A multi-objective optimization approach for green supply chain network design for the sea cucumber (Apostichopus japonicus) industry.

In China, aquatic supply chain network design does not include the green concept or the coordination of environmental and economic performance. Sea cucumber (Apostichopus japonicus) is an aquatic product of high economic value; however, studies on sea cucumber supply chain network optimization are lacking. This study is the first to design the sea cucumber supply chain and construct an optimization model. Considering the characteristics of the sea cucumber industry, LCA for Experts software and the CML-IA-Aug. 2016-world method were used to assess each aquaculture model's global warming potential (GWP), as the environmental performance indicator. In addition, multi-objective genetic algorithm (MOGA) coupled with Modified Technique for Order of Preference by Similarity to Ideal Solution (M-TOPSIS) integrates yield production, economic benefits, and environmental performance. The results demonstrated that cage seed rearing (CSR) combined bottom sowing aquaculture (BSA) represents the best production strategy upstream of the sea cucumber supply chain. In the downstream, the best proportion of sales channels in supermarkets, boutique stores and online shops accounted for 14.79 %, 58.02 % and 27.19 % of the production, respectively. The proposed optimization scenario 4 (S4) can increase product profit by 27.88 % and reduce GWP by 56.89 %. The following improvement measures are proposed: using sea cucumber aquaculture industry standards (cleaner production and green supplier selection) to regulate the behavior of enterprises, adopting an ecological and green production strategy, eliminating high-energy consumption and high emission production practices, and promoting widespread adoption of green consumption concepts. Finally, these measures may improve the sea cucumber supply chain, achieve coordinated environmental and economic performance development in the sea cucumber industry, and provide guidance for green optimization of other aquatic product supply chains in China.

Expression of Piwi, MMP, TIMP, and Sox during Gut Regeneration in Holothurian Eupentacta fraudatrix (Holothuroidea, Dendrochirotida).

Mesodermal cells of holothurian Eupentacta fraudatrix can transdifferentiate into enterocytes during the regeneration of the digestive system. In this study, we investigated the expression of several genes involved in gut regeneration in E. fraudatrix. Moreover, the localization of progenitor cells of coelomocytes, juvenile cells, and their participation in the formation of the luminal epithelium of the digestive tube were studied. It was shown that Piwi-positive cells were not involved in the formation of the luminal epithelium of the digestive tube. Ef-72 kDa type IV collagenase and Ef-MMP16 had an individual expression profile and possibly different functions. The Ef-tensilin3 gene exhibited the highest expression and indicates its potential role in regeneration. Ef-Sox9/10 and Ef-Sox17 in E. fraudatrix may participate in the mechanism of transdifferentiation of coelomic epithelial cells. Their transcripts mark the cells that plunge into the connective tissue of the gut anlage and give rise to enterocytes. Ef-Sox9/10 probably controls the switching of mesodermal cells to the enterocyte phenotype, while Ef-Sox17 may be involved in the regulation of the initial stages of transdifferentiation.

Ecological and reproductive characteristics of holothuroids Isostichopus badionotus and Isostichopus sp. in Colombia.

Isostichopus badionotus and Isostichopus sp. are two holothuroids exploited in the Caribbean region of Colombia. Until recently, they were considered a single species. During one year, 222 individuals of Isostichopus sp. and 114 of I. badionotus were collected in two bays of the Santa Marta region to study their reproductive biology and collect information on their size, weight and habitat. Both sea cucumber morphotypes showed an annual reproductive cycle, with a reproductive season from September to November, closely related to the increase in water temperature and rainfall. In both sea cucumbers the population structure exhibited a unimodal distribution composed of mature individuals and a sex ratio of 1:1. Isostichopus sp. had an average size and weight (193 ± 52 mm and 178 ± 69 g) and size and weight at first maturity (175 mm and 155 g) that was much lower than I. badionotus (respectively, 324 ± 70 mm and 628 ± 179 g; 220 mm and 348 g). While 98% of Isostichopus sp. individuals were collected in the upper 2.5 m, on rocky bottoms between cracks, 73% of I. badionotus individuals were found between 3 and 7.8 m depth, exposed on sandy bottoms. These differences imply that management measures (e.g. minimum catch size) should not be the same for both sea cucumbers morphotypes.

Molecular Aspects of Regeneration Mechanisms in Holothurians.

