animals, such as fishes, amphibians, reptiles, and mammals, but more closely resembled a semi-dormant state. It was observed that the shift in the A. japonicus physiological state from normal to dormancy was a chronic rather than acute process, indicated by the gradual depression of metabolic rate. While metabolic rates declined 44.9% for the estivation group and 71.7% for the hibernation group, relative to initial rates, during the 36 d culture period, metabolic rates were not maintained at constant levels during these states. The metabolic depression processes for sea cucumbers in hibernation and estivation appeared to be a passive and an active metabolic suppression, respectively. In contrast, the metabolic rates (128.90±11.70 μg/g h) of estivating sea cucumbers were notably higher (107.85±6.31 μg/g h) than in starving sea cucumbers at 17°C, which indicated that the dormancy mechanism here, as a physiological inhibition, was not as efficient as in higher animals. Finally, the principle metabolic substrate or energy source of sea cucumbers in hibernation was lipid, whereas in estivation they mainly consumed protein in the early times and both protein and lipid thereafter.

Keyword: Apostichopus japonicus; oxygen consumption rates; chemical composition; temperature, periods of inactivity

1 INTRODUCTION

The sea cucumber Apostichopus japonicus (Selenka), belonging to Echinodermata, Holothuroidea, has become an important aquaculture species in China (Chen, 2004). As a deposit feeder, A. japonicus plays an important role in the energy flow and nutrient cycling in aquaculture and natural systems (Yang et al., 2000a; 2000b).

A temperate species, A. japonicus is a dominant species along Asian coasts from 35°N to 44°N, vertically distributed from the intertidal zone to waters 20–30 m deep, and can survive conditions of 0–30°C (Yu et al., 1999; Chen, 2004). Temperature is the principal factor determining growth, with the

fastest growth rates in spring and autumn. Growth is obviously inhibited with low temperatures in winter or high temperatures in summer (Chang et al., 2004), with movement, metabolism, and feeding considerably reduced and hibernation initiated when temperatures are lower than 3°C (Tanikawa et al., 1955; Fish, 1967; Yu et al., 1999). In contrast, at summer high temperatures (>20°C), the organisms generally also show a state of estivation, especially for large individuals with body weight >25 g (Choe, Supported by the National Natural Science Foundation of China

(No.30400333) and the National Key Program of Science and Technology

of China (2006BAD09A01) Corresponding author: dongsl@ouc.edu.cn

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1963; Liu et al., 1996; Li et al., 1996). Estivation generally lasts for 4 months each year in most of the cucumber's range in China (Liu et al., 1996). In northern China, water temperature is maintained at 2°C–28°C in sea cucumber culture ponds and can be lower or higher in relatively shallow ponds (Chen, 2004).

Estivation is defined as a state of dormancy or torpor which allows animals to survive under extremely hot and/or arid conditions; hibernation is a similar state or behavior employed to deal with cold conditions. Estivation or hibernation periods vary between species and with differing ambient conditions, using the overall strategy to maintain vital physiological processes during these long periods of torpor. Animals can depress metabolism and/or cease feeding with cold temperature (e.g., hibernating mammals), anaerobiosis (intertidal mollusks, turtles, fishes, and diving mammals), and dehydration (brine shrimp, snails, frogs, and lungfish), as a strategy to extend an animal’s survival time with limited internal fuel supplies under environmental stresses (Guppy et al., 1994; Pedler et al., 1996; Wilz et al., 2000; Secor, 2005). The changes in behavior or physiology of A. japonicus under hibernation and estivation exhibit typical characteristics, including cessation of feeding, reduction in activities, and metabolic depression (Tanikawa et al., 1955; Yu et al., 1999; Yang et al., 2005; Yang et al., 2006).

Estivation, hibernation, and starvation are unavoidable inactive states of A. japonicus in culture. During estivation, the organism’s alimentary tract degenerates (Li et al., 1996), and high mortality and biomass loss from estivation of A. japonicus are severe problems for sea cucumber mariculture. To gain insight into A. japonicus physiological adaptations during inactive periods of hibernation, estivation, and starvation, the present study focused on biomass changes, metabolic depression, and changes in biochemical composition of A. japonicus tissue during hibernation at lower temperatures, starvation at normal temperatures, and estivation at higher temperatures.

