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Broodstock rearing is fundamental to produce high-quality offspring for aquaculture. However, reproduction requires large amounts of energy and nutrients, and physiological activities of digestive enzymes determine the absorption of energy and nutrients. However, the interaction between reproduction and digestive physiology is still unclear in sea cucumbers. In this study, we used laboratory experiments and field investigations to assess the variations of digestive physiology and digestive function in the sea cucumber Apostichopus japonicus during reproduction. Unexpectedly, activities of both amylase and trypsin were depressed in adult sea cucumbers during reproduction under both laboratory and field conditions. As a physiological consequence, digestive efficiencies of energy and organic matter decreased significantly during reproduction. Although reproductive output of sea cucumbers varied greatly between laboratory and wild conditions, reproductive effort had no effect on the activities of numerous digestive enzymes. Together, these data indicated that reproductive status induced digestive function decline in A. japonicus. Our study provided very important information about the reproductive biology of sea cucumbers that could be applied to broodstock rearing and conservation of this commercially important species. [ABSTRACT FROM AUTHOR]

Simple Summary: The sea cucumber Apostichopus japonicus is an important aquatic invertebrate in the aquaculture industry. It has high nutritional and medicinal value and is popular with consumers, in particular in Asia. The high demand for the species has led to declines in wild populations and A. japonicus is listed as an endangered species by IUCN. Aquaculture of sea cucumber is important to safeguard the conservation of the species in the wild, and its successful reproduction in captivity is a prerequisite for industrial production. It is known that parental nutrient reserves, in particular of lipids, are directly related to the reproductive performance of sea cucumbers. In this study, we investigated the impact of a high-fat diet on the intestinal and gonadal metabolism of male and female A. japonicus. Diet lipid content significantly affected the intestinal microbiome and metabolite profile, and also interfered with gonadal metabolites. Dietary lipid supplementation enhanced the dominance of Proteobacteria in the intestines. Other microbes in the intestines responded to a high-fat diet, which may contribute to maintain intestinal homeostasis. The physiological and metabolic responses of sea cucumbers to a high-fat diet showed sex differences. Interestingly, a correlation between intestinal and gonadal tissue levels of polyunsaturated fatty acid was found. The results of this study indicate that the lipid content in diets can be differentially adjusted for male and female sea cucumbers to improve nutrition and promote reproduction. This data also contributes to a better understanding of the reproductive biology and sex differences of sea cucumbers. Parental nutrient reserves are directly related to reproductive performance in sea cucumbers. This study focused on the lipid requirements of male and female sea cucumbers Apostichopus japonicus during the reproductive stage and analyzed their physiological responses to a high-fat diet (HFD). The intestinal lipid metabolites and microbiome profile changed significantly in animals fed with the HFD, as given by an upregulation of metabolites related to lipid metabolism and an increase in the predominance of Proteobacteria in the microbiome, respectively. The metabolic responses of male and female sea cucumbers to the HFD differed, which in turn could have triggered sex-related differences in the intestinal microbiome. These results suggest that the lipid content in diets can be differentially adjusted for male and female sea cucumbers to improve nutrition and promote reproduction. This data contributes to a better understanding of the reproductive biology and sex differences of sea cucumbers. [ABSTRACT FROM AUTHOR]

Seed production plays an important role in aquaculture. The sea cucumber Apostichopus japonicus is an important marine aquaculture species in many Asian countries. By 2020, the annual total seed production of A. japonicus reached 551 billion individuals in China. However, the traditional method of culturing A. japonicus larvae is cost intensive and causes environmental pollution. In this study, we developed an eco‐friendly method for rearing sea cucumber larvae. We designed a cage system to provide a suitable living environment for larvae in open‐air outdoor ponds, and then, we identified 0.2 larvae per ml as the optimal stocking density for larval rearing. We also found that substrates made of oyster and scallop shells were optimal for larval settlement. A. japonicus larvae showed good survival, growth, development and settlement when cultured using this low‐cost and non‐polluting method. Our results can be applied to support sustainable development of the sea cucumber industry. [ABSTRACT FROM AUTHOR]

