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Functional food such as, quinoa, coix seed, wild rice and chickpea have experienced rapidly increasing demand globally and exhibit high economic values. Nevertheless, a method for rapid yet accurate detection of these source components is absent, making it difficult to identify commercially available food with labels indicating the presence of relevant components. In this study, we constructed a real-time quantitative polymerase chain reaction (qPCR) method for rapid detection of quinoa, coix seed, wild rice and chickpea in food to identify the authenticity of such food. Specific primers and probes were designed with 2S albumin genes of quinoa, SAD genes of coix seed, ITS genes of wild rice and CIA-2 genes of chickpea as the target genes. The qPCR method could specifically identify the four wild rice strains, yielding, LODs of 0.96, 1.14, 1.04 and 0.97 pg/µL quinoa, coix seed, wild rice and chickpea source components, respectively. Particularly, the method allowed the identification of the target component with content below 0.01%. A total of 24 commercially available food samples of different types were detected by using the method and the results indicate that the developed method is applicable to the detection of different food matrices, as well as authenticity verification in deeply processed food. [ABSTRACT FROM AUTHOR]

Sea cucumber Apostichopus japonicus is an important economic species in echinoderms; however, its culture frequently suffered from diseases. Environmental stress was found to be a dominant inducer of A. japonicus diseases, and thus, discovering the characteristics of immune system under environmental stress is of great significance for disease control in A. japonicus culture. Phenoloxidase (PO) is recognized as a crucial immune‐related enzyme in invertebrates. In order to preliminarily illustrate the different roles that A. japonicus PO isoforms play under environmental stresses, the transcriptional expression of three identified A. japonicus PO isoforms (named as AjPOI, AjPOII and AjPOIII respectively) after different in vivo environmental stress simulations was determined with quantitative real‐time PCR method. Meanwhile, the transcriptional expression analysis of other representational immune‐related factors under environmental stresses was also performed for the evaluation of relationships between AjPOs and other immune‐related factors. The correlations between AjPOs and other immune‐related factors were analysed using Pearson correlation coefficient, and the effects of AjPOs on other immune‐related factors were then tested by dsRNA interference method. The results of transcriptional expression analysis indicated that AjPOI was involved in the resistance to low salinity, low temperature and hypoxia; AjPOII and AjPOIII were both required for the resistance to low salinity, high temperature, low temperature and hypoxia; and all three AjPOs were excluded from the response against high salinity. In addition, three AjPOs were found to take part in different periods of resistance to environmental stresses, and this property made A. japonicus PO system capable of outputting resistance activities continuously. The correlation analysis combined with dsRNA interference test revealed that AjPOI was capable of regulating AjCTSD, AjI‐LYZ and AjMTF expression; AjPOII and AjPOIII both played important roles in the regulation of AjC‐LYZ expression; and AjPOIII was also able to regulate AjToll expression. These findings suggested that A. japonicus PO isoforms function in innate immunity not only via catalysing the oxidative reactions, but also by broadly regulating the expression of other immune‐related factors. On the whole, three AjPOs showed obvious differences among each other in the resistance to environmental stresses and the regulation of other immune‐related factors, but comparatively, AjPOII and AjPOIII shared more similarity. [ABSTRACT FROM AUTHOR]

The immune-related enzymes in marine animals are very sensitive to divalent metal ions. To investigate the roles divalent metal ions play in the influence on the immunity of sea cucumber Apostichopus japonicus, one of the most important commercial species in Asian countries, the effects of eight divalent metal ions at concentrations of 2.5, 5, 10, 15, 20, 25 and 30 mmol L−1 on the activities of superoxide dismutase (SOD), phenoloxidase (PO), acid phosphatase (ACP), alkaline phosphatase (AKP) and myeloperoxidase (MPO) in coelomic fluid were determined with the nitro blue tetrazolium chloride (NBT) method, dopachrome formation method, p-nitrophenyl phosphate ( pNPP) method and 3,3′,5,5′-tetramethyl benzidine (TMB) method. The results indicated that Mg2+ enhanced the activities of SOD, PO, ACP and AKP significantly and showed no obvious effect on MPO activity; Zn2+ increased the activities of SOD, ACP and AKP, and showed no obvious effect on the activities of PO and MPO; Cu2+ enhanced the activities of ACP, AKP and MPO and activated SOD and PO at a certain concentration range; Ca2+ and Mn2+ inhibited the activities of ACP and AKP; Fe2+ had strong inhibitory effect on SOD activity; Pb2+ showed inhibitions on the activities of SOD, PO, ACP and AKP; and Cd2+ inhibited MPO activity greatly. The data obtained in this study collectively suggest that Mg2+, Zn2+ and Cu2+ have potential in promotion of A. japonicus immunity, while Ca2+, Fe2+, Pb2+, Cd2+ and Mn2+ might be limiting factors to the immune response of A. japonicus. [ABSTRACT FROM AUTHOR]

