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The sea cucumber Apostichopus japonicus has important nutritional and medicinal value. Unfortunately, we know little of the source of active chemicals in this animal, but the plentiful pigments of these animals are thought to function in intriguing ways for translation into clinical and food chemistry usage. Here, we found key cell groups with the gene activity predicted for the color morphology of sea cucumber body using single-cell RNA-seq. We refer to these cell populations as melanocytes and quinocytes, which are responsible for the synthesis of melanin and quinone pigments, respectively. We integrated analysis of pigment biochemistry with the transcript profiles to illuminate the molecular mechanisms regulating distinct pigment formation in echinoderms. In concert with the correlated pigment analysis from each color morph, this study expands our understanding of medically important pigment production, as well as the genetic mechanisms for color morphs, and provides deep datasets for exploring advancements in the fields of bioactives and nutraceuticals. [ABSTRACT FROM AUTHOR]

Simple Summary: The sea cucumber Apostichopus japonicus is an ecologically significant echinoderm with considerable nutritional and therapeutic value. Color variation of A. japonicus is one of its most important cultivation traits, as color affects its bioactive compounds, taste, and market price. Microphthalmia-associated transcription factor (MITF) is one of the most critical genes in melanocyte development and melanin synthesis pathways. However, how MITF regulates body color and differentiation in sea cucumbers is poorly understood. Here, we analyzed the expression level and location of MITF in white, purple, and green sea cucumbers and identified the genes regulated by MITF using chromatin immunoprecipitation followed by sequencing. Our findings suggested that MITF contributed to the body color differentiation of green, purple, and white sea cucumbers A. japonicus through expression differences and regulation of downstream genes. These findings give researchers a foundation for further research into the processes that underlie the development of body color in sea cucumbers and provide light on how genes are regulated. The results will also provide support for sea cucumber breeding and a useful theoretical basis in order to contribute to the development of the sea cucumber industry. Melanin, which is a pigment produced in melanocytes, is an important contributor to sea cucumber body color. MITF is one of the most critical genes in melanocyte development and melanin synthesis pathways. However, how MITF regulates body color and differentiation in sea cucumbers is poorly understood. In this study, we analyzed the expression level and location of MITF in white, purple, and green sea cucumbers and identified the genes regulated by MITF using chromatin immunoprecipitation followed by sequencing. The mRNA and protein expression levels of MITF were all highest in purple morphs and lowest in white morphs. In situ hybridization indicated that MITF mRNA were mainly expressed in the epidermis. We also identified 984, 732, and 1191 peaks of MITF binding in green, purple, and white sea cucumbers, which were associated with 727, 557, and 887 genes, respectively. Our findings suggested that MITF contributed to the body color differentiation of green, purple, and white sea cucumbers through expression differences and regulation of downstream genes. These results provided a basis for future studies to determine the mechanisms underlying body color formation and provided insights into gene regulation in sea cucumbers. [ABSTRACT FROM AUTHOR]

Abstract Pigmentation processes provide a traceable and relevant trait for understanding key issues in evolutionary biology such as adaptation, speciation and the maintenance of balanced polymorphisms. The sea cucumber Apostichopus japonicus , which has nutritive and medical properties, is considered the most valuable commercial species in many parts of Asia. Compared with the green morph, the purple morph is rare and has great appeal to consumers. However, little is currently known about the molecular mechanism of body color formation in A. japonicus , even in echinoderm. Here, we employ illumina sequencing to examine expression patterns of the gene network underlying body wall development in purple and green morphs of A. japonicus. Overall, the number of down-regulated genes in the green morph was significantly more than in the purple morph during the pigmentation stage. We observed dynamic expression patterns of a large number of pigment, regulation and growth genes from the "Melanogenesis", "Melanoma", "Wnt signaling pathway", "Notch signaling pathway", "epithelium development", "epidermal growth factor receptor binding","growth factor activity" and "growth", including contrasting expression patterns of these genes in green and purple morph. This study provides comprehensive lists of differentially expressed genes during body wall development in the green and purple morphs, revealing potential candidate genes that may be involved in regulating body color formation and polymorphism. These data will provide valuable information for future genetic studies on sea cucumbers elucidating the molecular mechanisms underlying pigmentation, and may support the culture of desirable color morphs. [ABSTRACT FROM AUTHOR]