Holothurians, or sea cucumbers, belong to the phylum Echinodermata. They show good regenerative abilities. The present review provides an analysis of available data on the molecular aspects of regeneration mechanisms in holothurians. The genes and signaling pathways activated during the asexual reproduction and the formation of the anterior and posterior parts of the body, as well as the molecular mechanisms that provide regeneration of the nervous and digestive systems, are considered here. Damage causes a strong stress response, the signs of which are recorded even at late regeneration stages. In holothurian tissues, the concentrations of reactive oxygen species and antioxidant enzymes increase. Furthermore, the cellular and humoral components of the immune system are activated. Extracellular matrix remodeling and Wnt signaling play a major role in the regeneration in holothurians. All available morphological and molecular data show that the dedifferentiation of specialized cells in the remnant of the organ and the epithelial morphogenesis constitute the basis of regeneration in holothurians. However, depending on the type of damage, the mechanisms of regeneration may differ significantly in the spatial organization of regeneration process, the involvement of different cell types, and the depth of reprogramming of their genome (dedifferentiation or transdifferentiation).

A roadmap for intestinal regeneration.

Regeneration of lost or injured organs is an intriguing process in which numerous cellular events take place to form the new structure. Studies of this process during reconstitution of the intestine have been performed in echinoderms, particularly in holothurians. Many cellular events triggered during regeneration have been described using the sea cucumber Holothuria glaberrima as a research model. More recent experiments have targeted the molecular mechanisms behind the process, a task that has been facilitated by the new sequencing technologies now available. In this review, we present studies involving cellular processes and the genes that have been identified to be associated with the early events of gut regeneration. We also present ongoing efforts to perform functional studies necessary to establish the role(s) of the identified genes. A synopsis of the studies is given with the course of the regenerative process established so far.

Genome-Wide DNA Methylation Signatures of Sea Cucumber Apostichopus japonicus during Environmental Induced Aestivation.

Organisms respond to severe environmental changes by entering into hypometabolic states, minimizing their metabolic rates, suspending development and reproduction, and surviving critical ecological changes. They come back to an active lifestyle once the environmental conditions are conducive. Marine invertebrates live in the aquatic environment and adapt to environmental changes in their whole life. Sea cucumbers and sponges are only two recently known types of marine organisms that aestivate in response to temperature change. Sea cucumber has become an excellent model organism for studies of environmentally-induced aestivation by marine invertebrates. DNA methylation, the most widely considered epigenetic marks, has been reported to contribute to phenotypic plasticity in response to environmental stress in aquatic organisms. Most of methylation-related enzymes, including DNA methyltransferases, Methyl-CpG binding domain proteins, and DNA demethylases, were up-regulated during aestivation. We conducted high-resolution whole-genome bisulfite sequencing of the intestine from sea cucumber at non-aestivation and deep-aestivation stages. Further DNA methylation profile analysis was also conducted across the distinct genomic features and entire transcriptional units. A different elevation in methylation level at internal exons was observed with clear demarcation of intron/exon boundaries during transcriptional unit scanning. The lowest methylation level occurs in the first exons, followed by the last exons and the internal exons. A significant increase in non-CpG methylation (CHG and CHH) was observed within the intron and mRNA regions in aestivation groups. A total of 1393 genes were annotated within hypermethylated DMRs (differentially methylated regions), and 749 genes were annotated within hypomethylated DMRs. Differentially methylated genes were enriched in the mRNA surveillance pathway, metabolic pathway, and RNA transport. Then, 24 hypermethylated genes and 15 hypomethylated genes were Retrovirus-related Pol polyprotein from transposon (RPPT) genes. This study provides further understanding of epigenetic control on environmental induced hypometabolism in aquatic organisms.

Human and climatic drivers affect spatial fishing patterns in a multiple-use marine protected area: The Galapagos Marine Reserve.

