© 2018. Published by The Company of Biologists Ltd | Journal of Experimental Biology (2018) 221, jeb177451. doi:10.1242/jeb.177451 RESEARCH ARTICLE Effect of water temperature on diel feeding, locomotion behaviour and digestive physiology in the sea cucumber Apostichopus japonicus Jiamin Sun1, Libin Zhang1,2,*, Yang Pan1,3, Chenggang Lin1, Fang Wang4 and Hongsheng Yang1,2 ABSTRACT important commercial aquaculture species. The A. japonicus This study used controlled laboratory conditions to directly assess the industry has now overtaken the traditional shrimp and fish role of water temperature in controlling diel feeding and locomotion aquaculture industries, and A. japonicus generates the highest behaviours, and digestive physiology, in the sea cucumber single-species output value and profit in China (Yang et al., 2015). Apostichopus japonicus. The results revealed that both the It is distributed mainly along the coasts of Northeastern Asia, proportion of feeding individuals and ingestion rate were highest at including eastern Russia, northern China, Japan, North Korea and 16°C. Regardless of water temperature, sea cucumbers appeared to South Korea (Hamel and Mercier, 2008; Okorie et al., 2008). be nocturnal and their peak feeding activity occurred at 00:00 h to Usually, A. japonicus is cultured in shallow seas (sea ranching) or 04:00 h. Tentacle insertion rate was not significantly correlated ponds (cofferdam). In recent years, intensive indoor culture has with water temperature (<24°C). In all temperature treatments expanded rapidly, becoming an important rearing method in China except 24°C, the proportion of moving sea cucumbers was also (Yang et al., 2015). Understanding the behavioural and physiological observed to be higher at night than during the day. The water features of A. japonicus under different environmental conditions is temperature above thermal threshold (24°C) for aestivation may alter beneficial not just to understand the natural behaviour of the species, the diel locomotion rhythm. The highest lipase and amylase activities but also to improve the rearing techniques for sea cucumbers. were both observed at 20°C. The highest activities of lipase and Water temperature represents the most pervasive aspect of the amylase at all temperature treatments were observed at 22:00 h to environment affecting marine ectotherms, varies markedly on a 02:00 h, which was slightly earlier than the feeding peak. In variety of spatial and temporal scales and is one of the most conclusion, even in total darkness, A. japonicus showed more important environmental factors affecting growth and physiological active feeding and moving activities, and higher digestive enzyme performance in aquatic animals (Buentello et al., 2000). Previous activities, at night than during the day. These results demonstrated studies have demonstrated the effects of water temperature on that diel feeding and locomotion behaviours, at least in the short term, growth (An et al., 2007; Dong and Dong, 2006; Dong et al., 2006; were not controlled by light or low water temperature (<24°C) but by Yang et al., 2005), immune response (Wang et al., 2008) and body an endogenous rhythm, and A. japonicus had the ability to optimize composition (Dong et al., 2006) of A. japonicus. However, our the digestive function for the coming feeding peak. These findings understanding of the behavioural responses of A. japonicus to should provide valuable information for the development of the temperature variation lags well behind our understanding of aquaculture of this species. physiological responses. The diel cycles of sea cucumbers vary among different species, KEY WORDS: Apostichopus japonicus, Water temperature, Feeding, with maximal activity being diurnal, nocturnal or crepuscular Locomotion, Digestive enzyme (Wheeling et al., 2007). However, the activity may be scheduled to coincide with regular changes in many environmental factors. The INTRODUCTION effect of light on rhythms and diel cycles in sea cucumbers has been Trade in sea cucumbers dates back several centuries owing to their well described (Chen et al., 2007; Dong et al., 2011; Dong et al., health benefits and medicinal properties. In response to a dwindling 2010; Mercier et al., 1999), but the effect of water temperature on supply from wild stocks and increasing demands causing a surge in their behavioural rhythms has received less attention. Temperature- market prices, commercial aquaculture of sea cucumbers has begun induced changes in diel activity have been observed mainly in fish recently in many countries (Purcell et al., 2014; Toral-Granda et al., (Brown and Mackay, 1995; Fraser et al., 1993; Hurst and Duffy, 2008). Apostichopus japonicus (Selenka 1867) is one of the most 2005; Reebs, 2002). To our knowledge, relevant studies on sea cucumbers have been conducted mainly in the tropical species Holothuria scabra (Mercier et al., 2000; Purcell, 2010; 1Key Laboratory of Marine Ecology and Environmental Sciences, Institute of Wolkenhauer, 2008). Oceanology, Chinese Academy of Sciences, 7 Nanhai Road, Qingdao 266071, Shandong, China. 