Sea cucumbers seek refuge to crevices in adverse environments. However, the mechanism by which crevices mitigate the adverse impacts of stress on sea cucumbers remains totally unknown. The present study investigated the detrimental effects of mechanical perturbation on behavior and physiology of sea cucumbers (Apostichopus japonicus). We subjected sea cucumbers to mechanical perturbation by placing them in a sieve submerged in water and stirring the sieve parallel to the water surface to simulate common mechanical perturbation in seed production. We found that mechanical perturbation significantly reduced cortisol, glucose, crawling frequency, and feeding duration of A. japonicus. These results suggest that mechanical perturbation decreases cortisol and glucose levels, potentially impacting feeding and crawling behaviors of A. japonicus. This study further investigated how crevices mitigated the negative effects of mechanical perturbation. There was a significant increase in the residence time of A. japonicus within crevices, while A. japonicus without crevices exhibited significantly increased movement distances after mechanical perturbation. These results indicate a preference among A. japonicus for inhabiting crevices or actively seeking such habitats following mechanical perturbation. Following mechanical perturbation, A. japonicus exhibited a significantly decrease in feeding time and crawling frequency compared to those that had been inhabiting crevices for 7 h. Sea cucumbers in the crevice showed a significantly higher crawling frequency after being exposed to 7 h of mechanical perturbation, compared to the individuals not in the crevice. These results indicate that crevices serve as an efficacious mechanism for ameliorating the negative impacts of mechanical perturbation on sea cucumber behaviors. Further, the glucose level increased significantly with the increase of time in the crevice in sea cucumbers subjected to mechanical perturbation. In summary, crevices attenuate the deleterious impacts of mechanical perturbation on sea cucumbers by enhancing glucose level, crawling, and feeding behaviors, thereby probably promoting the production efficiency in aquaculture of A. japonicus. The study elucidates the mechanism by which crevices alleviate stress in A. japonicus, providing valuable insights into the seed production.

Crevices serve as a natural congregation site for sea cucumbers. Short-term exposure to the crevices enhances feeding and defecation behaviors of sea cucumbers. However, the long-term effects on digestive ability and growth remain entirely unknown. In this study, we investigated the effects of crevices on the growth and digestive ability of juvenile sea cucumber over a 60-day cultivation period. Crevices significantly increased the muscle layer thickness, mucosal layer thickness, fold length, and fold width in the intestines of sea cucumbers. These findings suggest that crevices improved the intestinal micromorphology of sea cucumbers. Sea cucumbers reared within crevices exhibited significantly elevated pepsin activity compared to those cultured without crevices. Crevices probably play a beneficial role in food digestion by improving intestinal micromorphology and pepsin activity in sea cucumbers. Furthermore, this study found a significant increase in relative weight gain rate (WGR) and relative body wall rate (BWR) among sea cucumbers inhabiting crevices, in contrast to those devoid of such habitats. In summary, the present study reveals that crevice is an effective approach to enhancing the digestive ability and growth of juvenile sea cucumbers.

Seawater temperature change is an important concern for seed production and pond culture of sea cucumbers. The present study found that tentacle activity frequency was significantly lower in sea cucumbers exposed to continuous and rapid temperature increases than that of those at ambient temperature. Feeding behavior directly determines food intake, and further affects physiology and growth efficiency of sea cucumbers. This means that the decline in feeding caused by continuous and rapid temperature increases needs to be addressed in sea cucumber aquaculture. However, a sudden temperature change of 5 degrees C had no significant effect on behaviors of sea cucumbers. This indicates that continuous temperature increases, rather than a sudden increase, result in behavioral impacts on sea cucumbers. Therefore, we recommend aqua-farmers reduce the feeding amount for sea cucumbers during continuous and rapid temperature increases. In the present study, feeding behavior was significantly higher in sea cucumbers fed with 3% dietary tryptophan than that of those fed with 0% and 5% dietary tryptophan. This indicates that 3% dietary tryptophan increases the food intake of sea cucumbers, and mitigates the feeding decline caused by continuous and rapid temperature increase. This indicates that tryptophan has the potential to promote the feeding of sea cucumbers in seed production and pond culture. Adhesion capacity of sea cucumbers fed with 5% dietary tryptophan was significantly higher than that of individuals fed with 0% and 3% dietary tryptophan. This suggests that dietary tryptophan increases the feeding of sea cucumbers exposed to continuous and rapid temperature increases in pond culture and seed production. In addition, this study found that sea cucumbers fed with 3% dietary tryptophan had higher intestinal colony richness under the continuously rapid temperature change. The present study provides an effective method to improve adhesion behavior and to alleviate the impacts on feeding behavior for seed production and pond culture of sea cucumbers exposed to continuous and rapid temperature increases.