Assessments of the effectiveness of marine protected areas (MPAs) usually assume that fishing patterns change exclusively due to the implementation of an MPA. This assumption increases the risk of erroneous conclusions in assessing marine zoning, and consequently counter-productive management actions. Accordingly, it is important to understand how fishers respond to a combination of the implementation of no-take zones, and various climatic and human drivers of change. Those adaptive responses could influence the interpretation of assessment of no-take zone effectiveness, yet few studies have examined these aspects. Indeed, such analysis is often unfeasible in developing countries, due to the dominance of data-poor fisheries, which precludes full examination of the social-ecological outcomes of MPAs. In the Galapagos Marine Reserve (Ecuador), however, the availability of long-term spatially explicit fishery monitoring data (1997-2011) for the spiny lobster fishery allows such an analysis. Accordingly, we evaluated how the spatiotemporal allocation of fishing effort in this multiple-use MPA was affected by the interaction of diverse climatic and human drivers, before and after implementation of no-take zones. Geographic information system modelling techniques were used in combination with boosted regression models to identify how these drivers influenced fishers' behavior. Our results show that the boom-and-bust exploitation of the sea cucumber fishery and the global financial crisis 2007-09, rather than no-take zone implementation, were the most important drivers affecting the distribution of fishing effort across the archipelago. Both drivers triggered substantial macro-scale changes in fishing effort dynamics, which in turn altered the micro-scale dynamics of fishing patterns. Fishers' adaptive responses were identified, and their management implications analyzed. This leads to recommendations for more effective marine and fishery management in the Galapagos, based on improved assessment of the effectiveness of no-take zones.

Age determination in echinoderms: first evidence of annual growth rings in holothuroids.

While age is fundamental in animal biology, forming the basis of critical concepts such as life-history strategies, longevity and population structures, measuring this variable in some taxa remains problematic. Such is the case of holothuroid echinoderms, which play key roles in marine benthic communities from the shore to the abyss, and which are extensively fished in many regions across the globe. Here, we present and validate a promising ageing technique using the cold-water species Psolus fabricii. The method involves the extraction of the oldest dermal plates (largest dorsal ossicles) to preserve their original pigments and structure. While plates initially appear to have a uniform texture, polishing and dying them reveals layered ring patterns. A study of laboratory-reared juveniles, from settlement to 40 months of age, confirmed that one layer is added annually, making plates both larger and thicker, and generating successive light and dark rings, the latter representing the transition (overlap) between two layers. Therefore, each pair of rings represents an annual growth band. Size-at-age data obtained using this method revealed that growth of P. fabricii is slow and that wild individuals collected at diving depths had reached an age of several decades.

Transcriptome analysis of body wall reveals growth difference between the largest and smallest individuals in the pure and hybrid populations of Apostichopus japonicus.

Long-term inbreeding of sea cucumber has resulted in a decrease in its growth rate, which has severely affected yield and economic efficiency. In this study, three Apostichopus japonicus families were constructed and screened into the weight of smallest and largest, which included Russian, Chinese, and their hybrids (RC). We examined the transcriptional profiles of hybrid (RC) and purebred (CC and RR). A total of 49.69 Gb clean reads were obtained, and the Q30 base percentage was above 90.47%. A total of 5191 novel genes were discovered, of which 2592 genes were annotated. Differentially expressed genes (DEGs) were identified, and functional annotation and enrichment analysis were performed. Approximately 1874 DEGs were screened in the Chinese sea cucumber (CC) difference group; 2591 DEGs were obtained in the hybrid sea cucumber difference group (RC), and 3006 DEGs were obtained in the Russian sea cucumber difference group (RR). In Gene Ontology (GO) analysis, highest DEG enrichment was observed for the functional categories of cellular process and metabolic process. In terms of cellular components, DEG enrichment was observed in cell part, cell; for molecular function, DEG enrichment was detected in catalytic activity, binding, hydrolase activity, transferase activity. According to the differential expression analysis, we found that 15 heat shock protein (HSP) genes that have the same expression trends, which were upregulated in the smallest weight of three sea cucumber lines. In addition, COG analysis of defense genes was conducted. All defense genes (ATP-binding cassette transporters (ABCs), multidrug resistance protein (MRPs), and beta-lactamase) showed the same expression trend, which was significantly upregulated in smallest individuals compared to that of largest individuals in RC lines, which implied the smallest individuals are exposed to more pressure during growth. These results may lead to the smallest individuals showing slow growth. Additionally, we selected 12 DEGs to validate the result by qPCR. Those DEGs were included in growth-related and resistance genes. Sequencing of the A. japonicus transcriptome improves our understanding of the transcriptional regulatory apparatus that controls individual development and growth.

