Chemical Comparison and Identification of Xanthine Oxidase Inhibitors of Dioscoreae Hypoglaucae Rhizoma and Dioscoreae Spongiosae Rhizoma by Chemometric Analysis and Spectrum-Effect Relationship.

Dioscoreae hypoglaucae Rhizoma (DH) and Dioscoreae spongiosae Rhizoma (DS) are two similar Chinese herbal medicines derived from the Dioscorea family. DH and DS have been used as medicines in China and other Asian countries for a long time, but study on their phytochemicals and bioactive composition is limited. This present study aimed to compare the chemical compositions of DH and DS, and explore the anti-xanthine oxidase components based on chemometric analysis and spectrum-effect relationship. Firstly, an HPLC method was used to establish the chemical fingerprints of DH and DS samples, and nine common peaks were selected. Then, hierarchical clustering analysis, principal component analysis and orthogonal partial least squares discriminant analysis were employed to compare and discriminate DH and DS samples based on the fingerprints data, and four steroidal saponins compounds (protodioscin, protogracillin, dioscin, gracillin) could be chemical markers responsible for the differences between DH and DS. Meanwhile, the anti-xanthine oxidase activities of these two herbal medicines were evaluated by xanthine oxidase inhibitory assay in vitro. Pearson correlation analysis and partial least squares regression analysis were subsequently used to investigate the spectrum-effect relationship between chemical fingerprints and xanthine oxidase inhibitory activities. The results showed that four steroidal saponins, including protodioscin, protogracillin, methyl protodioscin and pseudoprogracillin could be potential anti-xanthine oxidase compounds in DH and DS. Furthermore, the xanthine oxidase inhibitory activities of the four selected inhibitors were validated by anti-xanthine oxidase inhibitory assessment and molecular docking experiments. The present work provided evidence for understanding of the chemical differences and the discovery of the anti-xanthine oxidase constituent of DH and DS, which could be useful for quality evaluation and bioactive components screening of these two herbal medicines.

Identification and functional analysis of Dmrt1 gene and the SoxE gene in the sexual development of sea cucumber, Apostichopus japonicus.

Members of the Doublesex and Mab-3-related transcription factor (Dmrt) gene family handle various vital functions in several biological processes, including sex determination/differentiation and gonad development. Dmrt1 and Sox9 (SoxE in invertebrates) exhibit a very conserved interaction function during testis formation in vertebrates. However, the dynamic expression pattern and functional roles of the Dmrt gene family and SoxE have not yet been identified in any echinoderm species. Herein, five members of the Dmrt gene family (Dmrt1, 2, 3a, 3b and 5) and the ancestor SoxE gene were identified from the genome of Apostichopus japonicus. Expression studies of Dmrt family genes and SoxE in different tissues of adult males and females revealed different expression patterns of each gene. Transcription of Dmrt2, Dmrt3a and Dmrt3b was higher expressed in the tube feet and coelomocytes instead of in gonadal tissues. The expression of Dmrt1 was found to be sustained throughout spermatogenesis. Knocking-down of Dmrt1 by means of RNA interference (RNAi) led to the downregulation of SoxE and upregulation of the ovarian regulator foxl2 in the testes. This indicates that Dmrt1 may be a positive regulator of SoxE and may play a role in the development of the testes in the sea cucumber. The expression level of SoxE was higher in the ovaries than in the testes, and knocking down of SoxE by RNAi reduced SoxE and Dmrt1 expression but conversely increased the expression of foxl2 in the testes. In summary, this study indicates that Dmrt1 and SoxE are indispensable for testicular differentiation, and SoxE might play a functional role during ovary differentiation in the sea cucumber.

Sexually dimorphic expression of foxl2 in the sea urchin (Mesocentrotus nudus).