The purpose of this study was to investigate the effects of allicin on behavior, intestinal health, and behavioral resistance to handling stress in sea cucumbers in summer. Behavioral analysis found that 3

The selection of sea cucumbers characterized by resilience to temperature changes, handling stress, and disease is the key to enhancing the efficiency of Apostichopus japonicus production. Color morphs were devised to distinguish the resilience of A. japonicus. The objective of the present study was to evaluate the behavioral and physiological differences of seeds of A. japonicus in different colored under the condition of acute drop of temperature, seawater washing, and Vibrio infection. The dark-colored individuals showed significantly higher food consumption, fecal output, and glycogen phosphorylase activity under the condition of acute temperature drop. This connotates that dark-colored seeds demonstrate a swifter resumption of feeding activities and exhibit superior adaptability in rapidly cooling environments. Significantly longer foraging distance and foraging time, as well as faster foraging speed, and higher cortisol concentration were found in dark-colored individuals after seawater washing. This indicates that dark-colored individuals exhibit a swift responsiveness to handling stress, enabling prompt resumption of foraging activities. Dark-colored seeds have more survived number and higher catalase activity after Vibrio infection. This indicates that dark-colored individuals manifest a heightened resistance to Vibrio infection. In conclusion, dark-colored seeds of A. japonicus have a stronger ability to resist temperature changes, handling stress, and disease.

Selecting high-quality seeds with long-term advantages in behavior, intestinal health, and growth are the key to improve production efficiency of sea cucumber aquaculture. It is proposed to distinguish the seed quality of sea cucumbers by color morphs. In the present study, we carried out a 6-week experiment to investigate behavior, intestinal health, and growth of small sea cucumbers Apostichopus japonicus in different color morphs. We found that dark-colored seeds of sea cucumber were significantly more adhesive than those with light-colored seeds. This indicates that the dark-colored seeds of A. japonicus are more adaptive in complex environments in stock enhancement. Food consumption and defecation outputs of dark-colored seeds were significantly higher than those of light-colored seeds. In addition, the feces of dark-colored seeds of sea cucumber had significantly lower crude protein content and better intestinal morphology, but there was no advantage in digestive enzyme activities. This suggests that there are potential digestive benefits in dark-colored seeds. Further, dark-colored seeds of A. japonicus showed significantly better intestinal microbiota composition and faster growth rate than that of light-colored seeds. In conclusion, the present results prove that dark-colored seeds of sea cucumber have long-term advantages in behavior, intestinal health and growth. Overall, this study provides important information for the early selection of seeds and the consequent production efficiency in sea cucumber aquaculture.

Regulating movement direction is essential in the locomotion of animals. Sea cucumbers, as eyeless animals, do not have eyes for the perception of the surrounding environment and food. They have a unique way of swinging their bodies when a food cue is detected, although they lack an important perceptual tool. The present study investigated the movement patterns of the sea cucumber Apostichopus japonicus in the absence of a food cue (experiment 1) and in the presence of a food cue (experiment 2). In experiment 1, we found that the movement of sea cucumbers was close to a linear motion (motion linearity 0.91 ± 0.01). In experiment 2, sea cucumbers most frequently adjusted the movement direction when being exposed to food (84 times/216 min), indicating that sea cucumbers adjusted the direction of movement in the swing state but not the motion state. In experiment 2, we found significantly lower time in the immobility state in the sea cucumbers in the presence of food cues compared to that of those without being exposed to food cues, and the frequency of the motion state in response to food cues was 1.6 times than that of those without food cue. This suggests that food cues cause the change in motion state in sea cucumbers. Swing frequency was 1.7 times higher in sea cucumbers exposed to food cues than that of those not exposed to food cues. Further, sea cucumbers in the presence of food showed significantly better performances in swing angle and swing velocity compared to those not exposed to food cues. This suggests that food cue significantly affects the swing state of sea cucumbers. Notably, the present study described the movement patterns of sea cucumbers when they detected food cues, and other factors (such as the detection of predators) need to be further studied. The present study provides new insights into the regulation of movement direction in eyeless organisms.