Physiological plasticity allows animals to adjust their physiological function to abiotic and biotic variations. It has been mostly studied in the context of response to external factors and not much is known on how animals adjust their physiology to cope with variations in internal conditions. The process of reproduction implies gonadal maturation and other internal changes, bringing various challenges to the animal such as an increased demand for energy and oxygen. Here, the capacity of the sea cucumber, Apostichopus japonicus to adjust its respiratory function and physiological mechanisms during reproduction was studied using a time-lapse videography and metabolomics approach. The results showed that reproduction caused a significant increase in oxygen consumption in A. japonicus. Interestingly, breeding sea cucumbers can accommodate the high oxygen demand by accelerating respiratory rate. However, to maintain a necessary high level of respiratory activity during reproduction, sea cucumbers need consume large amounts of adenosine triphosphate (ATP). In addition, the metabolomic data suggests that oxidative stress and hormone regulation are the physiological mechanisms linking reproduction and respiratory function. Altogether, these findings suggest that plasticity of respiratory function is an effective tactic to cope with high oxygen demand during reproduction. [ABSTRACT FROM AUTHOR]

Abstract Economically important marine organisms face severe environmental challenges, such as high temperature and low dissolved oxygen, from global climate change. Adverse environmental factors impact the survival and growth of economically important marine organisms, thereby negatively influencing the aquaculture industry. However, little is known about the responses of sea cucumbers to combined environmental co-stressors till now. In this study, ultra-performance liquid chromatography (UPLC) was utilized to obtain metabolic profiles of sea cucumbers. Changes in the concentrations of 84, 68, and 417 metabolites related to the responses of sea cucumbers to heat (26 °C), hypoxia (2 mg/L) and the combined stress, respectively, were observed and analyzed. Representative biomarkers were discussed in detail, including deltaline, fusarin C, halichondrin B and rapanone. The concentration of metabolites involved in the regulation of energy metabolism, including amino acid, carbohydrate and lipid metabolism were significantly changed, and the tricarboxylic acid (TCA)-cycle was significantly altered under heat plus hypoxia. We interpreted these changes partly as an adaptation mechanism in response to environmental stress. Based on the decreased accumulation of glutamine, we hypothesized that heat stress is the main factor that interferes with the process of glutamic acid-glutamine metabolism. The present study showed that combined environmental stressors have a more extensive impact on the metabolites of the respiratory tree in sea cucumbers than single stress. These results would facilitate further development of the sea cucumber as an echinoderm model to study mechanisms of response to adverse environments, as well as to help advance knowledge of the adaptation of marine organisms to global climate change. Highlights • UPLC was utilized to obtain metabolic profiles of sea cucumbers under heat, hypoxia and the combined stress. • Synergistic effect of environmental stressors has an additive impact on the primary metabolome in sea cucumbers. • Ten representative potential biomarkers of heat and hypoxia in sea cucumber are illustrated. • The levels of metabolites involved in the regulation of energy metabolism were significantly changed. • The tricarboxylic acid (TCA)-cycle was significantly altered under the combined stress of heat and hypoxia. [ABSTRACT FROM AUTHOR]

Abstract: As an intertidal species, Apostichopus japonicus is subject to diel and seasonal temperature fluctuations. Color variation is a distinct characteristic of A. japonicus, and a new color morph, purple A. japonicus, was recently found on the coast of Rushan, Weihai City. This study was conducted to compare the performance of green, white, and purple color morphs of A. japonicus at different water temperatures to help improving aquaculture technology and management. In this study, green, white, and purple color morphs of juvenile A. japonicus (1.5 ± 0.1 g) were cultured at 10, 14, 18, 22, and 26 C for 60 d, according to the suitable temperature range for sea cucumber growth. Temperature was shown to have a significant effect on growth, energy budget, and physiology of sea cucumbers (P < 0.05). Specific growth rates, feed conversion efficiencies, and the proportion of energy allocated to growth of all three color morphs were optimal at 18 C. Activities of the enzymes related with oxidative stress, immune response, and digestion were determined. Superoxide dismutase (SOD) and trypsin activities generally reached a maximum in all three color morphs at 18 C. The purple color morph of A. japonicus was higher in growth rate, food conversion efficiency, and proportion of energy allocated to growth than green and white color morphs at 18 and 22 C, consistent with its higher trypsin and SOD activity, indicating the mutual effect of growth and physiology. At 18, 22, and 26 C, the green color morph had the highest level of lysozyme activity and purple color morph had the lowest, indicating possible infection by external pathogens of green color morph, which might be caused by high temperature. These results suggested that the optimum temperature for culture of the three color morphs was approximately 18 C, the purple had a better adaption to high temperature, and the green and white color morphs may be more susceptible to thermal stress than the purple color morph. [ABSTRACT FROM AUTHOR]