Sea urchin (Mesocentrotus nudus) is an important economically mariculture species in several Asian countries, and gonads are the sole edible parts for people. In addition to commercial value, it is an excellent model for studying gonadal development, sex determination and sex differentiation. Identify sex-related genes is an effective way to reveal the molecular mechanism of gonadal development. In the present study, the foxl2 homologous gene was identified in M. nudus. Foxl2 is not a maternal factor, and is detected for the first time in two-arm stages. Additionally, the expression of foxl2 in the testis is higher than in the ovaries at the same developmental stages. The foxl2 transcripts were specifically enriched in the cytoplasm of germ cellsboth in the ovary and testis, but their proteins were more concentrated in the area near the oocyte nucleus. Overall, this study contributes to our understanding of the dynamic and sexually dimorphic expression pattern of foxl2 and provide a useful germ cell marker during gametogenesis in sea urchin.

Chemical comparison and discrimination of two plant sources of Angelicae dahuricae Radix, Angelica dahurica and Angelica dahurica var. formosana, by HPLC-Q/TOF-MS and quantitative analysis of multiple components by a single marker.

INTRODUCTION: Angelica dahurica(BZ) and Angelica dahurica var. formosana(HBZ) are two plant sources of Angelicae dahuricae Radix. Although BZ and HBZ are commonly used herbal medicines with great medicinal and dietary values, study on their phytochemicals and bioactive compositions is limited. OBJECTIVE: To compare the chemical compositions of BZ and HBZ and find the chemical makers for discrimination and quality evaluation of the two botanical origins of Angelicae dahuricae Radix. METHODOLOGY: A high-performance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry method was established for chemical profiling of BZ and HBZ. Then, a quantitative analysis of multiple components by a single marker method was developed for simultaneous determination of nine bioactive coumarins (xanthotoxol, oxypeucedanin hydrate, byakangelicin, xanthotoxin, bergapten, oxypeucedanin, phellopterin, imperatorin and isoimperatorin). Moreover, chemometrics were performed to compare and discriminate BZ and HBZ samples. RESULTS: A total of 30 coumarins compounds were identified, and the chemical compositions in BZ and HBZ were quite similar. The quantitative analysis showed that there were significant differences in the contents of bioactive coumarins, and the chemometric analysis indicated five coumarins (xanthotoxol, xanthotoxin, bergapten, phellopterin and isoimperatorin) were responsible for the significant differences between BZ and HBZ, which could be used as chemical markers to distinguish the two original plant sources of Angelicae dahuricae Radix. CONCLUSION: The present work provided useful information for understanding the chemical differences between BZ and HBZ and also provided feasible methods for quality evaluation and discrimination of herbal medicines originating from multiple botanical sources.

Identification and functional analysis of foxl2 and nodal in sea cucumber, Apostichopus japonicus.

Sea cucumber (Apostichopus japonicus) is an important mariculture species in China. To date, the mechanisms of sex determination and differentiation in sea cucumber remain unclear. Identifying sex-specific molecular markers is an effective method for revealing the genetic basis of sex determination and sex differentiation. In this study, foxl2 and nodal homologous genes were identified in A. japonicus. Foxl2 exhibited dynamic and sexually dimorphic expression patterns in the gonads, with prominent expression in the ovaries and minimal expression in the testis according to real-time quantitative PCR (RT-qPCR) study. As nodal was specifically expressed in the ovary, it could serve as an ovary-specific marker in sea cucumber. Additionally, knockdown of foxl2 or nodal using RNA interference (RNAi) led to the down-regulation of piwi, germ cell-less, and dmrt1, suggesting that foxl2 and nodal may play important roles in gonad maintenance of sea cucumber. Overall, this study adds to our understanding of the roles of foxl2 and nodal in the gonadal development of A. japonicus, which provides further insight into the mechanisms of sea cucumber sex determination and differentiation.

[Dynamic changes of volatile components in Forsythia suspensa at different harvest periods based on GC-MS and chemometrics analysis].

