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Feeding behavior and digestive physiology in sea cucumber Apostichopus japonicus
PHB-10594; No of Pages 8 Physiology & Behavior xxx (2014) xxx–xxx
Contents lists available at ScienceDirect
Physiology & Behavior journal homepage: www.elsevier.com/locate/phb
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Jiamin Sun a,c, Libin Zhang a,⁎, Yang Pan a,b, Chenggang Lin a, Fang Wang c, Rentao Kan d, Hongsheng Yang a,⁎⁎
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Feeding behavior and digestive physiology in sea cucumber Apostichopus japonicus
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Keywords: Sea cucumber Apostichopus japonicus Size Tentacle Feeding rhythm Digestive enzyme
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of sea cucumber culture, such as ecological conditions, stocking density and feed nutrition have been solved to some extent [2–5]. However, relatively little is known about sea cucumber feeding behavior in an aquaculture setting. Such information could be useful for designing optimal feeding schedules to increase production and decrease food waste. Sea cucumber collects food by extending its tentacles surrounding its mouth. The type of tentacle is closely linked to the mode of feeding employed by each species [6]. For example, holothurians, Psychropotes longicauda have stubby peltate tentacles that collect sediment particles by sweeping, whereas holothurians, Oneirophanta mutabilis have branched digitate tentacles that collect sediment particles by raking [7]. The tentacle structure and feeding strategy vary not only in different species but also within same species. Cameron and Fankboner [8] observed that the pentacula larva, juvenile, and adult life stages of Parastichopus californicus had subtle differences in tentacle structure
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The feeding behavior and digestive physiology of the sea cucumber, Apostichopus japonicus are not well understood. A better understanding may provide useful information for the development of the aquaculture of this species. In this article the tentacle locomotion, feeding rhythms, ingestion rate (IR), feces production rate (FPR) and digestive enzyme activities were studied in three size groups (small, medium and large) of sea cucumber under a 12 h light/12 h dark cycle. Frame-by-frame video analysis revealed that all size groups had similar feeding strategies using a grasping motion to pick up sediment particles. The tentacle insertion rates of the large size group were significantly faster than those of the small and medium-sized groups (P b 0.05). Feeding activities investigated by charge coupled device cameras with infrared systems indicated that all size groups of sea cucumber were nocturnal and their feeding peaks occurred at 02:00–04:00. The medium and large-sized groups also had a second feeding peak during the day. Both IR and FPR in all groups were significantly higher at night than those during the daytime (P b 0.05). Additionally, the peak activities of digestive enzymes were 2–4 h earlier than the peak of feeding. Taken together, these results demonstrated that the light/dark cycle was a powerful environment factor that influenced biological rhythms of A. japonicus, which had the ability to optimize the digestive processes for a forthcoming ingestion. © 2014 Published by Elsevier Inc.
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Article history: Received 3 October 2014 Received in revised form 13 November 2014 Accepted 14 November 2014 Available online xxxx
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• The tentacle insertion rates depended on the body size. • The feeding peak of all size groups of sea cucumber occurred at 02:00–04:00. • The peak activities of digestive enzymes were 2–4 h earlier than the peak of feeding.
1. Introduction
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Sea cucumber Apostichopus japonicus is distributed mainly in northern China, eastern Russia, Japan, and Korea, which has become a dominant mariculture species because of its relatively high economic value [1]. Usually the sea cucumbers are cultured in shallow seas or ponds without supplement feeds in China. In recent years, industrial aquaculture of sea cucumbers has expanded rapidly becoming an important culturing mode, in which artificial diets are needed. Some basic issues
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Key Laboratory of Marine Ecology and Environmental Sciences, Institute of Oceanology, Chinese Academy of Sciences, 7 Nanhai Road, Qingdao 266071, Shandong, China University of Chinese Academy of Sciences, 19A Yuquan Road, Beijing 100049, China Ocean University of China, 5 Yushan Road, Qingdao 266003, Shandong, China d Shandong Blue Ocean Science and Technology Co., Ltd., Laizhou 261400, Shandong, China b
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⁎ Correspondence to: L. Zhang, Institute of Oceanology, Chinese Academy of Sciences, 7 Nanhai Road, Qingdao 266071, Shandong, China. Tel./fax: +86 532 82896096. ⁎⁎ Correspondence to: H. Yang, Institute of Oceanology, Chinese Academy of Sciences, 7 Nanhai Road, Qingdao 266071, Shandong, China. Tel./fax: +86 532 82898610. E-mail addresses: [email protected] (L. Zhang), [email protected] (H. Yang).
http://dx.doi.org/10.1016/j.physbeh.2014.11.051 0031-9384/© 2014 Published by Elsevier Inc.
Please cite this article as: Sun J, et al, Feeding behavior and digestive physiology in sea cucumber Apostichopus japonicus, Physiol Behav (2014), http://dx.doi.org/10.1016/j.physbeh.2014.11.051
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2. Materials and methods
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2.1. Animals and housing
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Sea cucumbers (A. japonicus) during juvenile period were collected from Tianheng Sea Cucumber Farm (Qingdao, China) and were acclimated in several fiberglass tanks at 16 °C for 2 weeks prior to the experiment. After being fasted for 24 h [21], the domesticated sea cucumbers were divided into three groups: small, with a body length of 5.0 ± 1.1 cm and a wet body weight of 17.80 ± 2.37 g; medium, with a body length of 8.4 ± 1.3 cm and a wet body weight of 40.00 ± 4.64 g; and large, with a body length of 13.6 ± 1.5 cm and a wet body weight of 89.21 ± 8.30 g. The ages of the small, medium and large-sized sea cucumbers were ten months, fourteen months and eighteen months respectively.
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2.2. Experimental conditions
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Aerated water in a 4000 L fiberglass tank was heated to 16.5 °C with a 2000 W electric heater controlled via a thermostat and then pumped into the experimental tanks. The temperature of the experimental room was maintained at 16.0 ± 1.0 °C with air-conditioning. Water salinity was 30–32 ppt, pH was 7.8–8.2, and dissolved oxygen was N5.0 mg L− 1 and maintained by continuous aeration. The animals were maintained on a 12 h light/12 h dark cycle, with the 12 h light period beginning at 08:00. In this study, 08:00–20:00 was defined as “day” and 20:00–08:00 was defined as “night”. The light intensity used in the experiment was 50 lx and was controlled by a light emitting diode with a dimmer and measured with an illumination photometer (TES1332A, Taishi Instruments, Taiwan, China). The sea cucumbers were fed a self-made diet in cylindrical form (diameter = 1 mm) containing 40% Sargassum powder and 60% sea mud during acclimation and all experiments except the tentacle observations. The approximate composition of the diet was 3.49 ± 0.16% crude protein, 0.58 ± 0.01% crude lipid, and 78.25% ± 0.76% ash (dry diet).
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2.3.2. Experiment 2: feeding rhythms Twenty-five small, eleven medium, and five large A. japonicus (similar densities) were separately placed into three glass aquaria (60 cm × 60 cm × 50 cm). Each size group had four replicates. The animals were fed with pellet diets once per day, and half of the water was exchanged at 08:00. After 3–4 days acclimation in the experimental aquaria, feeding behavior of A. japonicus was observed with charge coupled device (CCD) cameras (Hikvision, DS-2CC11A2P-IR3, China) suspended above the aquaria. The camera could record sea cucumber behavior in complete darkness using an infrared system. The video data were stored in a video recorder, and feeding behavior could be observed at any time from the monitor (Fig. 1). Feeding behavior was judged by observing the animals' position in the aquarium and the decreasing of food on the aquarium bottom. The number of sea cucumbers that were feeding was recorded four times every 2 h for 4 consecutive days and evaluated.
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2.3.3. Experiment 3: feeding and ingestion To ensure similar rearing densities in each tank, ten small, five medium and two large gut-evacuated sea cucumbers were stocked separately in three tanks (45 cm × 35 cm × 30 cm) with six replicates. The sea cucumbers were fed to satiation twice per day at 08:00 and 20:00 for 4 days. The remaining uneaten feed and feces were siphoned before exchanging water twice per day, followed by gentle rinsing with freshwater to remove salt. The uneaten food and feces were dried separately at 60 °C to constant weight. Both ingestion rate (IR) and feces production rate (FPR) were calculated as described by Maxwell et al. [22]:
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IR (mg g−1 h−1) = ((Wo − Wu) / Wsc) / t
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2.3.1. Experiment 1: tentacle locomotion To record and compare the fine-scale mechanistic feeding behavior of A. japonicus of different sizes, tentacle locomotion during feeding was observed using an upward-looking digital camera (Canon IXUS 125HS) located below the glass aquarium (60 cm × 60 cm × 50 cm) as described by Hudson et al. [6]. Six individuals of each size were observed. A pinch of sediment particles, insufficient to obstruct the view of camera, were provided to encourage feeding activity. The number of tentacles involved in feeding at any one time and the rates of tentacle insertion were analyzed frame-by-frame based on 100 separate tentacle movements.