Abstract: Two groups of Apostichopus japonicus, a thermotolerant strain (group G3: wet weight, 64.22 ± 13.16 g/ind.), which was selected focusing on the performance of thermo‐tolerance through three generations for 10 years, and a control group (group C: wet weight, 62.08 ± 12.01 g/ind.) were collected from July to October in a seawater pond in northern China. Differences in relative faecal mass (RFM), relative intestinal mass (RIM), percentage of individuals with faeces (PIWF) and intestinal metabolites were investigated and compared between the two groups. The temperatures of terminating aestivation of the two groups were assessed according to the values of RFM and RIM. A regression analysis showed that the corresponding group G3 temperatures were 0.56–1.34°C and 0.70–1.49°C higher than those of group C when RFM and RIM were 0.01–0.05 and 0.005–0.025 respectively. The PIWF values in group G3 were 11.5%–21.2% higher than that in group C from 20 July to 22 September. Significant differences at the concentrations of 52 metabolites were detected between the two groups, 36 were higher in group G3 and 16 were higher in group C. The concentrations of threitol, 2‐methylglutaric acid, N‐acetyl‐D‐galactosamine, N‐acetyl‐L‐leucine, lactose, oxoproline, 2,3‐dimethylsuccinic acid and d‐glucoheptose were significantly different between groups G3 and C and were considered metabolic markers distinguishing group G3 from group C. Metabolism of A. japonicus in group G3 was more active than that in group C. These results provide new insight for understanding ingestion and intestinal metabolism in the thermotolerant strain of A. japonicus under high summer temperature. [ABSTRACT FROM AUTHOR]

The ‘cost of reproduction’ refers to a series of negative effects that reproduction has on females. One behavioural cost of reproduction in vertebrates is the loss of or decrease in motility. Diminished locomotion in gravid individuals has serious potential effects on various behaviours, including locomotion, predation, antipredator, migration and the patrolling of territories. However, there have not been comparable studies on marine invertebrates. To address the cost of reproduction in marine invertebrates, we studied the sea cucumber, Apostichopus japonicus , as a model system. Using time-lapse technology and behavioural analysis software, we analysed the locomotor behaviour of A. japonicus from the nonbreeding to the mature stage. In addition, metabolism in muscle tissue of animals between the nonbreeding and growth stage was tested with ultraperformance liquid chromatography and quadrupole time-of-flight mass spectrometry (UPLC–Q–TOF–MS). The results showed that reproduction had no negative effects on locomotion frequency or maximum velocity. However, the total distance moved and cumulative duration of moving gradually decreased. Therefore, the results suggest that loss of locomotor endurance is the behavioural cost of reproduction in female A. japonicus . Additionally, we found 10 significant metabolic changes in the muscle tissue of animals in the growth stage. These results suggest that oxidative stress and lipid metabolism are potential physiological mechanisms linking reproduction and depressed locomotory performance. [ABSTRACT FROM AUTHOR]

Reproduction of the sea cucumber Apostichopus japonicus is critical for aquaculture production. Gonadal development is the basis of reproduction, and lipids, which are among the main nutrients required for gonadal development, directly affect reproduction. We investigated whether gonadal and intestinal lipid metabolism differed between male and female A. japonicus. Transcriptome analysis of the intestines of sexually mature male and female wild-caught individuals revealed differences in gene expression, with 27 and 39 genes being up-regulated in females and males, respectively. In particular, the expression of the fatty acid synthase gene was higher in males than in females. Metabolome analysis of the gonads identified 141 metabolites that were up-regulated and 175 metabolites that were down-regulated in the testes compared with the ovaries in the positive/negative mode of an LC-MS/MS analysis. A variety of polyunsaturated fatty acids were found at higher concentrations in the testes than in the ovaries. 16 s rDNA sequencing analysis showed that the composition and structure of the intestinal microbiota were similar between males and females. These results suggest that sex differences in intestinal metabolism of A. japonicus are not due to differences in the microbiota, and we speculate that gonadal metabolism may be related to intestinal morphology. This information might be useful in improving the reproductive efficiency of sea cucumbers in captivity. • Male and female Apostichopus japonicus may have different lipid metabolic characteristics. • The expression of FASN in male sea cucumber intestines was up-regulated. • The content of PUFAs (DHA, EPA, and ARA) was higher in sea cucumber testes than in ovaries. • Sex differences in intestinal metabolism of Apostichopus japonicus were not affected by microbiota. [ABSTRACT FROM AUTHOR]

