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1 W3-42, Department of Biological Sciences, Graduate School of Bioscience & Biotechnology, Tokyo Institute of Technology. O-okayama 2-12-1, Meguro, Tokyo 152-8551, Japan
2 Science and Education Center, Ochanomizu University. Ohtsuka 2-1-1, Bunkyo, Tokyo 112-8610, Japan
* To whom correspondence should be addressed, at Research Team for Zoonotic Diseases, National Institute of Animal Health, National Agriculture and Food Research Organization. 3-1-5 Kannondai, Tsukuba, Ibaraki, 305-0856, Japan. E-mail: ntakemae{at}affrc.go.jp
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Abbreviations: CCT, catch connective tissue • KASW, artificial seawater with the potassium concentration elevated to 100 mmol l–1 • LBWM, longitudinal body-wall muscle • VO2, oxygen consumption rate per unit of body weight
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Introduction |
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TOPAbstractIntroductionMaterials and MethodsResultsDiscussionLiterature Cited |
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Sea cucumbers are exceptional among echinoderms in not having massive calcite skeletons, and thus their low energy expenditure is not attributable to a large calcite content (Webster, 1975). We hypothesized that the large content of connective tissue, instead of calcite ossicles, and the exceptionally small content of muscle might contribute to the low energy expenditure. We measured the connective tissue content and the muscle content in sea cucumbers to test this hypothesis.
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Materials and Methods |
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TOPAbstractIntroductionMaterials and MethodsResultsDiscussionLiterature Cited |
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Sea cucumbers were collected near Sesoko Station, Tropical Biosphere Research Center, University of the Ryukyus, shipped to Tokyo Institute of Technology, and kept in an aquarium of circulating natural seawater at 23–25 °C. An animal, unfed for more than 2 days, was placed on a plate in the air for 10 min to let it evacuate seawater from the cloaca. The body weight was measured after the body surface was blotted with a piece of filter paper. Twelve animals with wet weight 291–562 g were used for the measurement of tissue-oxygen consumption.
Two samples were dissected from each animal: a piece of the LBWM with a wet weight of about 0.4 g and a column of dermis from the interambulacral area of the dorsal body wall. Dermis samples were trimmed so that their weight was about 2 g. Both samples were rested in artificial seawater (ASW) for more than 1 h at 20 °C. The ASW (My Sea, practical salinity 33) was purchased from Jamarine, Japan. A sample was placed in an air-tight trough of 14-ml volume filled with air-saturated ASW. The trough was placed in a temperature-controlled water bath at 20 °C. A polarographic oxygen electrode (5331 oxygen probe, Yellow Spring Instrument Co., USA), connected to an oxygen monitor (Model 5300, Yellow Spring Instrument Co., USA), was inserted into the trough. A magnetic stirrer ball just in front of the electrode provided a vigorous current to the electrode tip and a gentle stirring current to the rest of the trough. The electrode was calibrated with a zero solution (sodium sulphite) and air-saturated ASW (Nakaya et al., 2003). Data were collected through an analog-to-digital converter (A/D) board (LAB Stack, KGL, Japan) connected to a personal computer.
The oxygen content in a trough decreased linearly with time. The rate of decrease was calculated using a simple linear regression. The rate of oxygen consumption of the tissue (VO2) was the value thus obtained from which the rate measured in the trough filled with ASW without tissue samples was deducted; the latter was less than 1/10 of the former. VO2 was given as a wet-weight-specific value. VO2 values before and after chemical stimulation were compared. The oxygen consumption was recorded for 1 h in ASW, and VO2 was measured as the average slope of the oxygen consumption curve between 10 min before and just before chemical stimulation. Muscles and dermis rested in ASW were regarded as being in the relaxed state and in the standard state, respectively.