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2.3. Experimental protocol
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and feeding strategy. In the present study, we firstly investigated the mechanisms of locomotion and food capture in different size groups of A. japonicus at the tentacle level through real-time photography. Diurnal activity rhythms, particularly feeding rhythm, are ubiquitous throughout Phylum Echinodermata, such as holothurians and echinoids [9–12]. In aquatic animals, photoperiod is considered as the most important synchronizer of biological rhythm. Some studies have demonstrated that A. japonicus is a nocturnal sea cucumber that hides during daytime and comes out to feed at night [13,14]. However, the specific feeding rhythm and its causes in sea cucumber remain unknown. A close relationship exists between physiological function and biological rhythms in aquatic animals [15]. The organisms have the ability to anticipate the approaching meal and then optimize the digestive and metabolic processes. Several studies have been carried out into this physiological adaption. For example, some enzymatic activities have been observed to increase in the digestive system of fishes prior to the expected intake of the daily food [16–18]. The activities of digestive enzymes in sea cucumber have been extensively studied in relation to the influence of diet composition [4], food processing method [19] and temperature [20]. Nevertheless, the digestive rhythms of sea cucumber have scarcely been investigated. In the present experiment, we studied the effect of 12 h light/12 h dark cycle on feeding behavior and digestive physiology of A. japonicus in three size groups (small, medium and large). Our objectives were to: 1) observe and quantify A. japonicus tentacle locomotion; 2) determine their feeding rhythms; and 3) reveal the relationship between the feeding behavior and the digestive physiology of A. japonicus.
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FPR (mg g−1 h−1) = (Wf / Wsc) / t
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where Wo = dried weight of the offered food (mg); Wu = dried weight 175 of the uneaten feed (mg); Wsc = wet weight of the sea cucumber in the 176 tank (g); Wf = dried weight of the feces (mg) and t = time (h). 177 2.3.4. Experiment 4: digestive physiology Sea cucumbers were maintained under the same experimental conditions as Section 2.3.3 Experiment 3: feeding and ingestion for two weeks. Subsequently, six individuals of each size group (small, medium and large) were sampled to obtain intestines every 2 h during a 24 h cycle (12 sampling points). The intestines were cut longitudinally and washed thoroughly in ice-cold 0.84% normal saline. After rinsing, all samples were frozen quickly in liquid nitrogen and then stored at −80 °C until analysis. One intestine sample of sea cucumbers was insufficient to determine three digestive enzyme activities. So we mixed two replicate samples into one sample. In order to avoid the effect of different feeding times, all animals were fed excessively at 08:00 on the sampling day. Sampling during the dark phase was performed under a dim light.
Please cite this article as: Sun J, et al, Feeding behavior and digestive physiology in sea cucumber Apostichopus japonicus, Physiol Behav (2014), http://dx.doi.org/10.1016/j.physbeh.2014.11.051
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Fig. 1. Diagram of the experimental setup to observe Apostichopus japonicus feeding behavior. The signals from the charge-couple-device (CCD) camera are stored in the net-hard disk video recorder (NVR). The network switch provides ports for additional CCD cameras.
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Data analyses were performed with SPSS 19.0 for Windows statistical package. Prior to analysis, raw data were diagnosed for normality of distribution and homogeneity of variance using Kolmogorov–Smirnov test and Levene test respectively. The tentacle insertion rate, IR and FPR were subjected to one-way analysis of variance (ANOVA). The differences between day and night of IR and FPR were compared using independent sample T-test. A two-way followed by Tukey's multiple range tests for post-hoc pairwise comparisons to test the feeding proportion and three digestive enzymes activities between different times and sea cucumber size groups. Differences were considered significant at P b 0.05, and all data were presented as means ± standard deviation.
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3. Results
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3.1. Feeding behavior
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The frame-by-frame analysis of the video footage revealed that sea cucumbers of different sizes used similar strategy in feeding. Each tentacle contained several tree-like branches, and each branch comprised more sub-branches. The number and size of the tentacle branches
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increased with sea cucumber growth (Fig. 2). The feeding process involved several continuously coordinated movements. Some of the tentacles were outstretched from the oral crown during feeding and moved towards the sediment particles. As the tentacle approached the sediment surface, each branch was stretched outwards to maximize the available surface coverage for feeding. The tentacles spread out and collected sediment with a grasping motion. Once the tentacle had grasped a certain number of particles, it would retract inward. The tentacle was then inserted into the mouth and the food particles were rubbed onto the oral crown by the oral muscle and other tentacles (Fig. 3). The same action was repeated continuously during the sea cucumber feeding period. Each individual had 20 tentacles, but only 50% of all tentacles (10) were in contact with the sediment and actively feeding at any one time. There was no specific order in which tentacles were to stretch out or draw back. It was generally the tentacles on either side of shrinking tentacles that would stretch. The total time taken for a tentacle to be placed onto the sediment, collect food, pass to the mouth, and feed again was 47.07 ± 1.51 s, 45.16 ± 1.48 s, and 42.41 ± 1.85 s for the small, medium, and large sea cucumbers, respectively. Tentacle insertion rates by the large size group were significantly faster than those by the small and medium-sized groups (P b 0.05). However, no significant difference in the feeding rates was observed between the smallsized and the medium-sized groups of sea cucumbers (P N 0.05; Table 1). The length of time for a tentacle to be in contact with the sediment surface was 11.37 s ± 0.45 s, 11.05 ± 0.10 s, and 9.30 ± 0.39 s for the small, medium, and large-sized sea cucumbers, respectively. The length of time that the tentacle was left in contact with the sediment in the large-sized group was significantly shorter than that for the
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The activities of lipase, amylase and pepsin were determined by colorimetric analysis following the instruction of commercial assay kits (Nanjing Jiancheng, Bioengineering Institute, Nanjing, China), and were expressed as U g protein−1, U mg protein−1 and U mg protein−1, respectively. Protein was determined according to Bradford using bovine serum albumin (Sigma A-2153) as a standard [23].
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Fig. 2. Fine structure of the tentacle in Apostichopus japonicus (A: small; B: medium; and C: large). The number and size of the tentacle branches increase with sea cucumber growth (Ta–Tc, T = tentacle).
Please cite this article as: Sun J, et al, Feeding behavior and digestive physiology in sea cucumber Apostichopus japonicus, Physiol Behav (2014), http://dx.doi.org/10.1016/j.physbeh.2014.11.051
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small and medium-sized groups (P b 0.05). Similarly, no significant difference in the time that a tentacle was in contact with the sediment was observed between the small and medium-sized groups (P N 0.05; Table 1).
3.2. Feeding rhythms
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The medium and large sea cucumbers responded similarly to the daily light cycle (two-way analysis of variance, P N 0.05), whereas
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Table 1 Feeding behavior observations for three sizes of sea cucumber Apostichopus japonicus as tested by video observations in an aquarium. Size
No.
Tentacle insertion (s)
t1:5 t1:6 t1:7 t1:8 t1:9 t1:10 t1:11 t1:12 t1:13 t1:14 t1:15 t1:16 t1:17 t1:18 t1:19 t1:20 t1:21 t1:22 t1:23 t1:24 t1:25
Small
1 2 3 4 5 6 Mean 1 2 3 4 5 6 Mean 1 2 3 4 5 6 Mean
46.69 48.08 49.26 45.32 47.47 45.59 47.07 44.75 44.72 42.95 45.82 47.45 47.29 45.16 43.56 40.44 39.99 43.45 44.65 42.38 42.41
Large
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3.50 4.07 3.87 2.81 3.68 3.46 1.51a 2.86 3.09 3.29 3.81 3.70 3.51 1.48a 1.96 2.37 1.95 3.88 2.27 3.08 1.85b
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Medium
± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±
Duration in contact with sediment (s)
11.19 11.67 12.00 10.84 11.57 10.97 11.37 11.13 11.06 10.88 11.03 11.15 11.22 11.05 9.13 9.03 8.86 9.71 9.85 9.19 9.30
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dissimilarities arose between the small size and the other two size classes (Fig. 4). Regardless of size, a distinct feeding pattern was observed under the 12 h light/12 h dark cycle, as indicated by feeding proportions. More sea cucumbers fed at night (20:00–08:00) than during the day (08:00–20:00). The small and medium-sized groups had a feeding peak at 02:00–04:00 and the feeding peak of large sea cucumbers was between 02:00 and 06:00. In addition, the medium and large-sized groups had a second feeding peak at 10:00–16:00 (Fig. 4). The results also revealed a significant interaction between time and size with respect to feeding proportion (Table 2).