We evaluated the effect of rearing density on the yield of a fast-growing and heat-resistant strain of Apostichopus japonicus using a full-sib family. We analysed the relationship between density and key growth parameters during factory seedling of A. japonicus and developed a model to explain the variation in growth. From 130 to 220 days, the rearing density of A. japonicus had a significant effect on body weight, specific growth rate, coefficient of variation and survival rate ( P < 0.05). We obtained predicted integral regression equations for yield and growth rate, density fluctuation, variable coefficient fluctuation and survival rate. Specific growth rate had a significant effect on the yield of A. japonicus and, therefore, can be used as a primary indicator of yield. We recommend that stocking density should be adjusted from age 130-166 days to avoid frequent seedling separation to different pools of different seedling sizes during the fast growth period of A. japonicus seedlings, a procedure that causes yield decline. The optimal initial stocking density was 24.4 g m−2 (0.2 g L−1). [ABSTRACT FROM AUTHOR]

We evaluated the effect of high temperature on Apostichopus japonicus gametes. Gametes were exposed to the optimal temperature (18°C; control) or one of six high temperature treatments (26, 27, 28, 29, 30 and 31°C) and the cleavage rate ( CR) of fertilized eggs was measured as an indication of fertilization ability. At control temperatures (18°C), the male and female gametes had high fertilization ability ( CR ≥95.0%) in 5 h and the time at which 50% of gametes lost the ability to successfully fertilize ( LT50) was 7.03 h. All gametes were non-viable after 9 h. When male and female gametes were both exposed to high temperature, the LT50 was lower than that when female or male gametes were treated individually at the same high temperature ( P < 0.05). The LT50 was lower for male gametes treated alone than for female gametes treated alone. In conclusion, exposure to high temperature results in a decline in fertilization ability of A. japonicus gametes, and this decline was more rapid at higher temperature. The female gametes were more resistant to high temperatures than male gametes. [ABSTRACT FROM AUTHOR]

Techniques for in vitro oocyte maturation are important for studying maturation regulatory mechanisms, cell differentiation, and embryonic development. In this study, after Apostichopus japonicus gonads were dissected, oocytes, together with gonadal fluid extracted from gonads, were dried in shade. The effects of three temperatures (17, 21, and 25 °C) on oocyte maturation and following development were studied. The results showed that germinal vesicle breakdown (GVBD) of most oocytes occurred after desiccation, and those oocytes with GVBD could be fertilized and develop normally. There were no significant differences in the percentages of GVBD and individuals in blastula and gastrula stages between 17 and 21 °C (p >.05). The percentages of GVBD reached a maximum (17 °C: 98.6%, 21 °C: 97.1%) after 2 h of desiccation. The percentages of blastulae and gastrulae reached the maximum of more than 80% after 2.5–3.5 h of desiccation. At 25 °C, the percentages of GVBD, blastulae, and gastrulae reached a maximum of 76.7, 61.0, and 59.9% after 3.5 h of desiccation, respectively, and were significantly lower than that at 17 and 21 °C (p <.01). This study not only provided an effective and simple technique for in vitro induction of A. japonicus oocyte maturation, but also furthered understanding of gamete biology of this species. [ABSTRACT FROM AUTHOR]

Using incomplete diallels cross and North Carolina II design, this study established sea cucumber families with heat-resistant and fast-growing Apostichopus japonicus strains from different ecological areas. Then, this study analyzed the influence of paternal and maternal genetic effects on the growth of A. japonicus, evaluated the growth effect of different A. japonicus families in the early growth stage and estimated the combining ability and heritability. The study showed that, in the early growth stage of A. japonicus, the influence of paternal genetic effects on the growth was higher than that of maternal effects. The best five parents and eight offspring families in terms of genetic potential breeding and growth were selected. The 3-month weight, 9-month weight, 12-month weight, 5-month to 9-month specific growth rate, 9-month coefficient of variation (CV) and 12-month CV of A. japonicus are at moderate heritability level, in order, 28.18, 40.82, 43.56, 27.87, 44.39 and 38.59 %, respectively; these traits played a leading role in parental additive gene effect on the formation of hybrids offspring traits. [ABSTRACT FROM AUTHOR]