Sea urchins have long been used as research model organisms for developmental biology and evolutionary studies. Some of them are also important aquaculture species in East Asia. In this work, we report the construction of RAD-tag based high-density genetic maps by genotyping F1 interspecific hybrids derived from a crossing between a female sea urchin Strongylocentrotus nudus and a male Strongylocentrotus intermedius. With polymorphisms present in these two wild individuals, we constructed a female meiotic map containing 3,080 markers for S. nudus, and a male meiotic map for S. intermedius which contains 1,577 markers. Using the linkage maps, we were able to anchor a total of 1,591 scaffolds (495.9 Mb) accounting for 60.8% of the genome assembly of Strongylocentrotus purpuratus. A genome-wide scan resulted in the identification of one putative QTL for body size which spanned from 25.3 cM to 30.3 cM. This study showed the efficiency of RAD-Seq based high-density genetic map construction using F1 progenies for species with no prior genomic information. The genetic maps are essential for QTL mapping and are useful as framework to order and orientate contiguous scaffolds from sea urchin genome assembly. The integration of the genetic map with genome assembly would provide an unprecedented opportunity to conduct QTL analysis, comparative genomics, and population genetics studies. [ABSTRACT FROM AUTHOR]

DNA methylation is an important epigenetic modification that regulates gene expression in many biological processes, including immune response. In this study, whole-genome bisulfite sequencing (WGBS) was carried out on healthy body wall (HB) and skin ulceration syndrome (SUS) infected body wall (SFB) to gain insights into the epigenetic regulatory mechanism in sea cucumber Apostichopus japonicus. After comparison, a total of 116,522 differentially methylated regions (DMRs) were obtained including 67,269 hyper-methylated and 49,253 hypo-methylated DMRs (p < 0.05, FDR < 0.001). GO enrichment analysis indicated that regulation of DNA-templated transcription (GO: 0006355), where DNA methylation occurred, was the most significant term in the biology process. The integration of methylome and transcriptome analysis revealed that 10,499 DMRs were negatively correlated with 496 differentially expressed genes (DEGs). Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis showed that these DEGs were enriched in the phosphoinositide 3-kinase-protein kinase B (PI3K/Akt)/mammalian target of rapamycin (mTOR) signaling pathway. Interestingly, two serine/threonine-protein kinases, nemo-like kinase (NLK) and mTOR, were highlighted after functional analysis. The variations of methylation in these two genes were associated with SUS infection and immune regulation. They regulated gene expression at different levels and showed interaction during response process. The validation of methylation sites showed high consistency between pyrosequencing and WGBS. WGBS analysis not only revealed the changes of DNA methylation, but also presented important information about the regulation of key genes after SUS infection in A. japonicus. • WGBS were conducted on the healthy and SUS-infected body wall of Apostichopus japonicus. • A total of 116,522 DMRs were obtained including 67,269 hyper-methylated and 49,253 hypo-methylated DMRs. • The integration of methylome and transcriptome revealed that 10,499 DMRs had a negative correlation with 496 DEGs. • The variations of methylation in mTOR and NLK were associated with SUS infection and immune regulation. [ABSTRACT FROM AUTHOR]