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3.3. Ingestion and feces production rates
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3.3.1. Ingestion rate The IR of the small-sized group was significantly lower than that of the medium and large-sized groups (P b 0.05). However, no significant difference in IR was observed between the medium and large-sized sea
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Fig. 3. Feeding behavior in medium-sized Apostichopus japonicus. (A) The tentacle is extended from the oral crown (Ta). (B) The tentacle is placed onto the sediment surface (Tb). (C) The tentacle is pulled back towards the mouth (Tc). (D) The tentacle with some particles is passed upwards and inwards towards the mouth (Td).
± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±
1.64 1.57 1.26 1.34 1.56 1.34 0.45a 1.67 1.46 1.39 1.55 1.58 1.55 0.10a 1.17 1.01 1.03 1.14 1.60 1.14 0.39b
Data are means ± SD (n = 100). Different letters in the same column indicates a significant difference among different size groups of A. japonicus (P b 0.05).
Fig. 4. Diurnal feeding rhythms of three size groups of Apostichopus japonicus (n = 4). Vertical bars represent standard deviation of the means.
Please cite this article as: Sun J, et al, Feeding behavior and digestive physiology in sea cucumber Apostichopus japonicus, Physiol Behav (2014), http://dx.doi.org/10.1016/j.physbeh.2014.11.051
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J. Sun et al. / Physiology & Behavior xxx (2014) xxx–xxx Table 2 Results of the two-way ANOVA conducted to test the effect of different times and sizes on feeding proportion.
t2:4
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Feeding proportion
Time Size Time × size
542.242 531.48 45.277
11 2 22
49.147 48.168 4.104
b0.001 b0.001 b0.001
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3.4.1. Lipase activity The diurnal rhythm of lipase activity during the 24-h cycle was similar for all sizes of sea cucumbers (P N 0.05; Table 3). Lipase activity was highest at 00:00 and was relatively lower from 14:00 to 18:00 (Fig. 7A). Sea cucumbers of different sizes showed no difference in lipase activity during the diurnal cycle but significant differences were found among different times for all three sizes of sea cucumbers (Table 3).
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Fig. 6. (A) Mean feces production rate (FPR) of three size groups of Apostichopus japonicus. (B) Mean night and day feces production rates of three size groups of A. japonicus. Different letters within each treatment represent significant differences (n = 6, P b 0.05).
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3.4. Diurnal rhythms of digestive enzyme activities
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3.3.2. Feces production rate The FPR of the small-sized group was significantly lower than that of the medium and large-sized groups (P b 0.05). However, no significant difference in FPR was observed between the medium and large-sized groups (P N 0.05). The mean FPR values were 4.0 ± 0.8 mg g−1 h−1, 4.9 ± 0.4 mg g−1 h−1, and 4.8 ± 0.4 mg g−1 h−1 for the small, medium, and large-sized sea cucumbers respectively (Fig. 6A). The nocturnal FPR was significantly higher than the diurnal FPR within each size class (P b 0.05; Fig. 6B).
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cucumbers (P N 0.05). The mean IR values were 4.2 ± 0.8 mg g−1 h−1, 5.3 ± 0.4 mg g−1 h−1 and 5.3 ± 0.5 mg g−1 h−1 for the small, medium, and large-sized sea cucumbers, respectively (Fig. 5A). Significant differences were observed between day and night IR within each size class (P b 0.05; Fig. 5B). All sizes of sea cucumbers fed more actively at night, as IR was higher at night when compared with day values.
3.4.2. Amylase activity Peak amylase activity in the small and large-sized sea cucumbers occurred at 22:00; whereas, the highest amylase activity in medium-sized sea cucumbers appeared at 02:00. The lowest amylase activity for all sizes occurred at 16:00 (Fig. 7B). Both time-related and size-related differences in sea cucumber amylase activities were significantly different (P b 0.05). In addition, a significant interaction was detected between different times and sizes (Table 3).
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3.4.3. Pepsin activity The highest pepsin activity in all sizes of sea cucumbers occurred at 00:00–02:00. The lowest pepsin activity occurred at 16:00, 08:00, and 08:00 for the small, medium, and large-sized sea cucumbers, respectively (Fig. 7C). Both time-related and size-related differences in sea cucumber pepsin activities were significantly different (P b 0.05; Table 3).
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4. Discussion
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The video footage clearly showed that tentacles of different sizes had similar structure. The tentacles of the small-sized sea cucumbers were smaller than the other two with a less intricate branching pattern. The complexity of tentacle structure increased with the concomitant growth of the sea cucumbers, which was consistent with the study of Wang et al. [24]. Consequently, large-sized sea cucumbers might have a better
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Table 3 t3:1 Results of the two-way ANOVA conducted to test the effect of different times and sizes on t3:2 selected digestive enzyme activities. t3:3 Item
Source
MS
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t3:4
Lipase activity
Time Size Time × size Time Size Time × size Time Size Time × size
103.805 1.582 5.255 0.157 0.032 0.049 0.337 0.165 0.008
11 2 22 11 2 22 11 2 22
161.579 2.463 8.180 25.934 5.322 8.001 158.130 41.503 20.265
b0.001 0.092 b0.001 b0.001 0.007 b0.001 b0.001 b0.001 b0.001
t3:5 t3:6 t3:7 t3:8 t3:9 t3:10 t3:11 t3:12 t3:13
Amylase activity
Fig. 5. (A) Mean ingestion rate (IR) of three size groups of Apostichopus japonicus. (B) Mean night and day ingestion rates of three size groups of A. japonicus. Different letters within each treatment represent significant differences (n = 6, P b 0.05).
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Pepsin activity
Please cite this article as: Sun J, et al, Feeding behavior and digestive physiology in sea cucumber Apostichopus japonicus, Physiol Behav (2014), http://dx.doi.org/10.1016/j.physbeh.2014.11.051
J. Sun et al. / Physiology & Behavior xxx (2014) xxx–xxx
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Fig. 7. Diurnal variations in three digestive enzyme activities in the three size groups of Apostichopus japonicus (A: lipase; B: amylase; and C: pepsin). Vertical bars represent standard deviations of the means (n = 3).
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ability to collect food particles due to a larger capturing surface and a greater distance. The sea cucumbers picked up food particles from the sediment using the adhesive force and mechanical entrapment ability of the tentacles [8]. Sweeping and raking are the two main modes of tentacle locomotion in sea cucumbers [25]. These modes are found across a range of dendrochirota and aspidochirotid species. Video observations of the A. japonicus feeding strategy indicated that all sizes of sea cucumbers placed their tentacles on food sediments, extended them like “fingers”, and then used them to “grasp” the food particles and delivered them to the mouth. In spite of the similar tentacle structure and mechanisms of locomotion in A. japonicus of different sizes, their feeding rates vary. The time
taken for a tentacle to feed decreased as the sea cucumber body grew. The intraspecific differences between morphological structures and feeding rates may be associated with the physical conditions and energy requirements during different growth periods. Holtz and MacDonald [26] found a positive correlation between tentacle insertion rate and the amount of food ingested in the sea cucumber Cucumaria frondosa. The larger size sea cucumber needed more food to maintain normal life activities. As a result, the larger size sea cucumber would have more fine tentacles and feed more quickly. We observed the tentacle locomotion in the condition of illumination in this study; however, A. japonicus might prefer to move and feed in dark environments. Notwithstanding this limitation, we did quantify feeding behavior at the tentacle level.