Densidad poblacional y estructura de talla del pepino de mar Holothuria (Halodeima) grisea (Aspidochirotida: Holothuriidae) en aguas someras del sur del golfo de Morrosquillo, Caribe Colombiano / Density population and size structure of the sea cucumber Holothuria (Halodeima) grisea (Aspidochirotida: Holothuriidae) in shallow waters south of the gulf of Morrosquillo, Colombian Caribbean

Resumen Holothuria grisea, a pesar de no ser considerado un recurso comercial, ha sido objeto de extracciones ilegales en Colombia, hecho que se vuelve más grave debido al vació de información biológica de estos organismos. Por esta razón, se estimó la densidad poblacional y estructura de talla de H. grisea en tres sectores del sur del golfo de Morrosquillo de abril a noviembre 2015. Para ello, se ubicaron mensualmente tres transectos circulares en cada sector de muestreo abarcando un área de 300 m2, donde se contabilizaron y midieron in situ los individuos encontrados. La densidad media de H. grisea en el área estudiada fue 0.4 ind m-2, presentándose los mayores valores en el sector La Ahumadera (1.09 ± 0.11 ind m-2) y los menores valores en el Banco de Arena (0.02 ± 0.004 ind m-2). El análisis de varianza señalo una diferencia significativa en los valores de densidad y talla de H. grisea en los sectores estudiados. La especie presentó una distribución de tallas unimodal, registrándose una talla promedio de 13.5 ± 0.91 cm con una talla mínima de 2.5 y una máxima de 30 cm, en general las tallas intermedias presentaron una mayor representatividad a lo largo de este estudio (77.2 %). Los aspectos biológicos presentados en este documento son de gran importancia para la conservación de esta especie y corresponde a un avance en el conocimiento de la clase Holothuroidea en el Caribe Colombiano.

Abstract Holothuria grisea, despite not being considered a commercial resource has been the subject of illegal extractions in Colombia, a fact that becomes more serious due to the empty of biological information of these organisms. For this reason the density population and size structure of H. grisea were evaluated in three sectors south of the gulf of Morrosquillo from April to November 2015. To this end, three circular transects were located each month in each sampling site covering an area of 300 m2, where individuals were counted and measured in situ. The average density of H. grisea in the study area was 0.4 ind m-2, with the highest values in the La Ahumadera (1.09 ± 0.11 ind m-2) and the lowest values in Banco de Arena (0.02 ± 0.004 ind m-2). The analysis of variance showed a significance in the values of density and size of H. grisea in the studied sectors. The species presented a unimodal distribution of sizes with an average size of 13.5 ± 0.9 cm with a minimum size of 2.5 and a maximum of 30 cm, intermediate sizes generally showed a higher representativeness throughout this study (77.2 %). The biological aspects presented in this paper are of great importance for the conservation of this species and corresponds to an advance in the knowledge of the Holothuroidea class in the Colombian Caribbean. Rev. Biol. Trop. 66(2): 776-787. Epub 2018 June 01.

Transcriptome analysis of the sea cucumber (Apostichopus japonicus) with variation in individual growth.

The sea cucumber (Apostichopus japonicus) is an economically important aquaculture species in China. However, the serious individual growth variation often caused financial losses to farmers and the genetic mechanisms are poorly understood. In the present study, the extensively analysis at the transcriptome level for individual growth variation in sea cucumber was carried out. A total of 118946 unigenes were assembled from 255861 transcripts, with N50 of 1700. Of all unigenes, about 23% were identified with at least one significant match to known databases. In all four pair of comparison, 1840 genes were found to be expressed differently. Global hypometabolism was found to be occurred in the slow growing population, based on which the hypothesis was raised that growth retardation in individual growth variation of sea cucumber is one type of dormancy which is used to be against to adverse circumstances. Besides, the pathways such as ECM-receptor interaction and focal adhesion were enriched in the maintenance of cell and tissue structure and communication. Further, 76645 SSRs, 765242 SNPs and 146886 ins-dels were detected in the current study providing an extensive set of data for future studies of genetic mapping and selective breeding. In summary, these results will provides deep insight into the molecular basis of individual growth variation in marine invertebrates, and be valuable for understanding the physiological differences of growth process.

Molecular ecological network analysis reveals the effects of probiotics and florfenicol on intestinal microbiota homeostasis: An example of sea cucumber.