This work details the usage of EFIRM® (Electric Field Induced Release and Measurement) for PCR-free rapid electrochemical detection of mitochondrial DNA. EFIRM® was able to perform highly sensitive detection of animal species for meat contamination testing without multistep sample lysis, DNA extraction, or PCR amplification steps, demonstrating the capability to detect the presence of foreign meat species that only constituted 0.1% of the total mass of a food sample (achieving sensitivity equivalent to that of PCR). The EFIRM® strategy utilizes surface immobilized nucleic acid probes that complement to mitochondrial sequence of Ovis Aries, Sus Scrofa, and Bos Taurus and are immobilized in a polypyrrole matrix on a 96-electrode array. Quantification was performed through amperometric measurement of oxidation-reduction reactions on a streptavidin-peroxidase enzyme chain that completes the nucleic acid complex. All electrochemical procedures were performed using a high-throughput potentiostat system that allows parallelized electrochemical measurement and interfacing to the 96-electrode array. Image 1 • A novel electrochemical DNA biosensor method for detection of mitochondrial DNA was developed for identifying animal species. • This analysis method is simple, PCR-free, and only requires sample immersion in a 2% SDS solution. • Assay detection was rapid, requiring less than 2 hrs and as little as 40 mg of sample. [ABSTRACT FROM AUTHOR]

MicroRNAs (miRNAs) are small non-coding RNAs that mediate mRNA degradation or translation repression. Previous study showed that the expression of miRNAs was significantly changed in the body wall of sea cucumber Apostichopus japonicus after skin ulceration syndrome (SUS) infection, which is a dynamic process. However, the critical miRNAs from body wall that involved in different infection stages of SUS remain unknown. In this study, four cDNA libraries were constructed with the body wall from healthy and three SUS-infected stages of A. japonicus . A total of 248 conserved miRNAs and five novel miRNAs were identified through Illumina HiSeq 2000 platform. Compared to the control, 238 miRNAs showed significant differential expression at three stages of SUS progression. Totally, 3149 miRNA-mRNA pairs were identified by target prediction and 314 miRNA-mRNA pairs showed negative correlation. It is noteworthy that 15 miRNAs and four mRNAs were located at the crucial positions of the network built with the anti-correlated miRNA-mRNA pairs. GO and KEGG enrichment analysis indicated that the predicted targets were involved in many immune-related processes. Deep analysis of miR-31c-5p, miR-29b-3p, NF-kB, mucin 2 and titin showed that they may play important roles in the pathogens attachment and recognition, signaling transduction and lesions repair of A. japonicus after SUS infection. These results would be useful for further investigating the potential roles of critical miRNAs and mRNAs in A. japonicus immune regulation. [ABSTRACT FROM AUTHOR]

In order to preliminarily illustrate the functional differences of phenoloxidases (POs) in Apostichopus japonicus , the full-length cDNAs of two POs (named as AjPOⅡ and AjPOⅢ, respectively) were cloned from the coelomocytes of A. japonicus using 3′- and 5′-rapid amplification of cDNA ends method, and combined with the previously acquired full-length cDNA of a laccase-type PO from A. japonicus (Accession No. KF040052 , named as AjPOⅠ), the sequence structure and phylogenic status of POs from A. japonicus (AjPOs) were comparatively analyzed, and the transcriptional expression of AjPOs in different tissues, at different developmental stages and after different bacterial challenges was determined with quantitative real-time PCR method. Sequence analysis indicated AjPOⅡ and AjPOⅢ were both laccase-type POs, coincident to the results of phylogenic analysis. Sequence analysis also showed that AjPOⅠ had a transmembrane domain (J. Jiang et al., 2014), AjPOⅡ contained a signal peptide, and AjPOⅢ possessed a signal peptide and a transmembrane domain, implying that three AjPOs might play different roles in immune and physiological processes. Transcriptional expression analysis showed that AjPOⅡ and AjPOⅢ were most abundant in tube feet, while AjPOⅠhad the highest expression level in coelomocytes (J. Jiang et al., 2014), suggesting that AjPOⅠ may be mainly involved in immune response, while AjPOⅡ and AjPOⅢ are probably responsible for other physiological processes in addition to immune response. Besides, three AjPOs were determined to have different expression patterns during organism development and different spectrums of response against bacteria, which further indicated that there might be immune and physiological functional differentiation among three AjPOs. [ABSTRACT FROM AUTHOR]