Please cite this article as: Sun J, et al, Feeding behavior and digestive physiology in sea cucumber Apostichopus japonicus, Physiol Behav (2014), http://dx.doi.org/10.1016/j.physbeh.2014.11.051
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[1] Okorie OE, Ko SH, Go S, Lee S, Bae JY, Han K, et al. Preliminary study of the optimum dietary ascorbic acid level in sea cucumber, Apostichopus japonicus (Selenka). J World Aquacult Soc 2008;39:758–65. [2] Dong Y, Dong S, Ji T. Effect of different thermal regimes on growth and physiological performance of the sea cucumber Apostichopus japonicus Selenka. Aquaculture 2008; 275:329–34. [3] Li L, Li Q. Effects of stocking density, temperature, and salinity on larval survival and growth of the red race of the sea cucumber Apostichopus japonicus (Selenka). Aquacult Int 2010;18:447–60. [4] Xia S, Yang H, Li Y, Liu S, Zhou Y, Zhang L. Effects of different seaweed diets on growth, digestibility, and ammonia-nitrogen production of the sea cucumber Apostichopus japonicus (Selenka). Aquaculture 2012;338:304–8. [5] Yang H, Yuan X, Zhou Y, Mao Y, Zhang T, Liu Y. Effects of body size and water temperature on food consumption and growth in the sea cucumber Apostichopus japonicus (Selenka) with special reference to aestivation. Aquacult Res 2005;36: 1085–92. [6] Hudson IR, Wigham BD, Solan M, Rosenberg R. Feeding behaviour of deep-sea dwelling holothurians: inferences from a laboratory investigation of shallow fjordic species. J Mar Syst 2005;57:201–18. [7] Roberts D, Moore H. Tentacular diversity in deep-sea deposit-feeding holothurians: implications for biodiversity in the deep sea. Biodivers Conserv 1997;6:1487–505. [8] Cameron JL, Fankboner PV. Tentacle structure and feeding processes in life stages of the commercial sea cucumber Parastichopus californicus (Stimpson). J Exp Mar Biol Ecol 1984;81:193-209.30. [9] Reese ES. The complex behavior of echinoderms. In: Boolootian RA, editor. Physiology of Echinodermata. New York: John Wiley and Sons; 1966. p. 157–218. [10] Hammond L. Patterns of feeding and activity in deposit-feeding holothurians and echinoids (Echinodermata) from a shallow back-reef lagoon, Discovery Bay, Jamaica. Bull Mar Sci 1982;32:549–71. [11] Mercier A, Battaglene SC, Hamel JF. Daily burrowing cycle and feeding activity of juvenile sea cucumbers Holothuria scabra in response to environmental factors. J Exp Mar Biol Ecol 1999;239:125–56. [12] Lawrence J, Hughes-Games L. The diurnal rhythm of feeding and passage of food through the gut of Diadema setosum (Echinodermata: Echinoidea). Israel J Zool 1972;21:13–6. [13] Chang Y, Ding J, Song J, Yang W. Biological research and aquaculture of sea cucumber and sea urchin. Beijing, China: Ocean Press; 2004 3–89. [14] Dong G, Dong S, Wang F, Tian X. Effects of light intensity on daily activity rhythm of juvenile sea cucumber, Apostichopus japonicus (Selenka). Aquac Res 2010;4:1640–7. [15] Zhou X, Niu C, Li Q. Effects of light on feeding behavior, growth and survival of aquatic animals. Acta Hydrobiol Sin 2000;2:178–81 (in Chinese). [16] Montoya A, López-Olmeda JF, Yúfera M, Sánchez-Muros MJ, Sánchez-Vázquez FJ. Feeding time synchronises daily rhythms of behaviour and digestive physiology in gilthead seabream (Sparus aurata). Aquaculture 2010;306:315–21. [17] Vera LM, De Pedro N, Gómez-Milán E, Delgado MJ, Sánchez-Muros MJ, Madrid JA, et al. Feeding entrainment of locomotor activity rhythms, digestive enzymes and neuroendocrine factors in goldfish. Physiol Behav 2007;90:518–24. [18] del Pozo A, Montoya A, Vera LM, Sánchez-Vázquez FJ. Daily rhythms of clock gene expression, glycaemia and digestive physiology in diurnal/nocturnal European seabass. Physiol Behav 2012;106:446–50. [19] Xia S, Yang H, Li Y, Liu S, Xu Q, Rajkumar M. Effects of food processing method on digestibility and energy budget of Apostichopus japonicus. Aquaculture 2013;384: 128–33. [20] Gao F, Yang H, Xu Q, Wang F, Liu G. Effect of water temperature on digestive enzyme activity and gut mass in sea cucumber Apostichopus japonicus (Selenka), with special reference to aestivation. Chin J Oceanol Limnol 2009;27:714–22. [21] Dong Y, Dong S, Tian X, Wang F, Zhang M. Effects of diel temperature fluctuations on growth, oxygen consumption and proximate body composition in the sea cucumber Apostichopus japonicus Selenka. Aquaculture 2006;255:514–21. [22] Maxwell KH, Gardner J, Heath PL. The effect of diet on the energy budget of the brown sea cucumber, Stichopus mollis (Hutton). J World Aquacult Soc 2009;40: 157–70. [23] Bradford MM. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 1976; 72:248–54. [24] Wang J, Cong W, Jiang Y, Zhang J. Effects benthic diatom species and density on settlement and ontogenetic development of feeding organs in sea cucumber Apostichopus japonicus. J Dalian Ocean Univ 2010;4:298–307 (in Chinese, with English abstract).
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This work was supported by the Natural Science Foundation of China (41106134), the State Science and Technology Support Program (2011BAD13B02, 2011BAD13B05), the Funding of Shandong Postdoctoral Innovation, and the National Marine Public Welfare Research Project (2013418043).
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Daily activity rhythms have been reported in many echinoderm species [10,11,27,28]. The nocturnal increase in activity of Clypeaster rosaceus is clearly associated with feeding, whereas the nocturnal and activity of Meoma ventricosa was not associated with increased feeding [10]. The general feeding rhythm of the three sizes of A. japonicus under a 12 h light/12 h dark diurnal cycle coincided well with the daily activity rhythm described by Dong et al. [29], in which feeding behavior occurred more frequently at night than in the day. The rhythmic activities of some echinoderms might be affected by internal and external factors that cannot be explained easily [30]. Daily activity rhythms are a response to variations in other environmental factors, such as waves breaking and fluctuations in temperature, salinity, and food supply [6, 11,31,32]. In the present study, the experiments within the same size were conducted in a laboratory with a constant water environment, such as constant water temperature, salinity, pH, and sufficient dissolved oxygen, so the results were unlikely confounded by such physical and chemical factors except light. Therefore, the feeding rhythm of sea cucumbers within the same size group was mainly attributed to the light/dark cycle. However, the behaviors of sea cucumber may have originated from a biological clock reflecting a phylogenetic carry-out from ancestral holothurians. The reason for developing an internal clock is complex, but a major pressure for the evolution of nocturnal feeding is predation [33]. In addition, the small-sized sea cucumber had a relatively lower proportion of feeding and no second peak in feeding during the day. This difference in feeding rhythms between size classes suggested that small individuals become less sensitive to light as they grew. Body wall thickness increases with growth; thus, a large-sized sea cucumber would probably be less sensitive to light [11]. Regardless of size, A. japonicus had higher IR and FPR at night than those during the day, which was consistent with the results in feeding proportions. Light is one of the most important factors determining diurnal variations in digestive enzyme activities [34]. The relationship between the light cycle and the variations in digestive enzyme activities has been studied in many aquatic animals, such as copepods [35] and fish [16, 18,36]. In this study, the activities of selected digestive enzymes in all sizes of sea cucumbers were higher at night than those during the day, which meant that the light/dark cycle has affected digestive physiology as well as feeding rhythms. On the other hand, diets also have direct effects on digestive enzyme activities, and the diurnal rhythm of the digestive enzymes could be modified by feeding schedule. However, we could ignore the effect of diets because the animals were fed regularly once per day and diet was available at all times in this study. Lipase activity was lowest when compared with the activities of amylase and trypsin. This result could be due to the relatively low crude fat content (0.58 ± 0.01%) in the feed or because of the low demand of dietary lipid (5%) for A. japonicus [37]. Furthermore, digestive enzyme activities may be regulated by hormones; however, knowledge of hormones in sea cucumbers is still limited. Therefore, further research is needed to investigate the hormonal and molecular mechanisms of feeding behavior in A. japonicus. The digestive enzyme activities increased before the feeding peak, which was in agreement with previous investigations in fishes such as gilthead seabream Sparus aurata [16] and goldfish [17], suggesting that there was a possible preparation phase for feeding activity. The rhythm of digestive enzymes secretion could be controlled by an anticipatory mechanism when animals were expecting to be fed. This mechanism will make the organisms to optimize digestive and metabolic processes which allow them to concentrate on feeding in a short time period to lower the risks of predation in the wild [36]. From a physiological standpoint, this mode will also improve food acquisition and nutrient utilization. It can be concluded from the present study that the tentacle insertion rate in A. japonicus depended on the body size. The feeding rhythms and digestive physiology of A. japonicus were both size-related and photoperiod-related. In addition, A. japonicus has the ability to
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[32] Eckert GL. Spatial patchiness in the sea cucumber Pachythyone rubra in the California Channel Islands. J Exp Mar Biol Ecol 2007;348:121–32. [33] Nelson B, Vance R. Diel foraging patterns of the sea urchin Centrostephanus coronatus as a predator avoidance strategy. Mar Biol 1979;51:251–8. [34] Van Wormhoudt A. Activités enzymatiques digestives chez Palaemon serratus: variations annuelles de l'acrophase des rythmes circadiens. Biochem Syst Ecol 1977;5: 301–7. [35] Baars M, Oosterhuis S. Diurnal feeding rhythms in North Sea copepods measured by gut fluorescence, digestive enzyme activity and grazing on labelled food. Neth J Sea Res 1984;18:97–119. [36] Aranda A, Madrid JA, Sánchez-Vázquez FJ. Influence of light on feeding anticipatory activity in goldfish. J Biol Rhythms 2001;16:50–7. [37] Zhu W, Mai K, Zhang B, Wang F, Xu G. Study on dietary protein and lipid requirement for sea cucumber, Stichopus japonicus. Mar Sci 2005;3:54–8 (in Chinese, with English abstract).