2 MATERIALS AND METHODS

2.1 Sample collection and maintenance

Experiments were performed from November 5 to February 10, 2006, in the laboratory of the Institute of Aquaculture, Ocean University of China, Qingdao, People’s Republic of China. A. japonicus were

collected from a culture farm at Jiaonan City, Qingdao, China, transported to the laboratory, randomly divided into four groups, and divided into eight 400 L PVC tanks and acclimated for 20 d at 17ºC. The sea cucumbers were fed a formulated diet (16.6% crude protein, 5.4% fat, 47.4% ash, 2.3% moisture, and 8.42 kJ/g metabolic energy) and continuous aeration supplied at all times. Every day 1/2–2/3 of the recirculated water was replaced with fresh seawater preheated or cooled to the appropriate temperature. Temperature was controlled to ±0.5°C, salinity at 29–31, and the light cycle was 14 h light/10 h dark.

2.2 Experimental design

Three experimental groups of sea cucumbers were cultured at 3°C, 17°C, and 24°C, with a fourth group as a control cultured with normal feed supply (feed 10% of wet body weight/d) at 17°C. The experimental temperatures of 3°C for hibernation and 24°C for estivation were determined by a preliminary experiment, which established that A. japonicus initiated dormancy at these temperature extremes. Previous reports also suggested that A. japonicus gradually stopped feeding and started dormancy when seawater temperature was below 3°C (Tanikawa et al., 1955; Yu et al., 1999) or above 20°C (Choe, 1963; Li et al., 1996; Liu et al., 1996). After laboratory acclimation, two of the four groups were maintained at a constant 17°C, while the other two groups were acclimated to 3 and 24°C by means of gradual changes in temperature up or down by 1–2°C/d. Upon reaching the desired temperature regime, the cucumbers were fed normal rations until all individuals had entered the experimental state (cessation of feeding in hibernation or estivation), and then experimentation began. In these experiments, cucumbers in low and high temperature states were considered to be in simulated states of their natural inactive environmental states and, thus, would reflect their dormant physiological characteristics, despite these simulated states maybe differing from their natural environment to some extent.

2.3 Body weight

Sea cucumbers were maintained in glass aquaria (450 mm×250 mm×350 mm) and a total of 16 experimental groups, of three sea cucumbers each, were randomly allocated to four sets for four treatments: the control or feeding group (FD), hibernation group (HT), estivation group (AT), and

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starvation group (ST). The sea cucumbers’ wet body weights were determined at 0, 6, 12, 18, 26, and 36 d after the acclimation and inactive states were achieved, with the individual wet weights taken within 1 min of removal from seawater and external water removed by drying on sterile gauze (Dong et al., 2006; Battaglen et al., 1999).

2.4 Determination of oxygen consumption rate

The oxygen consumption rate (Vo2) was determined at 0, 3, 6, 9, 12, 18, 26, and 36 d for the fasting and feeding groups during the experiment. For each determination, six individuals were placed separately in 3 L respiration chambers (Yang et al., 2006), maintained in the chambers for 4 h for acclimation. After a 6 h test period, the final level of dissolved oxygen in each chamber was measured with a DO meter (YSI-5000, USA); three chambers without sea cucumbers were used as controls. The oxygen consumption rate (Vo2, μg O2/g

h) was calculated using the following equation (Dong et al., 2006):

2oV = (DOc – DOe) V/M T

where DOc and DOe are the DO levels (μg O2/h) of the control and experimental chambers, respectively, V the volume of chamber (L), T the experimental time (h), and M the wet body weight of the individual (g).

2.5 Determinations of biochemical composition

At day 0, 6, 12, 18, 26, and 36, four sea cucumbers each from the four treatments were weighed and frozen at -80°C for analysis of their dry weight and biochemical composition. The sea cucumbers were later freeze dried at -60°C to constant weights and the tissue ground to a fine powder with a pestle and sieve. Crude protein was estimated from nitrogen content (N×6.25), measured with an elemental analyzer (VarioEL 3, Elementar Analysensysteme GmbH, Germany), lipid content determined by the methods of Bligh et al. (1959) and Kates (1972), and carbohydrate content determined with the phenol-sulfuric method (Dubois et al., 1956).