Sea cucumbers exposed to stressful circumstances eviscerate most internal organs, and then regenerate them rapidly under favorable environments. Reversible protein phosphorylation and acetylation are major modifications regulating protein function. Herein, for the first time, we perform quantitative phospho- and acetyl proteomics analyses of intestine regeneration in a sea cucumber species Apostichopus japonicus. We identified 1,862 phosphorylation sites in 1,169 proteins, and 712 acetylation sites in 470 proteins. Of the 147 and 251 proteins differentially modified by phosphorylation and acetylation, respectively, most were related to cytoskeleton biogenesis, protein synthesis and modification, signal recognition and transduction, energy production and conversion, or substance transport and metabolism. Phosphorylation appears to play a more important role in signal recognition and transduction than acetylation, while acetylation is of greater importance in posttranslational modification, protein turnover, chaperones; energy production and conversion; amino acid and lipid transport and metabolism. These results expanded our understanding of the regulatory mechanisms of posttranslational modifications in intestine regeneration of sea cucumbers after evisceration. [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]

The sea cucumber Apostichopus japonicus (Selenka), which has nutritive and medical properties, is considered the most valuable commercial marine species in many parts of Asia. Of the different color morphs, the albino sea cucumber is rare and has great appeal to consumers. Identification of factors contributing to albinism in the sea cucumber is therefore likely to provide a scientific basis for improving the cultivability of this morph. In this study, illumina sequencing was performed on three pigmentation stages of the albino sea cucumber: the early pigmentation stage (Wa), the mid pigmentation stage (Wb) and the late pigmentation stage (Wc). In total, 120,365 unigenes were generated with an N50 length of 3360 bp and 60,224 unigenes were annotated from seven functional databases (NR, NT, GO, COG, KEGG, Swissprot and Interpro). In addition, 39,285 SSRs were detected, distributed on 26,156 unigenes. Compared with sea cucumbers during the early pigmentation stage, 842 genes were identified as differentially expressed in the body wall of albino sea cucumbers during the mid-pigmentation stage, including 331 upregulated and 511 downregulated genes. Additionally, compared with sea cucumbers in the mid-pigmentation stage, 247 genes were identified as differentially expressed in the body wall of albino sea cucumbers during the late-pigmentation stage, including 67 upregulated and 180 downregulated genes. GO and KEGG analyses revealed possible differentially expressed genes, including “melanogenesis”, “Ras-signaling pathway”, “cAMP signaling pathway” and “epithelium development” involved in albinism formation in albino sea cucumbers. In conclusion, these studies identified many candidate albinism-related and pigmentation-related genes and signaling pathways. In addition, a comparative analysis was conducted of the differentially expressed genes from albino sea cucumber body walls in different pigmentation stages. The results should be highly useful in further elucidating the mechanisms underlying albinism in sea cucumbers. [ABSTRACT FROM AUTHOR]

The body colour of sea cucumber, Apostichopus japonicus (Selenka), is an important trait affecting the price and taste of its products; the rare white and purple colour types are favoured and are more expensive in China. Identification of factors that may contribute to the differences among white, green and purple sea cucumbers will provide a scientific basis for improving the cultivability of the rare colour morphs. In this study, the growth, histology, ultrastructure and expression of microphthalmia-associated transcription factor ( MITF) and astacin of two newly discovered white and purple colour morphs were investigated and compared with the common green morph, in the same breeding environment. At 50 days old, the growth and survival rates and the extent of pigmentation were the highest in purple, then green colour morphs and were lowest in white morphs. The white morph had fewer, and less developed, epidermal melanocytes compared with the green and purple colour morphs. MITF and astacin expression, which are necessary for the formation of melanin and astaxanthin, respectively, were highest in purple morphs and lowest in white morphs. In addition, there were significant differences in growth rates and expression of MITF and astacin among early, mid and late pigmentation stages of each colour morph. The results suggested that growth and survival rates were highest in purple sea cucumber and lowest in white sea cucumber. Epidermis thickness and melanin content may be the newfound factors contributed to the differences in growth and survival rate among three colour morphs. [ABSTRACT FROM AUTHOR]