Chemical stimulation was applied 1 h after the onset of measurement. Chemical stimulation used for dermis was ASW with the K+ concentration elevated to 100 mmol l–1 (KASW). For LBWM. either KASW or 1 mmol l–1 acetylcholine (ACh) was used. KASW is known to change the dermis in the standard state to the stiff (catch) state through stimulation of cells controlling connective tissue catch (Motokawa and Tsuchi, 2003). A small amount of medium containing a high concentration of K+ was introduced into the trough, and the same amount of the original medium was removed to increase the K+ concentration in the trough to 100 mmol l–1. The composition of K+-rich medium introduced was as follows: KCl, 433.7 mmol l–1; CaCl2, 10.1 mmol l–1; MgCl2, 52.5 mmol l–1; NaHCO3, 2.5 mmol l–1 (pH 8.1). The amount introduced was 20% of the trough volume. Because the newly introduced medium was saturated with oxygen and thus the oxygen concentration was a little higher than that of the media already in the trough, the introduction caused a small increase in the oxygen concentration in the trough. The control experiment, in which ASW instead of K+-rich medium was introduced, showed that the rate of oxygen consumption by dermis was not affected by this small rise in oxygen concentration. The introduced volume of the ACh-containing medium was 10% of the trough volume to make the final ACh concentration 1 mmol l–1. Acetylcholine chloride was purchased from Nacalai Tesque, Japan. The VO2 under chemical stimulation was obtained as the averaged slope of the oxygen consumption measured between 5 min and 25 min after the application of stimulation.
Oxygen consumption of individuals
The energy consumption of live individuals of A. mauritiana was also measured. Three animals, with wet body weight 368 g, 369 g, and 470 g, were used. A sea cucumber was placed in a plastic bag filled with about 1000 ml of seawater. The bag was placed in a water bath whose temperature was maintained at 20 °C. The oxygen electrode was inserted into the bag and seawater in the bag was stirred by a magnetic stirrer bar. The animal was rested for at least 1 h before oxygen consumption was measured. Twenty minutes after the measurement had begun, we patted the dorsal surface of the animal by hand for 20 min. The behavior of the animal was video-recorded and the body length was measured from the video.
Mechanical tests
Dynamic mechanical tests were performed on dermal pieces dissected from the interambulacral dorsal body wall of A. mauritiana. The size of the test piece was 6 mm x 2 mm x 0.5 mm, with the long axis corresponding to that of the animal. The sample was rested in ASW for more than 1 h at 20 °C before experiments. One end of the sample was glued by commercial adhesives to the aluminum plate at the bottom of the trough filled with ASW maintained at 20 °C; the other end was glued to the lever of a force gauge (LTS-200G, Kyowa, Japan) mounted on a vibrator. The vibrator periodically stretched the sample in the direction of the long axis and compressed it at a frequency of 0.3 Hz. Strain imposed was ±2% of the initial length. This condition had been confirmed to obtain the stiffness changes readily when the dermis of a sea cucumber altered its mechanical properties (Motokawa and Tsuchi, 2003). The force data were collected with a computer at a rate of 8 Hz. Stiffness was expressed as the maximum stress divided by the maximum strain (i.e., 0.02); the maximum stress was observed at the maximum strain. As chemical stimulation, KASW was added to the trough to increase the K+ concentration to 100 mmol l–1 1 h after the beginning of oscillation.
Isometric contractions of LBWM of A. mauritiana were measured. The length of the muscle samples was 10 mm. The sample was rested in ASW for 1 h before it was placed in an experimental trough. One end of the sample was glued to the bottom of the trough, and the other end was glued to the lever of a force gauge. A small stretch was given to the sample by raising the position of the force gauge, and that position was then fixed. This resulted in a small passive tension that subsequently decreased. The muscle was rested in the trough for 10 min, and the K+ concentration was raised to 100 mmol l–1 to induce contraction.
Tensile tests of LBWM were also carried out both in the relaxed state and in a contracted state induced by KASW. The muscle strip, whose original length was 10 mm, was stretched at the rate of 0.1 mm/s with a motor-driven manipulator (Surugaseiki, Japan). This strain rate was comparable with that in the stretching phase in the dynamic mechanical tests on the dermis.