The sea cucumber, Apostichopus japonicus , is an important marine aquaculture species in Asian countries. The small body size but high fecundity of A. japonicus suggests that using income resources to support gonad development might be an appropriate life history strategy, but the physiological mechanisms involved in this approach remain unclear. In this study, we conducted three experiments to assess the physiological traits that underlie income breeding strategy of the A. japonicus. As inferred by the carbon and nitrogen stable isotope signatures, the results showed that income breeding allows all adults, even individuals with poor body condition and small maternal size, to reproduce annually by flexibly adjusting their reproductive output. In addition, functionally, we found that the sea cucumbers allocate income resources to the demands of reproduction, growth and storage simultaneously during reproduction. However, dynamic changes of activities of Na+/K+-ATPase, hexokinase and pyruvate kinase suggested that income breeding places a metabolic burden on the breeding adults. Our results provided new insights into the physiological mechanisms underlying income breeding strategy in sea cucumbers. • Apostichopus japonicus is an income breeder. • Income breeding allows all adults to reproduce annually by adjusting their reproductive output. • Income resources are simultaneously allocated to the demands of reproduction, growth and storage during reproduction. • Income breeding places metabolic burden on the breeding sea cucumbers. [ABSTRACT FROM AUTHOR]

Gender differences in physiological characteristics are widespread in animals. Herein, differentially expressed genes (DEGs) in gonads of the sea cucumber Apostichopus japonicus were analysed by transcriptomics, and the results showed that 19,973 genes were commonly expressed in the males and females, 4186 were female-biased, and 2540 were male-biased, 4695 genes were up-regulated in the females and 3436 genes were up-regulated in the males. These DEGs were mainly associated with metabolism, including lipid metabolism, amino acid metabolism, nucleotide metabolism, energy metabolism, and cofactor and vitamin metabolism. 29 Cytochrome P450 (CYP) superfamily genes with gender differential expression were selected, and performed gene identification, phylogenetic, and functional analyses. The results indicated significant roles in multiple metabolic pathways, such as steroid hormone biosynthesis, ovarian steroidogenesis, cortisol synthesis and secretion, arachidonic acid metabolism, linoleic acid metabolism, and retinol metabolism. The findings provide insight into the molecular characteristics of physiological gender differences in sea cucumbers, and will help lay the foundation for the establishment of effective sea cucumber breeding technologies. • A new insight into the molecular characteristics of Apostichopus japonicus physiological sex differences • Various metabolic pathways play an important role in the development of gonads of sea cucumber. • The first systematic study of the Cyp superfamily gene of sea cucumber [ABSTRACT FROM AUTHOR]

Sea cucumber Apostichopus japonicus is important aquatic economic species in Asia. In recent years, due to the decline of wild resources of A. japonicus , in order to ensure the sustainable supply of seedlings, the broodstock rearing is particularly important during seedling production, reproduction is also an essential stage in the life history of A. japonicus. However，the physiology of marine animals undergoes variations during the process of reproduction. It is well known that the energy demand of broodstock increases and their immunity decreases during the reproduction. Intestinal microorganisms participate in host life activities, while the presence of pathogens and probiotics will affect host health. However, the function of intestinal microbiota in the energy and immunity during the reproduction remains unclear. In this study, we performed a metagenomic analysis of the gut microbiota in A. japonicus to investigate variations in microbial composition and KEGG pathways associated with reproductive periods. Results suggested that Firmicutes/Bacteroidetes (F/B) was gradually increasing during reproduction, which increased host energy harvest, and was difference between male and female A. japonicus. Virulence Factors of Pathogenic Bacteria (VFBD) upregulated from the gonadal growth period. The higher expression of VFBD during the gonadal growth period than at other times may be caused by the fact that Flavobacteriaceae and Rhodobacteraceae are most abundant during this period. Lysozyme activity was upregulated during reproduction and peaked during the gonadal mature period, with a similar trend of variability as in VFBD, which carried out immunological protection against A. japonicus. The relative abundance of Saccharomycetaceae , and Bacillaceae was highest during the spawning period, and it has been identified that microorganisms strengthen immune protection against A. japonicus during this period, when A. japonicus was in a weakened state. Based on KEGG analysis, expression of oxidative phosphorylation and the fever pathway increased after spawning, indicating that reproduction causes oxidative stress in A. japonicus. Additionally, the differences in several KEGG pathways were related to sex. Overall, by analyzing the energy and immune systems, the study confirmed that intestinal microorganisms play vital physiological roles in broodstock of A. japonicus during reproduction. This study provided important information for breeding and the application of A. japonicus in aquaculture. • Sea cucumber Apostichopus japonicus , is one of the most economically important aquaculture species in Asia. This study confirmed that intestinal microorganisms play vital physiological roles in broodstock of A. japonicus during reproduction, microorganisms participate regulation of host energy metabolism, immune adjustment. • There are several pathogenic bacteria increased during reproduction. Post spawning, the number of probiotics increases, which can protect A. japonicus and provide immune compensation. • The composition of intestinal microbiome is also different in male and female individuals. • Several KEGG pathways changed, focused on energy metabolism and oxidative damage. • This study provided an important information for breeding and the application in aquaculture of A. japonicus. [ABSTRACT FROM AUTHOR]