Sea cucumbers have a striking capacity to regenerate most of their viscera after evisceration, which has drawn the interest of many researchers. In this study, the isobaric tag for relative and absolute quantitation (iTRAQ) was utilized to investigate protein abundance changes during intestine regeneration in sea cucumbers. A total of 4073 proteins were identified, and 2321 proteins exhibited significantly differential expressions, with 1100 upregulated and 1221 downregulated proteins. Our results suggest that intestine regeneration constitutes a complex life activity regulated by the cooperation of various biological processes, including cytoskeletal changes, extracellular matrix (ECM) remodeling and ECM-receptor interactions, protein synthesis, signal recognition and transduction, energy production and conversion, and substance transport and metabolism. Additionally, real-time PCR showed mRNA expression of differentially expressed genes correlated positively with their protein levels. Our results provided a basis for studying the regulatory mechanisms associated with sea cucumber regeneration. [ABSTRACT FROM AUTHOR]

Simple Summary: Slow growth and germplasm degradation have restricted the sustainable commercial development of the sea cucumber industry. To analyze the genetic mechanism of growth traits of sea cucumbers, we constructed a high-density genetic linkage map based on single nucleotide polymorphism (SNP) molecular markers and performed a quantitative trait loci (QTL) mapping analysis. We annotated a critical candidate gene related to growth traits and explored mRNA expression levels. The results showed that the gene was significantly highly expressed during the larval developmental stages. These results can be used to genetically improve the growth traits of sea cucumbers. Genetic linkage maps have become an indispensable tool for genetics and genomics research. Sea cucumber (Apostichopus japonicus), which is an economically important mariculture species in Asia, is an edible echinoderm with medicinal properties. In this study, the first SNP-based high-density genetic linkage map was constructed by sequencing 132 A. japonicus individuals (2 parents and 130 offspring) according to a genotyping-by-sequencing (GBS) method. The consensus map was 3181.54 cM long, with an average genetic distance of 0.52 cM. A total of 6144 SNPs were assigned to 22 linkage groups (LGs). A Pearson analysis and QTL mapping revealed the correlations among body weight, body length, and papillae number. An important growth-related candidate gene, protein still life, isoforms C/SIF type 2 (sif), was identified in LG18. The gene was significantly highly expressed during the larval developmental stages. Its encoded protein reportedly functions as a guanine nucleotide exchange factor. These results would facilitate the genetic analysis of growth traits and provide valuable genomic resources for the selection and breeding of new varieties of sea cucumbers with excellent production traits. [ABSTRACT FROM AUTHOR]

The sea cucumber Apostichopus japonicus is an important economic species owing to their high nutritive and medicinal value. Body color is one of the most important traits in the cultivation, which affects taste and market price of holothurian products. Pigmentation is an important stage of sea cucumber growth and development, in addition to achieving rare and beautiful coloration. In this study, UHPLC-QTOF/MS technique was performed to analyze the metabolome of white, green and purple A. japonicus body wall during the pigmentation process. A total of 2633 metabolites were identified. OPLS-DA clearly discriminated the body wall metabolites among the three color morphs. In addition, 13 annotated metabolites that could discriminate white, green and purple A. japonicus were screened out. KEGG metabolic pathway analysis revealed that "biosynthesis of unsaturated fatty acids" and "fatty acid biosynthesis" were closely related in the different color morphs. Furthermore, we performed comparative analysis of polysaccharide and saponin among white, green and purple A. japonicus. The results showed that the content of polysaccharide and saponin in purple A. japonicus was the highest, while that in white A. japonicus was the lowest. This study will provide valuable information for future studies on sea cucumber and the molecular mechanism underlying pigmentation and color polymorphism, and may contribute to support the culturing of desirable color morphs. [Display omitted] • UHPLC-QTOF/MS was used to analyze the metabolome of white, green and purple A. japonicus. • A total of 2633 metabolites were identified. • OPLS-DA clearly discriminated the body wall metabolites among the three color morphs. • Metabolites and pathways that contribute to pigmentation of A. japonicus were identified. • The content of polysaccharide and saponin in purple A. japonicus was the highest. [ABSTRACT FROM AUTHOR]