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[25] Roberts D, Gebruk A, Levin V, Manship BAD. Feeding and digestive strategies in deposit-feeding holothurians. Oceanogr Mar Biol Annu Rev 2003;38:257–310. [26] Holtz E, MacDonald BA. Feeding behavior of the sea cucumber Cucumaria frondosa (Echinodermata: Holothuroidea) in the laboratory and the field: relationships between tentacle insertion rate, flow speed, and ingestion. Mar Biol 2009;156: 1389–98. [27] Bakus G. Defensive mechanisms and ecology of some tropical holothurians. Mar Biol 1968;2:23–32. [28] Chesher RH. Contributions to the biology of Meoma ventricosa (Echinoidea: Spatangoida). Bull Mar Sci 1969;19:72–110. [29] Dong G, Dong S, Tian X, Wang F. Effects of photoperiod on daily activity rhythm of juvenile sea cucumber, Apostichopus japonicus (Selenka). Chin J Oceanol Limnol 2011;29:1015–22. [30] Yamanouchi T. Ecological and physiological studies on the holothurians in the coral reef of Palau Islands. Palao Trop Biol Station 1939;25:603–34. [31] Barkai A. The effect of water movement on the distribution and interaction of three holothurian species on the South African west coast. J Exp Mar Biol Ecol 1991;2: 241–54.
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Contents lists available at ScienceDirect
Physiology & Behavior journal homepage: www.elsevier.com/locate/phb
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Jiamin Sun a,c, Libin Zhang a,⁎, Yang Pan a,b, Chenggang Lin a, Fang Wang c, Rentao Kan d, Hongsheng Yang a,⁎⁎
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Feeding behavior and digestive physiology in sea cucumber Apostichopus japonicus
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Keywords: Sea cucumber Apostichopus japonicus Size Tentacle Feeding rhythm Digestive enzyme
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of sea cucumber culture, such as ecological conditions, stocking density and feed nutrition have been solved to some extent [2–5]. However, relatively little is known about sea cucumber feeding behavior in an aquaculture setting. Such information could be useful for designing optimal feeding schedules to increase production and decrease food waste. Sea cucumber collects food by extending its tentacles surrounding its mouth. The type of tentacle is closely linked to the mode of feeding employed by each species [6]. For example, holothurians, Psychropotes longicauda have stubby peltate tentacles that collect sediment particles by sweeping, whereas holothurians, Oneirophanta mutabilis have branched digitate tentacles that collect sediment particles by raking [7]. The tentacle structure and feeding strategy vary not only in different species but also within same species. Cameron and Fankboner [8] observed that the pentacula larva, juvenile, and adult life stages of Parastichopus californicus had subtle differences in tentacle structure
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The feeding behavior and digestive physiology of the sea cucumber, Apostichopus japonicus are not well understood. A better understanding may provide useful information for the development of the aquaculture of this species. In this article the tentacle locomotion, feeding rhythms, ingestion rate (IR), feces production rate (FPR) and digestive enzyme activities were studied in three size groups (small, medium and large) of sea cucumber under a 12 h light/12 h dark cycle. Frame-by-frame video analysis revealed that all size groups had similar feeding strategies using a grasping motion to pick up sediment particles. The tentacle insertion rates of the large size group were significantly faster than those of the small and medium-sized groups (P b 0.05). Feeding activities investigated by charge coupled device cameras with infrared systems indicated that all size groups of sea cucumber were nocturnal and their feeding peaks occurred at 02:00–04:00. The medium and large-sized groups also had a second feeding peak during the day. Both IR and FPR in all groups were significantly higher at night than those during the daytime (P b 0.05). Additionally, the peak activities of digestive enzymes were 2–4 h earlier than the peak of feeding. Taken together, these results demonstrated that the light/dark cycle was a powerful environment factor that influenced biological rhythms of A. japonicus, which had the ability to optimize the digestive processes for a forthcoming ingestion. © 2014 Published by Elsevier Inc.
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Article history: Received 3 October 2014 Received in revised form 13 November 2014 Accepted 14 November 2014 Available online xxxx
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• The tentacle insertion rates depended on the body size. • The feeding peak of all size groups of sea cucumber occurred at 02:00–04:00. • The peak activities of digestive enzymes were 2–4 h earlier than the peak of feeding.
1. Introduction
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Sea cucumber Apostichopus japonicus is distributed mainly in northern China, eastern Russia, Japan, and Korea, which has become a dominant mariculture species because of its relatively high economic value [1]. Usually the sea cucumbers are cultured in shallow seas or ponds without supplement feeds in China. In recent years, industrial aquaculture of sea cucumbers has expanded rapidly becoming an important culturing mode, in which artificial diets are needed. Some basic issues
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Key Laboratory of Marine Ecology and Environmental Sciences, Institute of Oceanology, Chinese Academy of Sciences, 7 Nanhai Road, Qingdao 266071, Shandong, China University of Chinese Academy of Sciences, 19A Yuquan Road, Beijing 100049, China Ocean University of China, 5 Yushan Road, Qingdao 266003, Shandong, China d Shandong Blue Ocean Science and Technology Co., Ltd., Laizhou 261400, Shandong, China b
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⁎ Correspondence to: L. Zhang, Institute of Oceanology, Chinese Academy of Sciences, 7 Nanhai Road, Qingdao 266071, Shandong, China. Tel./fax: +86 532 82896096. ⁎⁎ Correspondence to: H. Yang, Institute of Oceanology, Chinese Academy of Sciences, 7 Nanhai Road, Qingdao 266071, Shandong, China. Tel./fax: +86 532 82898610. E-mail addresses: [email protected] (L. Zhang), [email protected] (H. Yang).
http://dx.doi.org/10.1016/j.physbeh.2014.11.051 0031-9384/© 2014 Published by Elsevier Inc.
Please cite this article as: Sun J, et al, Feeding behavior and digestive physiology in sea cucumber Apostichopus japonicus, Physiol Behav (2014), http://dx.doi.org/10.1016/j.physbeh.2014.11.051
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2. Materials and methods
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2.1. Animals and housing
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Sea cucumbers (A. japonicus) during juvenile period were collected from Tianheng Sea Cucumber Farm (Qingdao, China) and were acclimated in several fiberglass tanks at 16 °C for 2 weeks prior to the experiment. After being fasted for 24 h [21], the domesticated sea cucumbers were divided into three groups: small, with a body length of 5.0 ± 1.1 cm and a wet body weight of 17.80 ± 2.37 g; medium, with a body length of 8.4 ± 1.3 cm and a wet body weight of 40.00 ± 4.64 g; and large, with a body length of 13.6 ± 1.5 cm and a wet body weight of 89.21 ± 8.30 g. The ages of the small, medium and large-sized sea cucumbers were ten months, fourteen months and eighteen months respectively.