With water temperature increasing in summer, large-sized sea cucumbers ( Apostichopus japonicus ) generally enter aestivation, while there still occurs a small portion of individuals that do not enter aestivation and remain active and ingesting. In this study we investigated the differences in immuno- and metabolic enzyme activities and metabolites between non-aestivating and aestivating groups of A. japonicus . Animals (wet weight > 150 g) were collected from July to October in a seawater pond, north China. The individuals that voided feces, or had intestine contents after dissection, were defined as non-aestivating group, whereas those without feces or intestine contents were defined as aestivating group. Relative intestine mass (RIM), immuno-enzyme activities (SOD, CAT, T-AOC) and metabolic enzyme activities (PK, HK, MDH, LDH) of the body wall, and metabolites in intestine in both groups were determined. Results showed that temperature had significant effects on RIM and all enzyme activities in the non-aestivating group ( P < 0.05). However, the significant effects of temperature on the T-AOC, PK and LDH activities were not found in the aestivating group ( P > 0.05). Meanwhile, the RIM and all enzyme activities in the non-aestivating group were significantly higher than those in the aestivating group before September 10. The metabolites including glutamate, 3-aminoisobutyrate, aspartate, choline, phosphocholine, and taurine were abundant in the aestivating group, whereas 12 kinds of amino acids, glucose, glycogen, dimethylamine, inosine, and homarine were abundant in the non-aestivating group. It was obvious that metabolism of the non-aestivating group was more active than that of the aestivating group. This study provided new insights for understanding the differences in intestine degradation, enzyme activities and metabolites between non-aestivating and aestivating A. japonicus under high temperature in summer. Meanwhile, the results might be helpful for high temperature resistant sea cucumber selection in aquaculture. Statement of relevance We state that the work has not been published and is not being considered for published elsewhere. [ABSTRACT FROM AUTHOR]

Abstract Multiple environmental stressors caused by global warming influence the regulation of proteins involved in various biological processes in aquatic organisms. As an important component of the marine ecosystem, sea cucumber is a suitable echinoderm species for researching stress responses. In this study, we have investigated the proteomic response of respiratory trees in the sea cucumber Apostichopus japonicus , challenged with environmental stresses by using the iTRAQ technique. Results showed that 262, 155 and 433 proteins were differentially regulated in response to heat, hypoxia, and heat plus hypoxia, respectively. Expression of key genes and proteins were measured by real-time PCR and western blot to validate the accuracy of the proteome files. Results showed that the interaction between these two stressors has an additive effect at the proteome level. Proteins involved in amino acid and carbohydrate metabolic processes were largely induced by heat while repressed by hypoxia. Exposure to multiple climate change stressors caused several proteins involved in lipid metabolic progress to be down-regulated and lipid catabolic processes were induced. Additionally, hypoxia and the combined stress induced proteins involved in iron homeostasis. ATP synthesis and gluconeogenesis were induced under heat and the combined stress, while ATP and glycogen synthesis were depressed under hypoxia. Proteins related with immune and defense response were largely induced and protein synthesis ability was depressed under all three stresses. Thus, sea cucumber may adopt different strategies to cope with varied environmental stress, and the situation in heat group is more similar with the combined treatment than hypoxia group. These proteomic changes in response to high-temperature and low-oxygen levels may provide insights into the defense strategies of sea cucumber in response to global climate changes. Significance of the study The study focused on comparative quantitative proteomics on Apostichopus japonicus respiratory tree using iTRAQ in responses to multiple climate change stressors: heat, hypoxia and the combined stress. Data showed that A. japonicus can have an acute reaction in diverse biological pathways to both individual and interacting environmental stress, including substance metabolism, signal transduction, protein synthesis, immune response and energy production. Results indicated that sea cucumber adopted different strategies to cope with varied environmental stress, and the interaction between these two stressors has an additive effect at the proteome level. These results offer insight into the molecular regulation of A. japonicus to multiple environmental stress and reveal possible molecular events in sea cucumber under climate changes. The understanding of adaptive variation under global climate changes in aquatic organisms could be improved. Graphical abstract Unlabelled Image Highlights • Comparative proteomic analysis was used to illustrated proteins responded to varied environmental stress in sea cucumber. • 262, 155 and 433 proteins were differentially regulated in response to heat, hypoxia, and heat plus hypoxia, respectively. • RT-qPCR and western blot confirmed key genes and proteins expressions. • The interaction between these two stressors has an additive effect at the proteome level. • Sea cucumber adopted different strategies to cope with varied environmental stress. [ABSTRACT FROM AUTHOR]