In order to preliminarily understand the immune difference between females and males in the sea cucumber Apostichopus japonicus , the activities assay of superoxide dismutase (SOD), catalase (CAT), peroxidase (POD), phenoloxidase (PO), acid phosphatase (ACP) and alkaline phosphatase (ALP) with biochemical methods, the detection of PO isozymes with native-PAGE and catechol staining, and the test of antibacterial activities with bacterial growth curve determination method were performed in this study using cell-free coelomic fluid (CCF) and coelomocyte lysate supernatant (CLS) from females and males as the samples. The PO activities were not detected in the CLS and showed no significant difference between the CCF from females and males. However, totally five PO isozyme bands were detected in the CLS of females while only four were detected in the CLS of males after zymogram analysis. These results implied that the PO isozymes in the coelomocytes of viripotent A. japonicus were inactive under natural condition and may be activated by some certain treatments during native-PAGE, and PO might play different immune and physiological roles between females and males. In addition, the activities of SOD, CAT, POD and ALP in the CCF and the activities of CAT, POD, ACP and ALP in the CLS from males were all significantly higher than those from females. The results collectively suggested that in viripotent A. japonicus , the gender had a remarkable effect on the immunity, and the immunocompetence of males might have an advantage over that of females. Furthermore, the activities of all determined enzymes except PO and the number of detected PO isozymes showed higher values in CLS than in CCF, implying that in viripotent A. japonicus , the coelomocytes might take more immune responsibility in comparison with CCF. [ABSTRACT FROM AUTHOR]

Myostatin (MSTN), also referred to as growth and differentiation factor-8 (GDF-8), is a member of the transforming growth factor-β superfamily (TGF-β) and an important negative regulator for skeletal muscle development and growth in vertebrates. However, its function is not clear in invertebrates. In this study, we cloned and analyzed the MSTN gene (Aj-MSTN) from sea cucumber ( Apostichopus japonicus ). The full-length cDNA sequence of Aj-MSTN gene was composed of 2912 bp, which contained a 5′ UTR of 487 bp, an ORF of 1356 bp encoding 452 amino acids and a 3′ UTR of 1069 bp. The structure of Aj-MSTN included a putative signal peptide, a TGF-β propeptide domain and a conserved TGF-β domain. Phylogenetic analysis showed that the Aj-MSTN gene was clustered in the same subgroup with the MSTN-like gene found in Strongylocentrotus purpuratus . Quantitative real-time PCR detection results indicated that the Aj-MSTN gene expressed widely in adult tissues and the highest expression level was observed in the body wall. At different developmental stages, the expression levels were increased significantly at early auricularia and doliolaria stages, and reached the peak at juvenile stage. Six SNPs were identified in 5′ flanking region and exons of the Aj-MSTN gene. Association analysis showed that SNP-1, SNP-2 and SNP-4 had significant effects on dry body weight. The results suggested that Aj-MSTN gene could be used as a candidate gene for the selective breeding of A. japonicus . [ABSTRACT FROM AUTHOR]

MicroRNAs (miRNAs) are small non-coding RNAs that are involved in many biological processes. To investigate the miRNAs related to skin ulceration syndrome (SUS) of Apostichopus japonicus , small RNA libraries of body wall, intestine, respiratory tree and coelomocytes from healthy and diseased A. japonicus were sequenced on Illumina Hiseq 2000 platform. A total of 247 conserved and 10 novel miRNAs were identified across all libraries. After pair-wise comparisons, 215 miRNAs in body wall, 36 in intestine, 2 in respiratory tree and 38 in coelomocytes showed significant expression differences. Further analyses were conducted on some tissue-specific differentially expressed miRNAs: miR-8 and miR-486-5p in body wall, miR-200-3p, let-7-5p and miR-125 in intestine, miR-278a-3p and bantam in respiratory, miR-10a and miR-184 in coelomocytes. Notably, these miRNAs in some species were reported to function in various physiological or pathological processes associated with immune regulations. Using stem-loop quantitative real time PCR, six representative miRNAs in four tissues were selected to validate the sequencing results. The Pearson's correlation coefficient ( R ) of the six miRNAs ranged from 0.777 to 0.948, which confirmed the consistency and accuracy between these two approaches. This study provides comprehensive expression and regulation patterns of functional miRNAs in different tissues and gives insights into the tissue-specific immune response mechanisms in SUS-infected A. japonicus . [ABSTRACT FROM AUTHOR]