Animal gut harbors diverse microbes that play crucial roles in the nutrition uptake, metabolism, and the regulation of host immune responses. The intestinal microbiota homeostasis is critical for health but poorly understood. Probiotics Paracoccus marcusii DB11 and Bacillus cereus G19, and antibiotics florfenicol did not significantly impact species richness and the diversity of intestinal microbiota of sea cucumber, in comparison with those in the control group by high-throughput sequencing. Molecular ecological network analysis indicated that P. marcusii DB11 supplementation may lead to sub-module integration and the formation of a large, new sub-module, and enhance species-species interactions and connecter and module hub numbers. B. cereus G19 supplementation decreased sub-module numbers, and increased the number of species-species interactions and module hubs. Sea cucumber treated with florfenicol were shown to have only one connecter and the lowest number of operational taxonomic units (OTUs) and species-species interactions within the ecological network. These results suggested that P. marcusii DB11 or B. cereus G19 may promote intestinal microbiota homeostasis by improving modularity, enhancing species-species interactions and increasing the number of connecters and/or module hubs within the network. In contrast, the use of florfenicol can lead to homeostatic collapse through the deterioration of the ecological network.

IBT-based quantitative proteomics identifies potential regulatory proteins involved in pigmentation of purple sea cucumber, Apostichopus japonicus.

Sea cucumbers are an important economic species and exhibit high yield value among aquaculture animals. Purple sea cucumbers are very rare and beautiful and have stable hereditary patterns. In this study, isobaric tags (IBT) were first used to reveal the molecular mechanism of pigmentation in the body wall of the purple sea cucumber. We analyzed the proteomes of purple sea cucumber in early pigmentation stage (Pa), mid pigmentation stage (Pb) and late pigmentation stage (Pc), resulting in the identification of 5580 proteins, including 1099 differentially expressed proteins in Pb: Pa and 339 differentially expressed proteins in Pc: Pb. GO and KEGG analyses revealed possible differentially expressed proteins, including"melanogenesis", "melanosome", "melanoma", "pigment-biosynthetic process", "Epidermis development", "Ras-signaling pathway", "Wnt-signaling pathway", "response to UV light", and "tyrosine metabolism", involved in pigment synthesis and regulation in purple sea cucumbers. The large number of differentially expressed proteins identified here should be highly useful in further elucidating the mechanisms underlying pigmentation in sea cucumbers. Furthermore, these results may also provide the base for further identification of proteins involved in resistance mechanisms against melanoma, albinism, UV damage, and other diseases in sea cucumbers.

Understanding regulation of microRNAs on intestine regeneration in the sea cucumber Apostichopus japonicus using high-throughput sequencing.

The sea cucumber, as a member of the Echinodermata, has the capacity to restore damaged organs and body parts, which has always been a key scientific issue. MicroRNAs (miRNAs), a class of short noncoding RNAs, play important roles in regulating gene expression. In the present study, we applied high-throughput sequencing to investigate alterations of miRNA expression in regenerative intestine compared to normal intestine. A total of 73 differentially expressed miRNAs were obtained, including 59 up-regulated miRNAs and 14 down-regulated miRNAs. Among these molecules, Aja-miR-1715-5p, Aja-miR-153, Aja-miR-252a, Aja-miR-153-5p, Aja-miR-252b, Aja-miR-2001, Aja-miR-64d-3p, and Aja-miR-252-5p were differentially expressed over 10-fold at 3days post-evisceration (dpe). Notably, real-time PCR revealed that Aja-miR-1715-5p was up-regulated 1390-fold at 3dpe. Moreover, putative target gene co-expression analyses, gene ontology, and pathway analyses suggest that these miRNAs play important roles in specific cellular events (cell proliferation, migration, and apoptosis), metabolic regulation, and energy redistribution. These results will provide a basis for future studies of miRNA regulation in sea cucumber regeneration.

Melanotransferrin: New Homolog Genes and Their Differential Expression during Intestinal Regeneration in the Sea Cucumber Holothuria glaberrima.