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Aerated water in a 4000 L fiberglass tank was heated to 16.5 °C with a 2000 W electric heater controlled via a thermostat and then pumped into the experimental tanks. The temperature of the experimental room was maintained at 16.0 ± 1.0 °C with air-conditioning. Water salinity was 30–32 ppt, pH was 7.8–8.2, and dissolved oxygen was N5.0 mg L− 1 and maintained by continuous aeration. The animals were maintained on a 12 h light/12 h dark cycle, with the 12 h light period beginning at 08:00. In this study, 08:00–20:00 was defined as “day” and 20:00–08:00 was defined as “night”. The light intensity used in the experiment was 50 lx and was controlled by a light emitting diode with a dimmer and measured with an illumination photometer (TES1332A, Taishi Instruments, Taiwan, China). The sea cucumbers were fed a self-made diet in cylindrical form (diameter = 1 mm) containing 40% Sargassum powder and 60% sea mud during acclimation and all experiments except the tentacle observations. The approximate composition of the diet was 3.49 ± 0.16% crude protein, 0.58 ± 0.01% crude lipid, and 78.25% ± 0.76% ash (dry diet).
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2.3.2. Experiment 2: feeding rhythms Twenty-five small, eleven medium, and five large A. japonicus (similar densities) were separately placed into three glass aquaria (60 cm × 60 cm × 50 cm). Each size group had four replicates. The animals were fed with pellet diets once per day, and half of the water was exchanged at 08:00. After 3–4 days acclimation in the experimental aquaria, feeding behavior of A. japonicus was observed with charge coupled device (CCD) cameras (Hikvision, DS-2CC11A2P-IR3, China) suspended above the aquaria. The camera could record sea cucumber behavior in complete darkness using an infrared system. The video data were stored in a video recorder, and feeding behavior could be observed at any time from the monitor (Fig. 1). Feeding behavior was judged by observing the animals' position in the aquarium and the decreasing of food on the aquarium bottom. The number of sea cucumbers that were feeding was recorded four times every 2 h for 4 consecutive days and evaluated.
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2.3.3. Experiment 3: feeding and ingestion To ensure similar rearing densities in each tank, ten small, five medium and two large gut-evacuated sea cucumbers were stocked separately in three tanks (45 cm × 35 cm × 30 cm) with six replicates. The sea cucumbers were fed to satiation twice per day at 08:00 and 20:00 for 4 days. The remaining uneaten feed and feces were siphoned before exchanging water twice per day, followed by gentle rinsing with freshwater to remove salt. The uneaten food and feces were dried separately at 60 °C to constant weight. Both ingestion rate (IR) and feces production rate (FPR) were calculated as described by Maxwell et al. [22]:
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2.3.1. Experiment 1: tentacle locomotion To record and compare the fine-scale mechanistic feeding behavior of A. japonicus of different sizes, tentacle locomotion during feeding was observed using an upward-looking digital camera (Canon IXUS 125HS) located below the glass aquarium (60 cm × 60 cm × 50 cm) as described by Hudson et al. [6]. Six individuals of each size were observed. A pinch of sediment particles, insufficient to obstruct the view of camera, were provided to encourage feeding activity. The number of tentacles involved in feeding at any one time and the rates of tentacle insertion were analyzed frame-by-frame based on 100 separate tentacle movements.
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and feeding strategy. In the present study, we firstly investigated the mechanisms of locomotion and food capture in different size groups of A. japonicus at the tentacle level through real-time photography. Diurnal activity rhythms, particularly feeding rhythm, are ubiquitous throughout Phylum Echinodermata, such as holothurians and echinoids [9–12]. In aquatic animals, photoperiod is considered as the most important synchronizer of biological rhythm. Some studies have demonstrated that A. japonicus is a nocturnal sea cucumber that hides during daytime and comes out to feed at night [13,14]. However, the specific feeding rhythm and its causes in sea cucumber remain unknown. A close relationship exists between physiological function and biological rhythms in aquatic animals [15]. The organisms have the ability to anticipate the approaching meal and then optimize the digestive and metabolic processes. Several studies have been carried out into this physiological adaption. For example, some enzymatic activities have been observed to increase in the digestive system of fishes prior to the expected intake of the daily food [16–18]. The activities of digestive enzymes in sea cucumber have been extensively studied in relation to the influence of diet composition [4], food processing method [19] and temperature [20]. Nevertheless, the digestive rhythms of sea cucumber have scarcely been investigated. In the present experiment, we studied the effect of 12 h light/12 h dark cycle on feeding behavior and digestive physiology of A. japonicus in three size groups (small, medium and large). Our objectives were to: 1) observe and quantify A. japonicus tentacle locomotion; 2) determine their feeding rhythms; and 3) reveal the relationship between the feeding behavior and the digestive physiology of A. japonicus.
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FPR (mg g−1 h−1) = (Wf / Wsc) / t
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where Wo = dried weight of the offered food (mg); Wu = dried weight 175 of the uneaten feed (mg); Wsc = wet weight of the sea cucumber in the 176 tank (g); Wf = dried weight of the feces (mg) and t = time (h). 177 2.3.4. Experiment 4: digestive physiology Sea cucumbers were maintained under the same experimental conditions as Section 2.3.3 Experiment 3: feeding and ingestion for two weeks. Subsequently, six individuals of each size group (small, medium and large) were sampled to obtain intestines every 2 h during a 24 h cycle (12 sampling points). The intestines were cut longitudinally and washed thoroughly in ice-cold 0.84% normal saline. After rinsing, all samples were frozen quickly in liquid nitrogen and then stored at −80 °C until analysis. One intestine sample of sea cucumbers was insufficient to determine three digestive enzyme activities. So we mixed two replicate samples into one sample. In order to avoid the effect of different feeding times, all animals were fed excessively at 08:00 on the sampling day. Sampling during the dark phase was performed under a dim light.
Please cite this article as: Sun J, et al, Feeding behavior and digestive physiology in sea cucumber Apostichopus japonicus, Physiol Behav (2014), http://dx.doi.org/10.1016/j.physbeh.2014.11.051
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Fig. 1. Diagram of the experimental setup to observe Apostichopus japonicus feeding behavior. The signals from the charge-couple-device (CCD) camera are stored in the net-hard disk video recorder (NVR). The network switch provides ports for additional CCD cameras.
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Data analyses were performed with SPSS 19.0 for Windows statistical package. Prior to analysis, raw data were diagnosed for normality of distribution and homogeneity of variance using Kolmogorov–Smirnov test and Levene test respectively. The tentacle insertion rate, IR and FPR were subjected to one-way analysis of variance (ANOVA). The differences between day and night of IR and FPR were compared using independent sample T-test. A two-way followed by Tukey's multiple range tests for post-hoc pairwise comparisons to test the feeding proportion and three digestive enzymes activities between different times and sea cucumber size groups. Differences were considered significant at P b 0.05, and all data were presented as means ± standard deviation.
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3.1. Feeding behavior
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The frame-by-frame analysis of the video footage revealed that sea cucumbers of different sizes used similar strategy in feeding. Each tentacle contained several tree-like branches, and each branch comprised more sub-branches. The number and size of the tentacle branches
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increased with sea cucumber growth (Fig. 2). The feeding process involved several continuously coordinated movements. Some of the tentacles were outstretched from the oral crown during feeding and moved towards the sediment particles. As the tentacle approached the sediment surface, each branch was stretched outwards to maximize the available surface coverage for feeding. The tentacles spread out and collected sediment with a grasping motion. Once the tentacle had grasped a certain number of particles, it would retract inward. The tentacle was then inserted into the mouth and the food particles were rubbed onto the oral crown by the oral muscle and other tentacles (Fig. 3). The same action was repeated continuously during the sea cucumber feeding period. Each individual had 20 tentacles, but only 50% of all tentacles (10) were in contact with the sediment and actively feeding at any one time. There was no specific order in which tentacles were to stretch out or draw back. It was generally the tentacles on either side of shrinking tentacles that would stretch. The total time taken for a tentacle to be placed onto the sediment, collect food, pass to the mouth, and feed again was 47.07 ± 1.51 s, 45.16 ± 1.48 s, and 42.41 ± 1.85 s for the small, medium, and large sea cucumbers, respectively. Tentacle insertion rates by the large size group were significantly faster than those by the small and medium-sized groups (P b 0.05). However, no significant difference in the feeding rates was observed between the smallsized and the medium-sized groups of sea cucumbers (P N 0.05; Table 1). The length of time for a tentacle to be in contact with the sediment surface was 11.37 s ± 0.45 s, 11.05 ± 0.10 s, and 9.30 ± 0.39 s for the small, medium, and large-sized sea cucumbers, respectively. The length of time that the tentacle was left in contact with the sediment in the large-sized group was significantly shorter than that for the
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The activities of lipase, amylase and pepsin were determined by colorimetric analysis following the instruction of commercial assay kits (Nanjing Jiancheng, Bioengineering Institute, Nanjing, China), and were expressed as U g protein−1, U mg protein−1 and U mg protein−1, respectively. Protein was determined according to Bradford using bovine serum albumin (Sigma A-2153) as a standard [23].