2.6 Statistical analysis

Data analyses were performed using the SPSS 11.0 software for Windows (SPSS Inc., 2001). Differences in body weight and biochemical compositions between the treatments were compared by one-way ANOVA followed by Duncan’s multiple range tests for post hoc pairwise comparisons. Prior to analysis, raw data were diagnosed for normality of distribution and homogeneity of variances by the

Kolmogorov-Smirnov test and Levene test, respectively (Zar, 1999). The relationship between dry body weight and feeding/fasting duration of A. japonicus in each experimental group was demonstrated by multiple regression analysis following standard least-square linear procedures.

3 RESULTS

3.1 Body weight

No mortality occurred during the experiment. Sea cucumber dry body weights (freeze-drying) during the fasting period showed that, while the FD weights increased by 16.9% after 36 d, the HT, AT, and ST treatments weights decreased significantly, by 9.0%, 33.3% and 31.2%, respectively (Fig.1). Quadratic functions relating dry body weight (freeze-drying, DW, g) to fasting/feeding duration (D, d) were fitted for each experimental group.

Fig.1 Dry body weight of Apostichopus japonicus at day 0, 6, 12, 18, 26, and 36 under conditions of feeding, hibernation, estivation, and starvation

Results expressed as mean±SE; n = 4 in all treatments; DW, dry body

weight; D, fasting or feeding duration; solid lines: 1, DW = 0.155 4 D2 –

0.548 2 D + 19.05 (r2 = 0.941 8, n = 12); 2, hibernation, DW = -0.031 9 D2

– 0.084 D + 18.768 (r2 = 0.975 5, n = 12); 3, estivation, DW = 0.240 5 D2 –

2.792 7 D + 19.529 (r2 = 0.997 6, n = 12); 4, starvation, DW = 0.145 8 D2 –

2.282 6 D + 21.361 (r2 = 0.981 6, n = 12)

3.2 Oxygen consumption rates

The Vo2 of the FD group increased by 21.0% after 36 d, while Vo2 remained at the highest level for the AT group, followed by the ST group, and the lowest for the HT group (Fig.2); the three fasting groups showed decreasing trends in Vo2 during this period. Notably, the HT group Vo2 decreased sharply during the initial 12 d period and then declined at a relatively slower rate thereafter. On average and compared to the initial values, Vo2 had decreased by 44.9%, 48.9% and 71.7% for the AT, ST, and HT groups, respectively, by the end of the experiment.

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Fig.2 Oxygen consumption rates of A. japonicus in feeding, hibernation, estivation, and starvation states

Results expressed as mean±SE, n=6 in all treatments

3.3 Biochemical composition

Though the initial weights and acclimation times of the four groups of sea cucumbers were the same, the initial protein and carbohydrate percentages for the AT group and lipid percentage for the HT group were initially lower than the other two groups, possibly due to differential tissue utilization during acclimation to different temperature regimes (Fig.3).

In the FD group, the protein and carbohydrate percentages increased significantly (P<0.05), while the lipid percentage decreased significantly compared with initial values (day 0, Fig.3). Between the beginning and end of fasting, the protein percentage in the HT group decreased, but not significantly (P=0.993). The lipid and carbohydrate percentages of this group, however, decreased significantly (P<0.05) from 7.0%–5.0% and 0.07%–0.02%, respectively, after 36 d. For the AT group, the protein and carbohydrate percentages decreased by day 6 from the initial 31.9% to 15.7% and 0.04% to 0.02%, respectively. No significant changes in lipid percentage was detected in the AT group before day 12 (P=0.673), but declined sharply henceforth and decreased to 4.1% by experiment's end. For the ST group, the protein, lipid, and carbohydrate percentages all decreased significantly (P<0.05) and simultaneously with fasting time, from 34.6%–16.2%, 7.3%–2.8%, and 0.07%–0.03%, respectively.