Ascorbic acid plays an important role in the growth and immunity of the sea cucumber, Apostichopus japonicus , a valuable commercial species in China, South Korea, and Japan. In this study, we investigated the dietary ascorbic acid requirements of two newly discovered color morphs of A. japonicus , white and purple, and compared them with that of the common green morph. Six test diets were formulated containing 0, 50, 100, 200, 400, and 800 mg ascorbic acid equivalent per kg, as l-ascorbyl-2-polyphosphate. The color morphs were fed the test diets for 60 days, and their growth, energy budget, and immunological performance recorded. Dietary ascorbic acid levels had a significant effect on the growth of A. japonicus ( P < 0.001). The green and purple morphs showed the greatest specific growth rate (SGR), ingestion rate (IR), and food conversion efficiency (FCE) at a dietary ascorbic acid level of 200 mg/kg, while maximum values for the white morph were recorded at 100 mg/kg, indicating a lower ascorbic acid requirement in the white morph than the green and purple morphs. This could be due to the lower growth and metabolic rates recorded in the white morph, whose SGR, IR, and FCE values and immune indicators were lower than the green and the purple morphs. Malonaldehyde (MDA) content of the coelomic fluid was significantly lower when dietary ascorbic acid was ≥200 mg/kg ( P < 0.05). Superoxide dismutase (SOD) and catalase (CAT) activities, and total anti-oxidative capacity (T-AOC) were generally higher at dietary ascorbic acid levels of 100, 200, and 400 mg/kg compared with the other treatments. SOD and CAT activities and T-AOC were significantly and positively correlated with SGR, indicating that supplementary dietary ascorbic acid had a beneficial effect on growth and immunity. SGR, energy intake, and the proportion of energy allocated to growth decreased significantly in the white and purple morphs when ascorbic acid levels reached 800 mg/kg ( P < 0.05); as did those of the green morph, but not to a significant level. Immune indicators decreased and MDA content increased slightly at 800 mg/kg, indicating that high levels of dietary ascorbic acid do not benefit growth or immunity. The SOD and CAT activities were higher in the purple morph than the green and white morphs, indicating its superior immune system capacity. Optimum growth and immunity of A. japonicus were achieved at ∼100 mg/kg of ascorbic acid in the white morph and 200 mg/kg in the green and purple morphs. [ABSTRACT FROM AUTHOR]