Mass mortality caused by skin ulceration syndrome (SUS) is the bottle-neck for the sustainable aquaculture of the sea cucumber Apostichopus japonicus. In the present study, probiotic Bacillus licheniformis (0.25 × 109 CFU/g) was used as the treatment for A. japonicus infected with the SUS that caused by Vibrio harveyi. We found that B. licheniformis significantly reduced the number of infected sea cucumbers 5 days and 7 days after the treatment (group B), compared to those without B. licheniformis treatment (group C) (P < 0.001; P < 0.001). Further, the sea cucumbers fed B. licheniformis had significantly lower mortality at the end of the experiment (<10%) than that of those without the B. licheniformis treatment (>60%) (P < 0.001). These results suggest that the treatment of B. licheniformis is an effective method to reduce the mass mortality resulted from SUS in sea cucumber aquaculture. Further, 3–5 days of treatment significantly improved the adverse symptoms of SUS on the physiology and behavior of sea cucumbers, including the righting behavior, adhesion behavior, food consumption, fecal output and mobility. This indicates B. licheniformis treatment has the advantage in the recovery of sea cucumbers after SUS. Moreover, there was no significant difference observed in the physiology and behavior of sea cucumbers between the SUS infected sea cucumbers after the 7-day treatment of B. licheniformis and the healthy individuals. SUS infected sea cucumbers effectively returned to a stage of normalcy. Further, we found a significantly lower infected rate in sea cucumbers exposed to the culture water of group B (∼5%) than that of those in exposure to the culture water of group C (∼60%). This indicates that the treatment of B. licheniformis efficiently controls the residual pathogenicity of SUS in culture water. The present study demonstrated the effectiveness of B. licheniformis treatment as an environmentally friendly approach to reducing mortality, improving symptoms, and controlling residual pathogenicity in sea cucumber aquaculture.

Mass mortality and low growth highly decrease the production efficiency and sustainable aquaculture development of the sea cucumber Apostichopus japonicus in summer. Sea urchin feces was proposed to address the summer problems. A laboratory study was conducted for ~ 5 weeks to investigate survival, food consumption, growth and resistance ability of A. japonicus cultured with the feces of sea urchins fed kelp (KF feces, group KF), the feces of sea urchins fed prepared feed (FF feces, group FF), and the prepared sea cucumber feed (group S) at high temperature (25 °C). The sea cucumbers of group KF had better survival (100%) than those of the group FF (~ 84%), higher CTmax (35.9 °C) than those of the group S (34.5 °C), and the lowest skin ulceration proportion (0%) when they were exposed to an infectious solution among the three groups. These results suggest that the feces of sea urchins fed kelp is a promising diet for improving the survival and enhancing the resistance in A. japonicus aquaculture in summer. Sea cucumbers fed significantly less FF feces after 24 h of ageing than the fresh FF feces, suggesting this kind of feces became unsuitable for A. japonicus in a short time (within 48 h). However, the 24 h of ageing at 25 °C for the high fiber feces of sea urchins fed kelp had no significant effects on the fecal consumption of sea cucumbers. In the present study, both fecal diets provide better individual growth to sea cucumbers than the prepared feed. Yet, the feces of sea urchins fed kelp provided the highest weight gain rate (WGR) to sea cucumbers. Therefore, the feces of sea urchins fed kelp is a promising food to reduce the mortality, to address the problems of summer, and to achieve higher efficiency in A. japonicus aquaculture in summer.

