Dietary tryptophan decreases the impacts of seawater temperature changes on behavior and gut health of the sea cucumber Apostichopus japonicus.

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 °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.

Behavior, intestinal health, and growth of small sea cucumbers Apostichopus japonicus in different color morphs.

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.

Bacillus licheniformis reduces mortality, improves symptoms, and controls residual pathogenicity in juvenile sea cucumbers Apostichopus japonicus infected with skin ulceration syndrome.

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.

Effects of circadian rhythm on behavior and physiology of the sea cucumber Apostichopus japonicus.

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.

Fitness costs of alarm cues in sea urchins: Fertilization, hatching, and larval size.

Sea urchins sense alarm cues extracted from crushed conspecifics and perform anti-predation behaviors in exposure to alarm cues. This indicates of the fitness benefits of alarm cues in sea urchins. The present study investigated whether fitness costs of alarm cues exist in fertilization, hatchability, deformity, and larval size of the sea urchin Mesocentrotus nudus. In the present study, we found that fertilization and hatching rates were significantly lower in the group with alarm cues than those in the group without alarm cues, indicating that fitness costs of alarm cues exist in sea urchins. However, there was no significant difference in deformity rate, larval length, stomach length, and stomach width of M. nudus with and without alarm cues. The group with alarm cues showed significantly shorter larval width than the group without alarm cues. This indicates that smaller larvae of sea urchins more probably survive in the environment with alarm cues. The present study reveals that fitness costs of alarm cues exist in sea urchins and sheds light on the ecological roles of alarm cues in kelp bed ecosystems.

Conspecific alarm cues are a potential effective barrier to regulate foraging behavior of the sea urchin Mesocentrotus nudus.

A cost-effective approach to controlling foraging and feeding behaviors of sea urchins is essential for the management of kelp beds. Laboratory experiments were designed to investigate whether alarm cues from crushed conspecific urchins can effectively prevent the foraging and feeding behaviors of the sea urchin Mesocentrotus nudus under the static seawater condition. The present study found that the number of M. nudus that foraged successfully was significantly lower when alarm cues were placed between the kelp and the sea urchins. This result indicates that alarm cues could play an important role in critical kelp-bed areas. It probably prevents sea urchins from foraging by acting as a barrier. Further, we found that alarm cues around the kelp significantly affected foraging behavior of M. nudus, indicating that the alarm cues around the kelp are a potential effective way to prevent sea urchins from foraging for the kelp. In addition, the number of sea urchins that stopped feeding was significantly higher in the group in the presence of alarm cues than that in the control group. This indicates that alarm cues may have an application in stopping sea urchins from feeding. However, there was no significant difference of Aristotle's lantern reflex between the groups with and without alarm cues. These results indicate that alarm cues greatly affect foraging behavior, but not Aristotle's lantern reflex of M. nudus. All together, the present study suggests that alarm cues have an application potential in the management of the kelp beds as green engineering. Future studies are essential to further investigate the chemical basis of the alarm cues of sea urchins for the application in large-scale.

Foraging behavior of the sea urchin Mesocentrotus nudus exposed to conspecific alarm cues in various conditions.

Conspecific alarm cues crushed from Mesocentrotus nudus prevent sea urchins from foraging the kelp, but do not repel them far away from the kelp. However, it remains largely unknown of whether this phenomenon was affected by conspecific alarm cues or by the attraction of the kelp. The present study found no significant difference in the duration in the danger area with or without the kelp around conspecific alarm cues. This suggests that the phenomenon is the strategy of sea urchins but not by the attraction of kelp. We found that conspecific alarm cues appearing between the kelp and sea urchins significantly affected foraging behavior of sea urchins fasted for 21 days. This indicates that conspecific alarm cues can effectively prevent fasted sea urchins from foraging the kelp. Further, there was no correlation between foraging velocity and the duration in the danger area. Pearson correlation analysis revealed no significant correlation between foraging velocity and the duration in the safety area close to different amounts of conspecific alarm cues, suggesting that conspecific alarm cues prevent sea urchins with strong foraging ability to forage. Collectively, the present results indicate that conspecific alarm cues as highly available biological barriers are cost-effective approaches to preventing overgrazing of sea urchins in the protection of kelp beds ecosystems. Notably, the present study is a short-term laboratory investigation that does not consider the complexity of natural conditions. Future studies are essential to test the present findings in the field.

Effects of eliminating interactions in multi-layer culture on survival, food utilization and growth of small sea urchins Strongylocentrotus intermedius at high temperatures.