Weight and volume of body components
To estimate muscle and connective-tissue content in the body of sea cucumbers, we took the following steps. We dissected sea cucumbers and weighed their organs. Then the muscle and connective tissue content in each organ was estimated from histological sections. The total number of tube feet in the body was estimated from the density of tube feet and the surface area of the body. From the data thus obtained, the total amounts of muscle and connective tissue were estimated. They were expressed as a percentage of body wet weight. The species used were A. mauritiana, Bohadschia argus (Jaeger), and Stichopus chloronotus (Brandt). Wet body weight of all animals is given in Table 4. All the sea cucumbers were collected in the same lagoon near Sesoko Station, Tropical Biosphere Research Center, University of the Ryukyus, in November when their gonads were undeveloped. The animals were kept in an aquarium of running seawater in Sesoko Station for more than 2 days to empty their gut contents. Animals were dissected and their organs were weighed. The organs and body fluids that were investigated were body wall with tube feet attached, perivisceral coelomic fluid, fluid from the water vascular system, circular body-wall muscle, longitudinal body-wall muscle, ampullae of tentacles, tentacles, ampullae of tube feet, calcareous ring, stone canal with madreporite attached, Polian vesicles, gut, cloaca, respiratory trees, Cuvierian tubules, hemal system, and gonad (Fig. 1).
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The surface area of the body and the density of the tube feet were measured in the pictures captured by an image scanner. We dissected the animal into dorsal and ventral halves and left them in seawater to relax before image capturing. The density was determined for anterior, middle, and posterior parts of the ventral surface, and the average of these values was taken as the average density for the ventral surface. The same procedure was adopted for the dorsal surface except in S. chloronotus, all of whose tube feet were counted.
The content of ossicles in the body was measured. The body was chopped into small pieces and immersed in commercial bleach to remove soft tissues. Ossicles were dried in an incubator maintained at 60 °C for more than 2 days and then weighed.
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Results |
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TOPAbstractIntroductionMaterials and MethodsResultsDiscussionLiterature Cited |
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Oxygen consumption of individuals
The oxygen-consumption rate of individuals of A. mauritiana in non-stimulated condition showed large variations. For example, it was 0.68–2.01 ml/h with the average 1.48 ml/h (n = 4) for the animal with a body weight of 369 g. Two other animals, one with body weight 368 g and the other 470 g, had average values of 1.23 ml/h and 1.61 ml/h respectively. The average oxygen consumption rate per unit weight for these three animals was 3.59 ± 0.38 µl · g–1 · h–1 (±SD). A typical response to mechanical stimulation is shown in Figure 2. The animal immediately shortened its body in response to patting and expelled seawater from the cloaca. At the same time, we could feel. by hand, the stiffening of the body. It took a few minutes for the body to be fully contracted, and during that period the oxygen consumption rate increased about 10 times. The animal remained shortened and stiff and kept the cloaca motionless as long as the patting continued. In a few minutes, however, the oxygen consumption rate decreased to a level about half of that before stimulation; and that low level, which averaged to be 2.09 ± 0.19 µl · g–1 · h–1 (± SD, n = 3), was maintained during patting.
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Typical stress-strain curves of LBWM in tensile tests are given in Figure 3c. The stress values at 2% and 15% in ASW were 0.47 ± 0.17 kPa and 2.23 ± 1.25 kPa (average ± SD, n = 8), respectively. The passive stress in KASW, which was obtained by subtracting the contractile stress at 0 strain from the total stress, was 6.38 ± 2.88 kPa (average ± SD, n = 11) at 2 % strain and 15.0 ± 6.00 kPa (average ± SD, n = 7) at 15 % strain. Four out of 11 samples ruptured before reaching 15% strain. The stiffness calculated from the slope of the stress strain curve at 15% strain in KASW was 69.6 ± 38.0 kPa (average ± SD, n = 7).
Connective tissue and muscle content
The fraction of the wet body weight that each organ occupied is given in Table 2. The body composition was similar in the three species, especially between A. mauritiana and B. argus, which belong to the same family (Holothuriidae). The largest component was the body wall, amounting to half of the body weight. The second and the third largest components were the body fluids: the coelomic fluid and the fluid that oozed out during dissection, mostly from the water vascular system, occupied 20%–30% and 8%–9% of the body, respectively. The top three components were common to all three species. The largest solid organ next to the body wall in the species in Holothuriidae was the circular and longitudinal body-wall muscle, occupying about 5%; in S. chloronotus; the respiratory trees were the next largest. The body-wall muscle of S. chloronotus, which occupied 3% of the body, was thinner than those of the other two species, and we had difficulty in separating longitudinal muscles from circular muscles by dissection. The hemal system of S. chloronotus was also poorly developed. Well-developed Cuvierian tubules were found only in B. argus, which was the only species examined in this study that is known to eject them in response to stimulation.