Protein is a necessary nutrient for the sea cucumber, Apostichopus japonicus , an important commercial species in China, South Korea and Japan. In this study, the dietary protein requirements of two newly discovered color morphs—white and purple was investigated and compared with the common, green morph. The green, white and purple morphs were fed a diet with five different protein levels (1, 6, 11, 16 and 21%) for 60 days with a mixture of soybean meal and fish meal (1:1) as the protein source, and their growth, energy budget, physiological and immunological performance were measured. Results showed that dietary protein levels had a significant effect on the growth performance of A. japonicus (P < 0.05). The maximum specific growth rate (SGR) and proportion of energy allocated for growth occurred at dietary protein levels of 6 and 11%, and the food conversion efficiency (FCE) was highest at the 11% protein level, indicating that the optimum dietary protein level for these color morphs is at ~ 11%. Apparent digestibility coefficients for dry matter (ADMD) and crude protein (ACPD) increased, but the ingestion rate decreased with increasing dietary protein levels, due perhaps to the decreased sea-mud content of the diets. The energy allocated to excretion, and the free ammonia and malonaldehyde content in coelomic fluid increased with increasing dietary protein levels, which can be explained by enhanced protein metabolism. Trypsin activity decreased significantly when dietary protein was > 16%, indicating that the protein content was beyond the digestive ability of sea cucumber, and that excess protein in the diet inhibits protein digestion. Catalase (CAT) and glutamic–oxaloacetic transaminase activity was positively and significantly correlated with SGR, indicating that rapid growth was supported by a robust immune system and effective protein metabolism. At a 1% dietary protein level, the purple color morph had significantly higher SGR, FCE, ACPD and CAT activity, and allocated more energy for growth than the green morph, indicating that the purple morph may be better adapted to a low protein diet than the green morph. The white morph had lower SGR, FCE, superoxide dismutase (SOD) and CAT activity, and allocated less energy to growth, but had a higher excretion energy and higher free ammonia content in coelomic fluid than the green or purple morphs, indicating a difference or physiological defect in protein metabolism in the white morph. Statement of relevance The results of this study would be invaluable in improving aquaculture technology and management strategies for green, white, and purple color morphs of Apostichopus japonicus . [ABSTRACT FROM AUTHOR]

The sea cucumber Apostichopus japonicus (Selenka) tolerates salinity fluctuations when inhabiting the intertidal zone and culture ponds. In this study, green, white and purple morphs of juvenile A. japonicus (1.0 ± 0.1 g) were cultured at salinities of 22, 26, 30, 34 and 38 for 60 days. The influence of salinity on the growth, energy allocation and physiological performance of A . japonicus was determined. The results indicated that the three color morphs tested could survive within the salinity range of 22–38. Salinity had a significant effect on the growth, energy allocation and physiological performance of sea cucumbers ( P < 0.05). Specific growth rates, ingestion rates and feed conversion efficiencies of A. japonicus were generally optimal at a salinity of 30. Similar results were obtained for energy ingested, and the proportion of energy allocated to growth. Significant changes in enzyme activities of sea cucumbers mainly occurred at salinities of 22 and 38, indicating that metabolism was affected by low and high salinities. Green A. japonicus grew faster than the white and purple color morphs at a salinity of 22. Growth rates were the highest in purple, then green and the lowest in white color morphs at salinities of 30, 34 and 38. At a salinity of 22, the green allocated more energy to growth and respiration. The purple morph allocated more energy to growth and respiration when water salinity reached 34 and 38. At salinities of 30, 34 and 38, the purple morph had higher trypsin and superoxide dismutase activities, which supported the higher specific growth rate and food conversion rates compared with the green and white color morphs. The results clearly showed that the green color morph performed better at low salinities, while the purple was better adapted to high salinities within the tolerance range. [ABSTRACT FROM AUTHOR]

Hsp10 and Hsp60 are important heat shock proteins (HSPs), which might be indispensable in the heat shock response and many other physiological processes. We obtained full-length cDNAs of genes hsp10 and hsp60 and classified their mRNA expression levels under thermal stress in the sea cucumber Apostichopus japonicus. Full-length hsp10 cDNA was 1528bp containing a 5′ untranslated region (UTR) of 83bp, a 3′ UTR of 1133bp and an open reading frame (ORF) of 312bp encoding 103 amino acid residues. Full-length hsp60 cDNA was 2560bp containing a 118-bp 5′ UTR, a 678-bp 3′ UTR and a 1764-bp ORF encoding 586 amino acid residues. The deduced amino acid sequences of Hsp10 and Hsp60 shared the highest identity with sequences of the sea urchin Strongylocentrotus purpuratus, and phylogenetic trees showed that the evolution of Hsp10 and Hsp60 was almost in accord with the evolution of species. Further analysis by real-time PCR showed that the expression of hsp10 and hsp60 mRNA was highly up-regulated at 26°C compared with other three groups (20, 22, 24°C), and their expression in the intestine was in a time-dependent manner at 26°C. The results suggested that hsp10 and hsp60 were involved in the heat-shock response in the sea cucumber A. japonicus. [ABSTRACT FROM AUTHOR]