Melanotransferrin (MTf) is a protein associated with oncogenetic, developmental, and immune processes which function remains unclear. The MTf gene has been reported in numerous vertebrate and invertebrate species, including echinoderms. We now report the finding of four different MTfs in the transcriptome of the sea cucumber Holothuria glaberrima. Sequence studies and phylogenetic analyses were done to ascertain the similarities among the putative proteins and their relationship with other transferrin family members. The genes were shown to be differentially expressed in various holothurian organs and to respond differently when the animals were challenged with the immune system activator lipopolysaccharide (LPS). Moreover, the four genes were found to be highly overexpressed during the early stages of intestinal regeneration. The finding of four different genes in the holothurian is particularly surprising, because only one MTf gene has been reported in all other animal species sequenced to date. This finding, combined with the increase expression during intestinal regeneration, suggests a new possible function of MTf in organ regenerative processes.

Anterior regeneration after fission in the holothurian Cladolabes schmeltzii (Dendrochirotida: Holothuroidea).

The regeneration of the anterior portion of the body after fission was studied in the holothurian Cladolabes schmeltzii using electron microscopy methods. Following fission, the posterior portion of the digestive tube, cloaca, and respiratory trees remain in the posterior fragment of the body. The regeneration comprises five stages. In the first stage, connective-tissue thickening (an anlage of the aquapharyngeal bulb) occurs on the anterior end between the torn-off ends of the ambulacra. Most of the lost anterior organs developed in the second and third stages. The structures of water-vascular system and nerve ring form through dedifferentiation, proliferation, and migration of cells of the radial water-vascular canals and the radial nerve cords, correspondently. The lost digestive system portion is restored through the formation and merging of two anlagen. The digestive epithelium of the esophagus and pharynx develops from lining cells of microcavities near the central portion of the connective-tissue thickening, which probably migrate from the epidermis. The second gut anlage develops through transformation of the anterior gut remnant portion. The enterocytes partly dedifferentiate, but the epithelium retains integrity. The gut anlage grows down the mesentery and joins the regenerating aquapharyngeal bulb. In the fourth and fifth stages, all lost organs are formed and have nearly normal structure. The regeneration was concluded to occur through morphallactic rearrangements of the remaining parts of organs. Epithelial morphogenesis is the key development mechanism of the digestive, water-vascular, and nervous systems.

A new genus and a new species in the sea cucumber subfamily Colochirinae (Echinodermata: Holothuroidea: Dendrochirotida: Cucumariidae) in the Mediterranean Sea.

A new genus Hemiocnus is here erected to accommodate the Mediterranean dendrochirotid sea cucumber Cladodactyla syracusana Grube, currently classified, with some doubt, in the cucumariid genus Pseudocnella. At the same time a new cucumariid species, Hemiocnus rubrobrunneus, is described from some Tunisian material, misidentified as Pseudocnella syracusana (Grube), received from the United States National Museum. The new genus appears most closely related to Pseudocnella than to any other genus within the Colochirinae. Although its body wall ossicles resemble those of Pseudocnella spp. it differs in that the two ventral-most tentacles are reduced and in the presence of rosettes in the tentacles. P. syracusana also cannot be classified in Ocnus because of the presence of multi-layered, fir-cone shaped plates in the body wall, often with one end denticulate; such ossicles are lacking in the type species of the latter genus. The new species, Hemiocnus rubrobrunneus, on the other hand, shows some resemblance to H. syracusanus in its characteristic buttons and incomplete baskets, differing in its softer body wall, lack of fir-cone-shaped plates and in the presence of rosettes and complete baskets in the body wall. There are also some resemblances of the new species to the Mediterranean species of Ocnus viz. O. brunneus, O. planci and O. lacteus, but the soft nature of the body wall, shallow quadrilocular instead of deep trilocular baskets, and the presence of large knobbed plates in the anal region precludes its inclusion in this genus.

Transcriptome analysis of tube foot and large scale marker discovery in sea cucumber, Apostichopus japonicus.