Low efficiency is one of the most serious problems of sea cucumber (Apostichopus japonicus) seed production in winter. Probiotics as dietary supplements have the potential to address this problem. The aim of this study was to investigate the effects of the application of dietary Bacillus amyloliquefaciens and B. methylotrophicus on foraging behaviors, food consumption, digestive enzyme activity, and defecation outputs of small A. japonicus that were cultured in sea water at 5 degrees C for 12 days. We found that sea cucumbers supplemented with Bacillus showed significantly longer foraging distance (N = 6, P < 0.001), higher foraging success rate (N = 6, P = 0.016), shorter foraging time (N = 6, P = 0.006), and faster foraging speed (N = 6, P < 0.001), compared to the control group. In addition, food consumption (N = 9, P < 0.001), amylase (N = 3, P = 0.039) and protease enzyme (N = 3, P = 0.025) activities, and defecation outputs (N = 9, P = 0.04) of sea cucumbers fed with a diet supplemented with Bacillus were significantly higher than those of the control group. These results indicate that dietary Bacillus has great potential to increase the aquaculture productivity of sea cucumbers in winter. It is advisable to be an environmentally friendly solution to resolve the overwintering problems in A. japonicus aquaculture. The present study further enriches our understanding of the potential role of Bacillus and provides valuable information for its application at low temperature and new insights into the overwintering management for A. japonicus aquaculture.

Sea cucumbers naturally prefer to inhabit crevices. Although this biological characteristic is widely used in sea cucumber aquaculture, the benefits of crevices remain largely unknown. The present study found that feeding and defecation rates significantly increased in sea cucumbers in the crevices for 24 h. After leaving the crevice, tentacle activity time of sea cucumbers in the experimental group was significantly more than that of those in the control group. Further, the present study found that sea cucumbers cultured in crevices for 21 days showed significantly longer adhesion time and tentacles activity time after air exposure and mechanical perturbation. Righting time of sea cucumbers cultured in crevices was significantly less than that in the control group after air exposure. In conclusion, the present study clarifies that crevice is beneficial to fitness-related behaviors of small A. japonicus. We consequently encourage aqua-farmers to increase the number of crevices in both pond culture and seed production, which is conducive to improve the feeding, defecation, adhesion, and righting behaviors of sea cucumbers.

Seed selection is an essential process in the production of the sea cucumber, Apostichopus japonicus, as it is important to assure sufficient survival and growth, as well as production efficiency in subsequent aquaculture operations. It was considered that using morph color as an index for selecting high-quality seed would be valuable in this regard. The purpose of the present study was to evaluate the differences in body size, foraging behavior, melatonin levels, and intestinal flora structure of seeds of A. japonicus in dark and light color morphs. Here, we found that dark-colored seeds of sea cucumbers showed significantly longer foraging distances both in the day and at night, compared to the light-colored seeds. This indicates that dark-colored seeds of A. japonicus have a significantly greater locomotor ability. Melatonin levels of dark-colored seeds of sea cucumber were significantly higher than those of light-colored seeds, suggesting that dark-colored A. japonicus has greater antioxidant ability. In addition, in the composition of intestinal microbiota, the higher Firmicutes to Bacteroidetes ratio (F/B ratio) and a higher abundance of the phylum Planctomycetes were detected in dark-colored seeds of sea cucumber. Further, dark-colored seeds of sea cucumber had greater body weight/body length and body wall weight/body length, indicating that dark-colored seeds have a better growth potential. On the basis of the obtained results, it is suggested that aqua-culturists select dark-colored seeds in order to improve production efficiency.

The present study investigated the behavioral and physiological responses to the circadian rhythm in the sea cucumber Apostichopus japonicus. We found that righting behavior of sea cucumbers was significantly faster at night than that in daytime (P < 0.001). We thus suggest aqua-farmers carry out seeding at night in stock enhancement. The number of tentacle swings was significantly higher at night than that in daytime (P = 0.005). Thus, we suggest aqua-farmers provide diets before the peak of sea cucumber feeding at night. There was no significant difference in foraging behavior and defecation behavior during the day and at night. This indicates that not all behaviors have different characteristics in circadian rhythm. In addition, we found that cortisol concentration was significantly higher at night than that in daytime (P = 0.021). This suggests that sea cucumbers are probably more prone to be stressed at night. However, there was no significant difference in 5-HT and melatonin during the day and at night, suggesting that 5-HT and melatonin are probably not affected by circadian rhythm. The present study clarifies the behavioral and physiological responses to circadian rhythm and provides valuable information into sea cucumber aquaculture.