4 DISCUSSION

Dormancy (hibernation or estivation) is a widely recognized behavioral and physiological state of animals that generally indicates reduced activity and metabolic rate and functions as a response to fluctuations in environmental conditions, such as temperature, water, or food. Previous studies have shown that the metabolic rates of animals drop

Fig.3 Biochemical compositions of A. japonicus at feeding, hibernation, estivation, and starvation states

The results are expressed as mean±SE, n=4 in all treatments

precipitously (depressed 60%–99%) in a few hours or days after estivation or hibernation are initiated (Guppy et al., 1999; Buck et al., 2000; Wilz et al., 2000; Jackson, 2002) and that metabolic rates during dormancy are maintained at constant, low levels independent of the ambient temperature (Nizielski et al., 1989; Arnold et al., 1991; Song et al., 1997; Buck et al., 2000; Milsom et al., 2007). The dormant state is beneficial to animals because lowered metabolic rates conserve energy and extend the animals’ survival times. As an aquatic, ectothermic species, A. japonicus exhibits both hibernation and estivation characteristics, indicated by cessation of feeding, reduction in activities, and depressed metabolism.

The present results show that the dormancy characteristics of A. japonicus were different from other higher animals (fishes, amphibians, reptiles, and mammalians) and more resembled a state of

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semi-dormancy. Initialization of the dormant state here was a long, gradual process, indicated by the slow depression of the metabolic rates and, although metabolic rates declined after 36 d by 44.9% and 71.7% in the AT and HT groups, respectively, they did not maintain a constant lowered rate. Dormancy resulted in obvious losses of body weight and apparent energy conservation, especially in the AT group. The metabolic rate of the AT group at 24°C was higher than that of the ST group at 17°C, and their average body weight decreased by 33.3% by day 36, finishing at 1.1% higher than the ST group.

The metabolic rates of ectothermic animals are related to temperature, and Q10 values are often calculated to determine whether observed changes in metabolic processes are the direct effect of temperature (indicated by Q10 values of 2–3) or independent of temperature (Q10>3, Snapp et al., 1981; Geiser, 1988; Guppy et al., 1999; Johanne et al., 2007). Observed here, the overall increase in Q10 values between 3°C and 17°C was 2.5 at 36 d, indicating that the metabolic depression of the sea cucumber in hibernation was a passive temperature-dependent process. This passive temperature-dependent metabolic depression in sea cucumber hibernation was the opposite of the temperature-independent active metabolic depression that has been observed in labrid fish (Tautogolabrus adspersus, Johanne et al., 2007), Antarctic fish (Notothenia coriiceps, Campbell et al., 2008), amphibians, reptiles, and mammals in hibernation (Snyder et al., 1990; Song et al., 1997; Lewis et al., 2007). In contrast, the Q10 value calculated here from the metabolic rates at 36 d between the 17°C and 24°C treatments was 1.3 for A. japonicus; therefore, the metabolic rate depression of these organisms in estivation was a result of temperature-independent active metabolic suppression. Ji et al. (2008) have reported a reduction in reactive oxygen species (ROS) and denatured protein in A. japonicus during estivation. However, the characteristics exhibited by A. japonicus indicated that the mechanism of its dormancy, functioning by physiological inhibition, was not as efficient as in higher animals; further experiments should be conducted to explore these differences.

Metabolic depression in dormancy allows animals to survive for extended periods with limited fuel reserves, and energy may be derived from the catabolism of protein, lipid, or carbohydrates for metabolic needs. In these experiments, the carbohydrate contribution as an energy source was

negligible due to the small quantities of carbohydrate in A. japonicus tissue. In hibernation, the sea cucumbers mainly utilized lipid for energy, which led to decreases in lipid proportions, with small changes in the tissue protein contents during the experiment. In estivation, the sea cucumbers mainly consumed protein for energy before day 12, with protein and lipid utilized simultaneously thereafter, probably due to the high metabolic activity (Fig.3).

In conclusion, during periods of inactive states, the body weight and metabolic rate declined and the biochemical composition changed in A. japonicus due to the stress of fasting. Accordingly, in practical sea cucumber aquaculture, it would be important to improve the feed composition to include suitable proportions of lipid and protein necessary to meet the nutritional requirements of sea cucumbers during estivation and hibernation. In addition, it appears possible in practice to lengthen the feeding season and increase production by avoiding the dormancy state through adjustments of the culture pool water levels to control water temperatures (i.e., lowering water level during late spring would raise water temperature or deeper water level during summer would decrease temperature).

5 ACKNOWLEDGMENTS

We would like to thank Hongbo JIANG and Guancang DONG for their assistance in experimental operations.

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