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Fig. 2. Fine structure of the tentacle in Apostichopus japonicus (A: small; B: medium; and C: large). The number and size of the tentacle branches increase with sea cucumber growth (Ta–Tc, T = tentacle).
Please cite this article as: Sun J, et al, Feeding behavior and digestive physiology in sea cucumber Apostichopus japonicus, Physiol Behav (2014), http://dx.doi.org/10.1016/j.physbeh.2014.11.051
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small and medium-sized groups (P b 0.05). Similarly, no significant difference in the time that a tentacle was in contact with the sediment was observed between the small and medium-sized groups (P N 0.05; Table 1).
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The medium and large sea cucumbers responded similarly to the daily light cycle (two-way analysis of variance, P N 0.05), whereas
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Table 1 Feeding behavior observations for three sizes of sea cucumber Apostichopus japonicus as tested by video observations in an aquarium. Size
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t1:5 t1:6 t1:7 t1:8 t1:9 t1:10 t1:11 t1:12 t1:13 t1:14 t1:15 t1:16 t1:17 t1:18 t1:19 t1:20 t1:21 t1:22 t1:23 t1:24 t1:25
Small
1 2 3 4 5 6 Mean 1 2 3 4 5 6 Mean 1 2 3 4 5 6 Mean
46.69 48.08 49.26 45.32 47.47 45.59 47.07 44.75 44.72 42.95 45.82 47.45 47.29 45.16 43.56 40.44 39.99 43.45 44.65 42.38 42.41
Large
N
3.50 4.07 3.87 2.81 3.68 3.46 1.51a 2.86 3.09 3.29 3.81 3.70 3.51 1.48a 1.96 2.37 1.95 3.88 2.27 3.08 1.85b
U
Medium
± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±
Duration in contact with sediment (s)
11.19 11.67 12.00 10.84 11.57 10.97 11.37 11.13 11.06 10.88 11.03 11.15 11.22 11.05 9.13 9.03 8.86 9.71 9.85 9.19 9.30
C
t1:4
O
R
R
E
249
t1:26 t1:27 t1:28
dissimilarities arose between the small size and the other two size classes (Fig. 4). Regardless of size, a distinct feeding pattern was observed under the 12 h light/12 h dark cycle, as indicated by feeding proportions. More sea cucumbers fed at night (20:00–08:00) than during the day (08:00–20:00). The small and medium-sized groups had a feeding peak at 02:00–04:00 and the feeding peak of large sea cucumbers was between 02:00 and 06:00. In addition, the medium and large-sized groups had a second feeding peak at 10:00–16:00 (Fig. 4). The results also revealed a significant interaction between time and size with respect to feeding proportion (Table 2).
252
3.3. Ingestion and feces production rates
262
3.3.1. Ingestion rate The IR of the small-sized group was significantly lower than that of the medium and large-sized groups (P b 0.05). However, no significant difference in IR was observed between the medium and large-sized sea
263
T
246 247
C
245
E
D
Fig. 3. Feeding behavior in medium-sized Apostichopus japonicus. (A) The tentacle is extended from the oral crown (Ta). (B) The tentacle is placed onto the sediment surface (Tb). (C) The tentacle is pulled back towards the mouth (Tc). (D) The tentacle with some particles is passed upwards and inwards towards the mouth (Td).
± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±
1.64 1.57 1.26 1.34 1.56 1.34 0.45a 1.67 1.46 1.39 1.55 1.58 1.55 0.10a 1.17 1.01 1.03 1.14 1.60 1.14 0.39b
Data are means ± SD (n = 100). Different letters in the same column indicates a significant difference among different size groups of A. japonicus (P b 0.05).
Fig. 4. Diurnal feeding rhythms of three size groups of Apostichopus japonicus (n = 4). Vertical bars represent standard deviation of the means.
Please cite this article as: Sun J, et al, Feeding behavior and digestive physiology in sea cucumber Apostichopus japonicus, Physiol Behav (2014), http://dx.doi.org/10.1016/j.physbeh.2014.11.051
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J. Sun et al. / Physiology & Behavior xxx (2014) xxx–xxx Table 2 Results of the two-way ANOVA conducted to test the effect of different times and sizes on feeding proportion.
t2:4
Item
Source
MS
df
F
P
t2:5 t2:6 t2:7
Feeding proportion
Time Size Time × size
542.242 531.48 45.277
11 2 22
49.147 48.168 4.104
b0.001 b0.001 b0.001
275 276 277 278 279 280 281
283
3.4.1. Lipase activity The diurnal rhythm of lipase activity during the 24-h cycle was similar for all sizes of sea cucumbers (P N 0.05; Table 3). Lipase activity was highest at 00:00 and was relatively lower from 14:00 to 18:00 (Fig. 7A). Sea cucumbers of different sizes showed no difference in lipase activity during the diurnal cycle but significant differences were found among different times for all three sizes of sea cucumbers (Table 3).
C
E
R R N C O U
288 289
Fig. 6. (A) Mean feces production rate (FPR) of three size groups of Apostichopus japonicus. (B) Mean night and day feces production rates of three size groups of A. japonicus. Different letters within each treatment represent significant differences (n = 6, P b 0.05).
T
3.4. Diurnal rhythms of digestive enzyme activities
286 287
F
3.3.2. Feces production rate The FPR of the small-sized group was significantly lower than that of the medium and large-sized groups (P b 0.05). However, no significant difference in FPR was observed between the medium and large-sized groups (P N 0.05). The mean FPR values were 4.0 ± 0.8 mg g−1 h−1, 4.9 ± 0.4 mg g−1 h−1, and 4.8 ± 0.4 mg g−1 h−1 for the small, medium, and large-sized sea cucumbers respectively (Fig. 6A). The nocturnal FPR was significantly higher than the diurnal FPR within each size class (P b 0.05; Fig. 6B).
282
284 285
O
273 274
R O
271 272
P
269 270
cucumbers (P N 0.05). The mean IR values were 4.2 ± 0.8 mg g−1 h−1, 5.3 ± 0.4 mg g−1 h−1 and 5.3 ± 0.5 mg g−1 h−1 for the small, medium, and large-sized sea cucumbers, respectively (Fig. 5A). Significant differences were observed between day and night IR within each size class (P b 0.05; Fig. 5B). All sizes of sea cucumbers fed more actively at night, as IR was higher at night when compared with day values.
3.4.2. Amylase activity Peak amylase activity in the small and large-sized sea cucumbers occurred at 22:00; whereas, the highest amylase activity in medium-sized sea cucumbers appeared at 02:00. The lowest amylase activity for all sizes occurred at 16:00 (Fig. 7B). Both time-related and size-related differences in sea cucumber amylase activities were significantly different (P b 0.05). In addition, a significant interaction was detected between different times and sizes (Table 3).
290
3.4.3. Pepsin activity The highest pepsin activity in all sizes of sea cucumbers occurred at 00:00–02:00. The lowest pepsin activity occurred at 16:00, 08:00, and 08:00 for the small, medium, and large-sized sea cucumbers, respectively (Fig. 7C). Both time-related and size-related differences in sea cucumber pepsin activities were significantly different (P b 0.05; Table 3).
298 299
4. Discussion
304
The video footage clearly showed that tentacles of different sizes had similar structure. The tentacles of the small-sized sea cucumbers were smaller than the other two with a less intricate branching pattern. The complexity of tentacle structure increased with the concomitant growth of the sea cucumbers, which was consistent with the study of Wang et al. [24]. Consequently, large-sized sea cucumbers might have a better
305 306
D
267 268
E
t2:1 t2:2 t2:3
5
293 294 295 296 297
300 301 302 303
307 308 309 310
Table 3 t3:1 Results of the two-way ANOVA conducted to test the effect of different times and sizes on t3:2 selected digestive enzyme activities. t3:3 Item
Source
MS
df
F
P
t3:4
Lipase activity
Time Size Time × size Time Size Time × size Time Size Time × size
103.805 1.582 5.255 0.157 0.032 0.049 0.337 0.165 0.008
11 2 22 11 2 22 11 2 22
161.579 2.463 8.180 25.934 5.322 8.001 158.130 41.503 20.265
b0.001 0.092 b0.001 b0.001 0.007 b0.001 b0.001 b0.001 b0.001
t3:5 t3:6 t3:7 t3:8 t3:9 t3:10 t3:11 t3:12 t3:13
Amylase activity
Fig. 5. (A) Mean ingestion rate (IR) of three size groups of Apostichopus japonicus. (B) Mean night and day ingestion rates of three size groups of A. japonicus. Different letters within each treatment represent significant differences (n = 6, P b 0.05).