Developing a cost-effective approach to effectively removing problematic (dead, diseased and asymptomatic) individuals is important to decrease the disease transmission in seed production and longline culture of the sea urchin Strongylocentrotus intermedius. A simple facility, which was divided into areas M (the upper 70 mm part) and W (the lower 70 mm part), was proposed to separate the problematic and healthy sea urchins based on the differences in their behavioral performances. The V-shaped plastic corridor was inside these two unseparated areas and connected by an upper platform. In experiment 1, fresh kelp was placed in the platform of area M, twenty-five healthy small sea urchins (similar to 10 mm of test diameter) and another twenty-five problematic individuals (previously exposed to the bacteria liquid) were put on the area W of the facility. The present study found that the average proportion of sea urchins distributed in areas M and W was 0.47/0.53. Significantly higher morbidity (similar to 9 times) and poorer performances in righting behavior, foraging behavior, Aristotle's lantern reflex and adhesion force were consistently found in area W than those in area M. The number of sea urchins with diseased Aristotle's lantern was significantly higher (similar to 24 times) than that of those without diseased Aristotle's lantern, among sea urchins without the black-month disease performances in area W. This approach, therefore, is effective in separating problematic sea urchins (including asymptomatic infectors). In experiment 2, five problematic sea urchins (previously exposed to the bacteria liquid but without disease symptoms) and thirty-five healthy sea urchins were maintained in both the new facility group (group 5) and the control group (group C) for 7 days. This study found significantly higher morbidity (similar to 2 times) and significantly poorer performances in adhesion force, righting behavior and Aristotle's lantern reflex occurred in group C compared to those in group S. These results indicate that the new facility greatly decreases disease transmission and leads to better performance in fitness-related behaviors of cultured sea urchins. The present study provides valuable information on effectively decreasing the disease transmission in seed production and longline culture of sea urchins.

Improving the aquaculture production efficiency by appropriate diets is an essential approach to meeting the increasing market demand for sea cucumbers. The feces of sea urchins, which contains various enzymes and microorganisms, is a potentially cost-effective food for sea cucumbers. To assess the usability of the fecal diet, a five-week laboratory simulation is conducted to investigate behaviors, digestion ability, growth and resistance ability of the sea cucumber Apostichopus japonicus fed with fecal diet at water temperatures of 15°C and 5°C. In the present study, A. japonicus fed with fecal diet shows an obvious preference to fecal diet rather than prepared feed at water temperatures of both 15°C and 5°C, which suggests that the feces is an applicable diet for A. japonicus. Furthermore, small A. japonicus fed with feces (group F) shows significant advantages in intestinal community richness, community diversity and intestine protease activity to A. japonicus fed with prepared feed (group S) at 15°C. These results indicate that the fecal diet provides benefits to digestion ability of small A. japonicus at 15°C. Weight gaining rate is significantly higher in the A. japonicus fed with feces than that in A. japonicus that were fed with feed or not fed with food (group C), which suggests that the direct improvement of the production efficiency at 15°C. The advantages in intestinal bacteria, protease activity, and growth are consistently found in group F compared with group S at 5°C. In addition, the composition of intestinal bacteria indicates that sea cucumbers may inherit the intestinal bacteria of sea urchins through fecal consumption. This suggests that the fecal diet enhances the digestion ability and enzyme activity at low water temperature and thus improves the growth of sea cucumbers. Furthermore, sea cucumbers fed with sea urchin feces have the highest survival rate among the three groups in exposure to an acute salinity decrease at both 5 and 15°C, indicating a better resistance to low salinity. This provides a new insight into the geographical expansion to low-salinity areas in sea cucumber aquaculture. In conclusion, the present study suggests that sea urchin feces have a great potential for the application in improving the production efficiency of sea cucumber aquaculture.