2Laboratory for Marine Ecology and Environmental Science, Another potential technique to study diurnal variations in feeding Qingdao National Laboratory for Marine Science and Technology, 1 Wenhai Road, is based on the measurement of digestive enzyme activities (Mata- Qingdao 266237, Shandong, China. 3Chinese Academy of Fishery Sciences, Sotres et al., 2016). Previous studies have indicated that the diel 150 Qingtacun Road, Beijing 100039, China. 4Ocean University of China, Fisheries College, 5 Yushan Road, Qingdao 266003, Shandong, China. digestive physiology of fish, such as sea bass (Tillner et al., 2014; del Pozo et al., 2012), sea bream (Zeytin et al., 2016; Montoya et al., *Author for correspondence ([email protected]) 2010) and goldfish (Vera et al., 2007), was mainly entrained by the L.Z., 0000-0002-4611-1852 light–dark cycle, food types and feeding times. Water temperature was one of the important factors affecting the digestive physiology Received 15 January 2018; Accepted 21 March 2018 of sea cucumbers (Gao et al., 2009). The diel digestive rhythms of A. Journal of Experimental Biology 1 RESEARCH ARTICLE Journal of Experimental Biology (2018) 221, jeb177451. doi:10.1242/jeb.177451 japonicus at different temperatures remain unclear, and it is crucial once per day, and half of the water was exchanged at 08:00 h. The to determine the optimum feeding time and frequency in water depth was maintained at 30 cm during the experiment. aquaculture practice. Charge-coupled device (CCD) cameras (Hikvision, DS- This study was carried out under controlled laboratory conditions 2CC11A2P-IR3, China) with the infrared systems were mounted to ensure that the effects of temperature were separate from that of 1.5 m above the experimental tank and connected to a video other environmental factors. In this study, we describe the recorder to record the feeding and locomotion behaviours of sea behavioural patterns and determine the digestive physiology of cucumbers. the sea cucumber A. japonicus under different water temperatures Feeding behaviour was determined by observing the individuals’ based on laboratory experiments. In particular, we examined diel position in the aquarium and the decrease of food on the aquarium feeding activity patterns and tentacle insertion rate, as well as the bottom. The proportion of feeding sea cucumbers was determined change of digestive enzyme activities, across the range of every 30 min based on the videos. Four consecutive feeding values temperatures likely encountered throughout the year by A. from 30 min intervals were used to generate a single mean value japonicus. In addition, diurnal and nocturnal ingestion rate (IR) every 2 h. Locomotion behaviour observations were based on and locomotion behaviour of A. japonicus were compared. analyses of the final 30 min of the video recordings made during each 2 h segment of the experiment. A grid dividing the tank into MATERIALS AND METHODS four quadrate sections was overlaid on the monitor during playback, Collection and maintenance and the number of line crossings was used as an index of horizontal Sea cucumbers were collected from Tianheng Sea Cucumber Farm, distance travelled along the bottom of the tank. A sea cucumber was Shandong Province, China, and transported to the laboratory considered actively moving if it made a horizontal movement of (Qingdao National Ocean Science Research Center). All individuals more than three body lengths during the 30 min. The mean were acclimated in six large tanks (1000 l−1)at6–14°C, 30–32 ppt proportion of moving sea cucumbers from 08:00 h to 20:00 h was salinity, a pH of 7.8–8.2, >5.5 mg l−1 dissolved oxygen and at a 14 h– used as the day value, and the mean from 20:00 h to 08:00 h was 10 h light–dark photoperiod. During the acclimation period, the sea used as the night value. Four consecutive days’ values of feeding cucumbers were fed once at 08:00 h using a homemade diet and locomotion behaviours were used to generate a single mean containing 40% Sargassum powder and 60% sea mud processed into value. a cylindrical form. Healthy undamaged sea cucumbers (body length: 5.2±0.6 cm, wet body weight: 22.7±1.2 g; means±s.d.) were selected Tentacle locomotion observation and used in the series of experiments. Tentacle locomotion during feeding was recorded using an upward- looking digital camera (Canon IXUS 125 HS) located below the Experimental design glass aquarium (60×60×50 cm) according to the method of Hudson Experimental conditions et al. (2005). A lamp with dim light beneath the tank allowed Five temperatures (8, 12, 16, 20 and 24°C) were selected to observations to be made in darkness. Four individuals of each approximately represent the overall temperature range (0–30°C) temperature treatment were observed and the rates of tentacle found in the natural habitat of A. japonicus (Dong and Dong, 2006). insertion were determined from 100 consecutive tentacle The experiments at different water temperatures were conducted movements.