Poor growth and disease transmission of small sea urchins Strongylocentrotus intermedius in summer greatly hamper the production efficiency of the longline culture. Reducing the adverse effects of high stocking density while maintaining high biomass is essential to address these problems. Here, we conducted a laboratory experiment to simulate the multi-layer culture for sea urchins at ambient high temperatures (from 22.2 to 24.5 °C) in summer for ~ 7 weeks. Survival, body size, lantern growth, gut weight, food consumption, Aristotle's lantern reflex, 5-hydroxytryptamine concentration, pepsin activity and gut morphology were subsequently evaluated. The present study found that multi-layer culture led to significantly larger body size than those without multi-layer culture (the control group). This was probably because of the greater feeding capacity (indicated by lantern growth and Aristotle's lantern reflex) and food digestion (indicated by morphology and pepsin activity of gut) in the multi-layer cultured sea urchins. These results indicate that multi-layer is an effective approach to improving the growth efficiency of sea urchins at high temperatures. We assessed whether eliminating interaction further improve these commercially important traits of sea urchins in multi-layer culture. This study found that eliminating interactions displayed greater body size and Aristotle's lantern reflex than those not separated in the multi-layer culture. This approach also significantly reduced the morbidity compared with the control group. These novel findings indicate that eliminating interactions in multi-layer culture greatly contributes to the growth and disease prevention of sea urchins at high temperatures. The present study establishes a new technique for the longline culture of sea urchins in summer and provides valuable information into the longline culture management of other commercially important species (e.g. scallops, abalones and oysters).

Carryover effects of long-term high water temperatures on fitness-related traits of the offspring of the sea urchin Strongylocentrotus intermedius.

It is important to study the fitness of marine invertebrates in exposure to high water temperature. We studied whether the long-term high temperatures work on the fitness-related traits (righting behavior, covering behavior, foraging behavior, Aristotle's lantern reflex, body size) of S. intermedius whose parents (males and females) were exposed to ambient or high temperatures (~3 °C higher than the ambient) for a long period of time. The present study found that test diameter, wet body weight and test weight of offspring were not significantly different between temperature treatments, indicating that the parental sea urchins in exposure to high temperatures develop no carryover effects on the body size of the offspring sea urchins. We found no significant difference in foraging behavior, Aristotle's lantern reflex, lantern length and lantern weight of sea urchins after their parents had experienced long-term high temperatures. In addition, no significant change was found in the righting and covering behaviors of sea urchins whose parents were at long-term high temperatures. These results indicate that no significant lasting effects exhibited in the fitness-related behaviors and tissue size after their parents were exposed to high temperatures for a long time. The crushing force of test and test thickness showed no significant difference in the offspring of S. intermedius, no matter whether their parents were exposed to long-term high temperatures or not. The current results enrich our understanding that the parental sea urchin experiencing long-term high temperatures probably develop no carryover effects on the test of their offspring. We found that sea urchins whose parents were exposed to long-term elevated temperatures showed a significantly higher lantern length/test diameter and a significantly lower test height/test diameter in offspring sea urchins due to the thermal experience of their parents, showing the plasticity of lantern and test of offspring sea urchins in response to the thermal experience of their parents. Together with our previous investigation, the present study indicates that small sea urchins are less susceptible to the carryover effects of high temperatures in comparison with the developmental stages of embryos and larvae.

[A multiple center, randomized, controlled, double-blinded and double-dummy trial of Yiqing Shuangjie Capsule and Tablet in treating acute upper respiratory tract infection with the syndrome of heat attacking the lung and Weifen].

OBJECTIVE: To evaluate the efficacy and safety of Yiqing Shuangjie Capsule and Tablet in treating acute upper respiratory tract infection with wind-warm syndrome. METHODS: The multiple center, double-blinded, double-dummy and randomized controlled method was conducted. Three hundred and sixty patients were randomly divided into the treatment group A (n=120, treated with Yiqing Shuangjie Capsule and Chaihuang analogues), treatment group B (n=120, treated with Yiqing Shuangjie Tablet and Chaihuang analogues) and the control group (n=120, treated with Chaihuang Tablet and Yiqing Shuangjie Capsule analogues). Every drug was administered 3 pills each time. Patients in the three groups were all treated for 5 days and three times daily. The accumulated scores of syndrome, clinical symptoms, adverse effect and body temperature were recorded before and after the treatment. The safety indexes, such as routine tests of blood, urine and stool, hepatic and renal function tests and electrocardiogram (ECG) were taken before and after the treatment. RESULTS: Three cases were excluded and eighteen cases lost to follow-up. There were 343 patients who entered to the intention-to-treat (ITT) analysis and 339 patients fitted in the per-protocol population set (PPS) analysis. After treatment, the therapeutic effects of respiratory tract infection were calculated by ITT and PPS analysis respectively. The rates of total obvious effect were 84.96% and 87.27% respectively in the treatment group A, and the total response rates were 96.46% and 97.27% respectively. The rates of total obvious effect were 85.47% and 86.20% respectively in the treatment group B, and the total response rates were 97.45% and 97.41% respectively. In the control group, the rates of total obvious effect both were 72.57%, and the total response rates both were 99.12%. There was significant difference among the three groups (P<0.05). The effects of traditional Chinese medicine syndrome were also detected by ITT and PPS analysis respectively. In the treatment group A, the total obvious effect rates were 84.07% and 85.46% respectively, and the total response rates were 96.46% and 97.27% respectively. In the treatment group B, the rates of total obvious effect were 88.89% and 89.65% respectively, and the total response rates were 97.44% and 97.41% respectively. In the control group, the rates of total obvious effect both were 72.57%, and total response rates both were 99.12%. There was also statistical significant difference among the three groups (P<0.05). ITT and PPS analysis had the same results. No adverse effect was found in the trial. CONCLUSION: Yiqing Shuangjie Capsule and Tablet are effective and safe in treating acute upper respiratory tract infection of wind heat syndrome without obvious adverse effect.