The energetic link in the benthic community is based on physiological characteristics of the low food absorption efficiency of sea urchins. Low food absorption efficiency of sea urchins is correlated with the activity of digestive enzymes and the duration of food in their gut. Thus, the digestive enzymes activities (pepsin and amylase enzyme activities) and gut emptying are important indicators in assessing nutrient digestion and absorption in sea urchins. In the present study, the relationship between these indicators and molecules related to digestive physiology were quantified in sea urchins. We found (1) an inter-regulatory relationship existed between Transient receptor potential cation channel, subfamily A, member 1 (TRPA1), and serotonin (5-hydroxytryptamine; 5-HT) in the gut of Strongylocentrotus intermedius; (2) digestive enzyme activities were negatively correlated with the TRPA1 and concentration of 5-HT in the gut of S. intermedius; (3) gut emptying rate was positively correlated with TRPA1 and concentration of 5-HT in the gut of S. intermedius. The present study revealed that the digestion and absorption of food are correlated with the TRPA1 and 5-HT in the gut of S. intermedius, which provides valuable information about the digestive physiology of sea urchins. This novel finding is relevant to understanding the low food digestibility of sea urchins. It also provides valuable information to the digestive physiology of sea urchins, which are key to maintaining the stability of food webs in the marine ecosystem.

The Integrated Multi-Trophic Aquaculture (IMTA) of sea cucumbers and sea urchins has high commercial values. The consumption of sea urchin feces by sea cucumbers is important but largely unknown. The present study aims to evaluate and subsequently improve the fecal consumption in sea cucumbers. Sea cucumbers ingested the feces of sea urchins at both 15 degrees C and 5 degrees C, suggesting that the feces of sea urchins has potential to be an appropriate diet for the aquaculture of A. japonicus. Because feces has a complex process after the egestion, the age and temperature significantly affected the fecal consumption by sea cucumbers. The optimal ageing time after egestion was 0-48 h at 15 degrees C, 0-72 h at 5 degrees C, respectively. Thus, we recommend aqua-farmers feed A. japonicus with feces at the optimal ageing hours and remove the feces in time. Further, light intensity significantly affected the fecal consumption by A. japonicus. Sea cucumbers had significantly more fecal consumption in low light intensity than that in high light intensity at both 5 and 15 degrees C. Further, sea cucumbers fed significantly more in low light intensity with shelters than those in low light intensity without shelter, suggesting that shelter provides extra benefits for the fecal consumption by sea cucumbers besides the low light intensity. In addition, significant feeding benefits were found in both low light intensity and shelter at low temperatures. This may solve the problem that sea cucumbers cease feeding at 5 degrees C in winter. Organizing IMTA in sheltered environment and low light intensity can improve fecal consumption at both 15 degrees C and 5 degrees C. The present study provides valuable information in evaluating and improving consumption of sea urchin feces by A. japonicus in the IMTA of sea cucumbers and sea urchins.

Seed production is essential for the profitable and sustainable aquaculture of the sea cucumber Apostichopus japonicus. Stresses are an inevitable consequence of handling in seed production. Here, we investigated the effects of different intensities of handling stresses (air exposure, medicine bath, mechanical perturbation and seawater washing) on the movement, foraging, feeding and defecation behaviors of small A. japonicus. The present study found that small A. japonicus exposed to mechanical perturbation showed significantly longer time to first reaching air-water interface, shorter movement distance and worse foraging ability among the groups. No significant difference of feeding behavior was found between small A. japonicus in exposure to mechanical perturbation and without handling stress. In contrast, small A. japonicus exposed to air, medicine bath and seawater washing showed significantly worse feeding ability compared with those without handling stress. There was no significant difference in foraging behavior of small A. japonicus exposed to air, medicine bath, seawater washing compared with those without handling stress. Further, high-intensity handling stresses significantly inhibited movement, foraging and feeding of small A. japonicus. Feeding behavior of small A. japonicus exposed to high-intensity air exposure had more serious carryover effects than those exposed to low-intensity air exposure. Moreover, air exposure showed serious carryover effects on feeding behavior of small A. japonicus. The present study provides valuable information for seed production of sea cucumbers.