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Data in Table 3, together with the total number of tube feet and the weight of circular and longitudinal muscle, were used to estimate the total muscle content and total connective tissue content in the body of A. mauritiana (Table 5). Circular muscle and longitudinal muscle were postulated to be purely muscular because we could not find connective tissue layers or other tissues of measurable thickness in their histological sections. Muscles used in locomotion, which included muscles in tube feet and body wall, occupied 5.1% of the body (Table 5). The total fraction of muscles—locomotory and non-locomotory muscles combined—was 6.6% of the body. The total fraction of connective tissue was 58% of the body; 90% of connective tissue was found in the body wall.
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Ossicles made up 1.67% ± 0.35% (average ± SD, n = 5) of the wet weight of the body wall in A. mauritiana, 1.90% ± 0.49% in B. argus, and 1.08% ± 0.59% in S. chloronotus. The ossicle content of the whole animal was 1.62% ± 0.34% (average ± SD, n = 5) of wet body weight in A. mauritiana, 1.42% ± 0.32% in B. argus, and 1.17% ± 0.64% in S. chloronotus.
Water content of the dorsal body wall dermis of A. mauritiana was 82.7% ± 1.9% (average ± SD, n = 19).
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Discussion |
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TOPAbstractIntroductionMaterials and MethodsResultsDiscussionLiterature Cited |
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The specific oxygen consumption rate of individuals of Actinopyga mauritiana (4 µl · g–1 · h–1) was comparable to the values reported for other sea cucumbers (Lawrence and Lane, 1982). However, on the basis of the equation for the relationship between oxygen consumption and body mass (Hemmingsen, 1960), we calculate that individual A. mauritiana used only 1/8 the energy (O2 consumption = 1.48 ml/h; 369 g animal) of a "standard" invertebrate of the same size (O2 consumption = 11.9 ml/h).
Echinoderms are known for their low energy consumption, which has been attributed to the large content of calcite ossicles and to the CCTs, which were supposed to maintain the body posture with little expenditure of energy (Emson, 1985; Motokawa, 1985, 1988). In sea cucumbers, however, calcite content is low: it was only 1.2%–1.7% of wet body weight in the present species. Below, we discuss evidence that, rather than the content of calcite, it is the content of connective tissue and the economical nature of the connective tissue that contribute to the low oxygen consumption of sea cucumbers.
Body composition
This study is the first detailed description of body components of sea cucumbers—in particular of the connective tissue and muscle of the whole body. On the basis of the wet weight of the body, connective tissues occupied 57.5% in A. mauritiana, 93% of which was found in the body-wall dermis, whereas muscles occupied only 6.6%. The estimated values for Bohadschia argus and Stichopus chloronotus were respectively 45% and 61% for connective-tissue content, and 7.7% and 5.4%, for muscle content. Giese (1966) found 30% for the body-wall connective tissue and 10% for body-wall muscles in Parastichopus californicus. The high content of connective tissue and low content of muscles seem to be characteristic of sea cucumbers among vermiform animals, most of which have body walls consisting mainly of muscles. For example, body-wall muscles occupy about half of the body weight of the earthworm Eisenia foetida (Motokawa, unpubl.). In mammals, an allometric study showed that muscles occupy 45% and skin occupies 14% of the body (Peters, 1983). Weight-specific oxygen consumption rate of skin is 10%–50% that of skeletal muscles in rats and 28% in dogs (Schmidt-Nielsen, 1984; Alexander, 1999). In the present study, the dermis also used far less energy than the muscles: VO2 of the dermis in the standard state was only 17% that of the resting muscles. From these data we could conclude that the higher content of energy-saving connective tissue and lower content of energy-consuming muscles contributed to the low energy expenditure of sea cucumbers.