• Differential metabolite and pathway of four A. japonicus varieties were identified. • Fatty acids, phospholipids and sugars were important in differentiating varieties. • Carbohydrate, protein and phospholipid metabolism were conserved among varieties. • Body wall metabolic profiles could be discriminated clearly according to varieties. The sea cucumber, Apostichopus japonicas , is an important economic species with high nutritive value. In recent years, driven by significant market demand, the sea cucumber breeding industry has developed rapidly. Body color and number of papillae are important factors that determine the value and price of sea cucumbers. In this study, metabolite profiling of four sea cucumber varieties (green, white, purple and spiny) was performed using ultra-performance liquid chromatography-quadrupole-time-of-flight-mass spectrometry (UPLC-QTOF-MS) combined with multivariate analysis. Orthogonal partial least-squares discriminant analysis (OPLS-DA) clearly discriminated the body wall metabolites of four sea cucumber varieties. Differential metabolites included fatty acids, phospholipids, and sugars. KEGG metabolic pathway analysis revealed that carbohydrate, protein, and phospholipid metabolism were highly conserved among the varieties. These results provide a comprehensive insight into differences in the metabolite profile of four A. japonicus varieties and a deeper understanding of sea cucumber varieties breeding. [ABSTRACT FROM AUTHOR]

Remodeling of extracellular matrix (ECM) regulated by matrix metalloproteinases (MMPs) is essential for tissue regeneration. In the present study, we used immunohistochemistry (IHC) techniques against ECM components to reveal changes of ECM during intestine regeneration of Apostichopus japonicus . The expression of collagen I and laminin reduced apparently from the eviscerated intestine, while fibronectin exhibited continuous expression in all regeneration stages observed. Meanwhile, we cloned two MMP genes from A. japonicus by RACE PCR. The full-length cDNA of ajMMP-2 like is 2733 bp and contains a predicted open reading frame (ORF) of 1716 bp encoding 572 amino acids. The full-length cDNA of ajMMP-16 like is 2705 bp and contains an ORF of 1452 bp encoding 484 amino acids. The predicted protein sequences of each MMP contain two conserved domains, ZnMc_MMP and HX. Homology and phylogenetic analysis revealed that ajMMP-2 like and ajMMP-16 like share high sequence similarity with MMP-2 and MMP-16 from Strongylocentrotus purpuratus , respectively. Then we investigated spatio-temporal expression of ajMMP-2 like and ajMMP-16 like during different regeneration stages by qRT-PCR and IHC. The expression pattern of them showed a roughly opposite trend from that of ECM components. According to our results, a fibronectin-dominate temporary matrix is created in intestine regeneration, and it might provide structural integrity for matrix and promote cell movement. We also hypothesize that ajMMP-2 like and ajMMP-16 like could accelerate cell migration and regulate interaction between ECM components and growth factors. This work provides new evidence of ECM and MMPs involvement in sea cucumber regeneration. [ABSTRACT FROM AUTHOR]

Wnt genes encode secreted glycoproteins that act as signaling molecules; these molecules direct cell proliferation, migration, differentiation and survival during animal development, maintenance of homeostasis and regeneration. At present, although the regeneration mechanism in Apostichopus japonicus has been studied, there is a little research on the Wnt signaling pathway in A. japonicus . To understand the potential role of the Wnt signaling pathway in A. japonicus , we cloned and sequenced the WntA gene in A. japonicus . Protein localization analysis showed that WntA protein was ubiquitously expressed in epidermal cells, the muscle and submucosa of the intestinal tissue. After stimulation and evisceration, the dynamic changes in expression of the WntA gene and protein showed that WntA was constitutively expressed during different stages of intestine regeneration in A. japonicus , with higher levels during the early wound healing stage and late lumen formation in the residual and nascent intestinal tissues, indicating its response to intestinal regeneration. Simultaneously, cell proliferation and apoptosis analysis showed that the patterns of cell proliferation were similar to the patterns of WntA protein expression during different intestinal regeneration stages in this organism. Taken together, these results suggested that WntA might participate in intestinal regeneration and may be connected with cell proliferation, apoptosis in different intestinal layers. This research could establish a basis for further examination of WntA functions in A. japonicus and Wnt genes in other echinoderms. [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]