Tube foot as one of the ambulacral appendages types in Aspidochirote holothurioids, is known for their functions in locomotion, feeding, chemoreception, light sensitivity and respiration. In this study, we explored the characteristic of transcriptome in the tube foot of sea cucumber (Apostichopus japonicus). Our results showed that among 390 unigenes which specifically expressed in the tube foot, 190 of them were annotated. Based on the assembly transcriptome, we found 219,860 SNPs from 34,749 unigenes, 97,683, 53,624, 27,767 and 40,786 were located in CDSs, 5'-UTRs, 3'-UTRs and non-CDS separately. Furthermore, 12,114 SSRs were detected from 7394 unigenes. Target genes of four specifically expressed miRNAs (miR-29a, miR-29b, miR-278-3p and miR-2005) in tube foot were also predicted based on the transcriptome, which contain immune-related factors (MBL, VLRA, AjC3, MyD88, CFB), skin pigmentation (MITF), candidate regeneration factor (TRP) and holothurians autolysis-related factor (CL). These results develop a relatively large number of molecular markers and transcriptome resources, and will provide a foundation for further analyses on the function and molecular mechanisms underlying A. japonicas tube foot.

Understanding mechanism of sea cucumber Apostichopus japonicus aestivation: Insights from TMT-based proteomic study.

Marine invertebrate aestivation is a unique strategy for summer survival in response to hot marine conditions. The sea cucumber, Apostichopus japonicus, is an excellent model marine invertebrate for studies of environmentally-induced aestivation. In the present study, we used a tandem mass tag (TMT)-coupled LC-MS/MS approach to identify and quantify the global proteome expression profile over the aestivation-arousal cycle of A. japonicus. A total of 3920 proteins were identified from the intestine of sea cucumber. Among them, 630 proteins showed significant differential expression when comparing three conditions of sea cucumbers: non-aestivating (active), deep-aestivation (at least 15days of continuous aestivation), and arousal after aestivation (renewed moving and feeding). Sea cucumbers in deep aestivation showed substantial differentially expressed proteins (143 up-regulated and 267 down-regulated proteins compared with non-aestivating controls). These differentially expressed proteins suggested that protein and phospholipid probably are major fuel sources during hypometabolism and a general attenuation of carbohydrate metabolism was observed during deep aestivation. Differentially expressed proteins also provided the first global picture of a shift in protein synthesis, protein folding, DNA binding, apoptosis, cellular transport and signaling, and cytoskeletal proteins during deep aestivation in sea cucumbers. A comparison of arousal from aestivation with deep aestivation, revealed a general reversal of the changes that occurred in aestivation for most proteins. Western blot detection further validated the significant up-regulation of HSP70 and down-regulation of methyltransferase-like protein 7A-like in deep-aestivation. Our results suggest that there is substantial post-transcriptional regulation of proteins during the aestivation-arousal cycle in sea cucumbers.

Effects of dietary nucleotides on growth, non-specific immune response and disease resistance of sea cucumber Apostichopus japonicas.

A 9-week feeding trial was conducted to investigate the effects of dietary nucleotides (NT) on growth, immune response and disease resistance of sea cucumber Apostichopus japonicas (initial weight: 5.87 ± 0.03 g). Four graded levels of dietary NT were designed as 0, 150, 375 and 700 mg/kg, respectively. After the feeding trial, sea cucumbers were challenged with Vibrio splendidus for the determination of disease resistance. The results showed that the specific growth rates were significantly higher in sea cucumber fed the diet with 375 mg/kg NT than those fed the basal diet without NT supplementation (P < 0.05). The highest total coelomocytes counts in coelomic fluid were found in the treatment with 150 mg/kg of dietary NT (P < 0.05). Compared to those fed with the basal diet, sea cucumber fed diets with nucleotides (≥ 375 mg/kg) had significantly higher phagocytic activities in coelomic fluid (P < 0.05). Respiratory burst activities in coelomic fluid significantly increased with increasing dietary NT supplementations up to 700 mg/kg (P < 0.05). No significant differences in the activities of superoxide dismutase, total nitric oxide synthase and acid phosphatase in coelomic fluid were found among all the treatments (P > 0.05). After being challenged with V. splendidus, the cumulative mortalities of sea cucumber fed diets with 150 and 375 mg/kg NT were significantly lower than that in the treatment without dietary nucleotide supplementation (P < 0.05). Under the experimental conditions, the present results confirmed that a diet supplemented with 375 mg/kg NT is able to enhance both non-specific immune response and growth of sea cucumber in vivo. In conclusion, it was showed that dietary NT does increase the growth performance, non-specific immunity and disease resistance of sea cucumber. The optimum dietary NT supplementation level for sea cucumber was found to be 375 mg/kg. The application of dietary NT may present a novel strategy for health management in sea cucumber's aquaculture.