291 292
Pepsin activity
Please cite this article as: Sun J, et al, Feeding behavior and digestive physiology in sea cucumber Apostichopus japonicus, Physiol Behav (2014), http://dx.doi.org/10.1016/j.physbeh.2014.11.051
J. Sun et al. / Physiology & Behavior xxx (2014) xxx–xxx
N
C
O
R
R
E
C
T
E
D
P
R O
O
F
6
U
Fig. 7. Diurnal variations in three digestive enzyme activities in the three size groups of Apostichopus japonicus (A: lipase; B: amylase; and C: pepsin). Vertical bars represent standard deviations of the means (n = 3).
311 312 313 314 315 316 317 318 319 320 321 322
ability to collect food particles due to a larger capturing surface and a greater distance. The sea cucumbers picked up food particles from the sediment using the adhesive force and mechanical entrapment ability of the tentacles [8]. Sweeping and raking are the two main modes of tentacle locomotion in sea cucumbers [25]. These modes are found across a range of dendrochirota and aspidochirotid species. Video observations of the A. japonicus feeding strategy indicated that all sizes of sea cucumbers placed their tentacles on food sediments, extended them like “fingers”, and then used them to “grasp” the food particles and delivered them to the mouth. In spite of the similar tentacle structure and mechanisms of locomotion in A. japonicus of different sizes, their feeding rates vary. The time
taken for a tentacle to feed decreased as the sea cucumber body grew. The intraspecific differences between morphological structures and feeding rates may be associated with the physical conditions and energy requirements during different growth periods. Holtz and MacDonald [26] found a positive correlation between tentacle insertion rate and the amount of food ingested in the sea cucumber Cucumaria frondosa. The larger size sea cucumber needed more food to maintain normal life activities. As a result, the larger size sea cucumber would have more fine tentacles and feed more quickly. We observed the tentacle locomotion in the condition of illumination in this study; however, A. japonicus might prefer to move and feed in dark environments. Notwithstanding this limitation, we did quantify feeding behavior at the tentacle level.
Please cite this article as: Sun J, et al, Feeding behavior and digestive physiology in sea cucumber Apostichopus japonicus, Physiol Behav (2014), http://dx.doi.org/10.1016/j.physbeh.2014.11.051
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This work was supported by the Natural Science Foundation of China (41106134), the State Science and Technology Support Program (2011BAD13B02, 2011BAD13B05), the Funding of Shandong Postdoctoral Innovation, and the National Marine Public Welfare Research Project (2013418043).
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anticipate the feeding activity and optimize digestive physiology to 401 make feeding more efficient. 402
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Daily activity rhythms have been reported in many echinoderm species [10,11,27,28]. The nocturnal increase in activity of Clypeaster rosaceus is clearly associated with feeding, whereas the nocturnal and activity of Meoma ventricosa was not associated with increased feeding [10]. The general feeding rhythm of the three sizes of A. japonicus under a 12 h light/12 h dark diurnal cycle coincided well with the daily activity rhythm described by Dong et al. [29], in which feeding behavior occurred more frequently at night than in the day. The rhythmic activities of some echinoderms might be affected by internal and external factors that cannot be explained easily [30]. Daily activity rhythms are a response to variations in other environmental factors, such as waves breaking and fluctuations in temperature, salinity, and food supply [6, 11,31,32]. In the present study, the experiments within the same size were conducted in a laboratory with a constant water environment, such as constant water temperature, salinity, pH, and sufficient dissolved oxygen, so the results were unlikely confounded by such physical and chemical factors except light. Therefore, the feeding rhythm of sea cucumbers within the same size group was mainly attributed to the light/dark cycle. However, the behaviors of sea cucumber may have originated from a biological clock reflecting a phylogenetic carry-out from ancestral holothurians. The reason for developing an internal clock is complex, but a major pressure for the evolution of nocturnal feeding is predation [33]. In addition, the small-sized sea cucumber had a relatively lower proportion of feeding and no second peak in feeding during the day. This difference in feeding rhythms between size classes suggested that small individuals become less sensitive to light as they grew. Body wall thickness increases with growth; thus, a large-sized sea cucumber would probably be less sensitive to light [11]. Regardless of size, A. japonicus had higher IR and FPR at night than those during the day, which was consistent with the results in feeding proportions. Light is one of the most important factors determining diurnal variations in digestive enzyme activities [34]. The relationship between the light cycle and the variations in digestive enzyme activities has been studied in many aquatic animals, such as copepods [35] and fish [16, 18,36]. In this study, the activities of selected digestive enzymes in all sizes of sea cucumbers were higher at night than those during the day, which meant that the light/dark cycle has affected digestive physiology as well as feeding rhythms. On the other hand, diets also have direct effects on digestive enzyme activities, and the diurnal rhythm of the digestive enzymes could be modified by feeding schedule. However, we could ignore the effect of diets because the animals were fed regularly once per day and diet was available at all times in this study. Lipase activity was lowest when compared with the activities of amylase and trypsin. This result could be due to the relatively low crude fat content (0.58 ± 0.01%) in the feed or because of the low demand of dietary lipid (5%) for A. japonicus [37]. Furthermore, digestive enzyme activities may be regulated by hormones; however, knowledge of hormones in sea cucumbers is still limited. Therefore, further research is needed to investigate the hormonal and molecular mechanisms of feeding behavior in A. japonicus. The digestive enzyme activities increased before the feeding peak, which was in agreement with previous investigations in fishes such as gilthead seabream Sparus aurata [16] and goldfish [17], suggesting that there was a possible preparation phase for feeding activity. The rhythm of digestive enzymes secretion could be controlled by an anticipatory mechanism when animals were expecting to be fed. This mechanism will make the organisms to optimize digestive and metabolic processes which allow them to concentrate on feeding in a short time period to lower the risks of predation in the wild [36]. From a physiological standpoint, this mode will also improve food acquisition and nutrient utilization. It can be concluded from the present study that the tentacle insertion rate in A. japonicus depended on the body size. The feeding rhythms and digestive physiology of A. japonicus were both size-related and photoperiod-related. In addition, A. japonicus has the ability to
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Please cite this article as: Sun J, et al, Feeding behavior and digestive physiology in sea cucumber Apostichopus japonicus, Physiol Behav (2014), http://dx.doi.org/10.1016/j.physbeh.2014.11.051
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[25] Roberts D, Gebruk A, Levin V, Manship BAD. Feeding and digestive strategies in deposit-feeding holothurians. Oceanogr Mar Biol Annu Rev 2003;38:257–310. [26] Holtz E, MacDonald BA. Feeding behavior of the sea cucumber Cucumaria frondosa (Echinodermata: Holothuroidea) in the laboratory and the field: relationships between tentacle insertion rate, flow speed, and ingestion. Mar Biol 2009;156: 1389–98. [27] Bakus G. Defensive mechanisms and ecology of some tropical holothurians. Mar Biol 1968;2:23–32. [28] Chesher RH. Contributions to the biology of Meoma ventricosa (Echinoidea: Spatangoida). Bull Mar Sci 1969;19:72–110. [29] Dong G, Dong S, Tian X, Wang F. Effects of photoperiod on daily activity rhythm of juvenile sea cucumber, Apostichopus japonicus (Selenka). Chin J Oceanol Limnol 2011;29:1015–22. [30] Yamanouchi T. Ecological and physiological studies on the holothurians in the coral reef of Palau Islands. Palao Trop Biol Station 1939;25:603–34. [31] Barkai A. The effect of water movement on the distribution and interaction of three holothurian species on the South African west coast. J Exp Mar Biol Ecol 1991;2: 241–54.
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Please cite this article as: Sun J, et al, Feeding behavior and digestive physiology in sea cucumber Apostichopus japonicus, Physiol Behav (2014), http://dx.doi.org/10.1016/j.physbeh.2014.11.051
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