Energetic advantage of catch connective tissue in posture maintenance
Stimulation by seawater containing a high K+ concentration (KASW) increased VO2 of dermis by 50%. The time course of stiffness change in KASW verified that the VO2 measured was that in the stiff state. KASW probably stimulated nerves and secretory cells involved in connective tissue catch by depolarization of the cell membrane (Motokawa, 1994). Because the dermis was occupied mostly by extracellular materials and thus the number of cells in the dermis was small, the contribution of cells other than those involved in connective tissue catch to this KASW-induced VO2 increase could be expected to be small.
KASW increased VO2 of the longitudinal body-wall muscle (LBWM) 2.6 times. The time course of contraction in KASW verified that measured VO2 in KASW was that in the contracted state. Acetylcholine (1 mmol l–1) doubled the VO2 of LBWM. That these two different muscle contractants increased VO2 by similar amounts strongly suggests that this increase was mostly associated with muscle contraction. Similarly, in LBWM of Stichopus mollis, Gay and Simon (1964) found that KASW containing the same amount of K+ as in the present experiment increased VO2 to 32.5 µl · g–1 · h–1 (22 °C), which was close to the present value of 23.5 µl · g–1 · h–1 (20 °C).
The VO2 of the dermis in the stiff state was 2.5 µl · g–1 · h–1, which was about 1/10 that of LBWM in contraction. The stress required to strain the dermis by 2% in the stiff state was 190 kPa, whereas the stress required to strain the contracting LBWM by the same amount was 26.8 kPa, which was the sum of the active tensile stress of 20.4 kPa and the passive tensile stress of 6.4 kPa. The stress required in the case of dermis was about 7 times greater than that of muscles. This implies that sea cucumbers could maintain their posture against the same external force with 1/7 tissue volume when they used connective tissue instead of muscle. Thus the volume advantage (1/7) and the energetic advantage (1/10) predict that sea cucumbers could maintain their posture with 1/70 the energy consumption. The extent of this energetic advantage changes, of course, with the applied force and thus the strain in the dermis. The energetic advantage would be greater at greater strains.
Patting individuals of A. mauritiana caused immediate shortening and stiffening of the body, forceful ejection of seawater from the cloaca, and an increase in the rate of oxygen consumption. Although muscular contraction and stiffening of the dermis no doubt contributed to this VO2 increase, the expelled seawater with low O2 content may have significantly contributed to this apparent increase. The shortened and stiff state of the body, and also the cessation of the cloacal pumping, lasted during the mechanical stimulation. The increase in VO2 was, however, only for the first few minutes, and then it decreased to a level half of that before mechanical stimulation. On one hand, this result was reasonable because respiratory trees would contribute half to the total oxygen uptake; the other half would be through body surfaces (Lawrence, 1987). On the other hand, we might have expected a sustained increase in VO2 because remaining contracted and stiff would need more energy. It is possible that some internal organs, including LBWM, shifted from aerobic to anaerobic metabolism (Shick, 1983), with the shortened body being maintained by the sustained contraction of the LBWM. The LBWM tolerates anaerobic condition in vitro (Ellington and Hammen, 1977; Shick, 1983). There are, however, no data on whether it generates active forces as large as those demonstrated under aerobic conditions. A more likely explanation, taking into account our results on the relative O2 consumption of muscle and connective tissue, is that the shortened and stiffened condition of the body was maintained by stiffened dermal connective tissue, which probably functioned aerobically because of its location near the body surface. The low energy requirement of CCT may give the sea cucumber the ability to prolong stiffening for mechanical defense and posture maintenance, even in adverse conditions when the supply of energy is limited.
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Acknowledgments |
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Footnotes |
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Literature Cited |
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TOPAbstractIntroductionMaterials and MethodsResultsDiscussionLiterature Cited |
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(VO2) of dermis and longitudinal body-wall muscle (LBWM) from Actinopyga mauritiana in artificial seawater (ASW), seawater with excess potassium (KASW), and acetylcholine (ACh)