Forsythiae Fructus is the dried fruit of Forsythia suspensa and the volatile compounds are its main bioactive components. According to the different harvest periods, F. suspensa can be divided into Qingqiao(mature F. suspensa) and Laoqiao(ripe F. suspensa). To investigate dynamic changes of volatile components in Qingqiao and Laoqiao samples collected at different periods, the present study extracted and analyzed the total volatile oils in Qingqiao and Laoqiao samples(four harvest periods for Qingqiao and two for Laoqiao) by steam distillation method. The results indicated that the content of volatile oils in F. suspensa samples at different harvest periods was significantly different. The content of volatile oils in Qingqiao samples(except those harvested in the first period) was higher than that of Laoqiao, and the content of volatile oils in both Qingqiao and Laoqiao increased with the harvest period. Furthermore, volatile compounds in F. suspensa were qualitatively analyzed by the gas chromatography-mass spectrometry(GC-MS), and 28 volatile compounds were identified. Chemometrics analyses including principal component analysis(PCA) and partial least squares discriminant analysis(PLS-DA) were further applied to explore differential markers and dynamic changes of volatile components in Qingqiao and Laoqiao samples at different harvest periods. Finally, four volatile compounds, including α-pinene, sabinene, ß-pinene, and 4-terpenol were selected as potential differential markers. The relative content of α-pinene and 4-terpenol was consistent with that of total volatile oils in the changing trend.

Application of SNP in Genetic Sex Identification and Effect of Estradiol on Gene Expression of Sex-Related Genes in Strongylocentrotus intermedius.

Sea urchin (Strongylocentrotus intermedius) is an economically important mariculture species in Asia, and its gonads are the only edible part. The efficiency of genetic breeding in sea urchins is hampered due to the inability to distinguish gender by appearance. In this study, we first identified a sex-associated single nucleotide polymorphism (SNP) by combining type IIB endonuclease restriction site-associated DNA sequencing (2b-RAD-seq) and genome survey. Importantly, this SNP is located within spata4, a gene specifically expressed in male. Knocking down of spata4 by RNA interference (RNAi) in male individuals led to the downregulation of other conserved testis differentiation-related genes and germ cell marker genes. We also revealed that sex ratio in this validated culture population of S. intermedius is not 1:1. Moreover, after a 58-day feeding experiment with estradiol, the expression levels of several conserved genes that are related to testis differentiation, ovary differentiation, and estrogen metabolism were dynamically changed. Taken together, our results will contribute toward improving breeding efficiency, developing sex-controlled breeding, and providing a solid base for understanding sex determination mechanisms in sea urchins.

Natural Deep Eutectic Solvents for the Extraction of Bioactive Steroidal Saponins from Dioscoreae Nipponicae Rhizoma.

In the present study, a simple and environmentally friendly extraction method based on natural deep eutectic solvents (NADESs) was established to extract four bioactive steroidal saponins from Dioscoreae Nipponicae Rhizoma (DNR). A total of twenty-one types of choline chloride, betaine, and L-proline based NADESs were tailored, and the NADES composed of 1:1 molar ratio of choline chloride and malonic acid showed the best extraction efficiency for the four steroidal saponins compared with other NADESs. Then, the extraction parameters for extraction of steroidal saponins by selected tailor-made NADES were optimized using response surface methodology and the optimal extraction conditions are extraction time, 23.5 min; liquid-solid ratio, 57.5 mL/g; and water content, 54%. The microstructure of the DNR powder before and after ultrasonic extraction by conventional solvents (water and methanol) and the selected NADES were observed using field emission scanning electron microscope. In addition, the four steroidal saponins were recovered from NADESs by D101 macroporous resin with a satisfactory recovery yield between 67.27% and 79.90%. The present research demonstrates that NADESs are a suitable green media for the extraction of the bioactive steroidal saponins from DNR, and have a great potential as possible alternatives to organic solvents for efficiently extracting bioactive compounds from natural products.

Identification and functional characterization of piwi1 gene in sea cucumber, Apostichopus japonicas.

The sea cucumber (Apostichopus japonicus) is an economically important mariculture species in Asia. However, the genetic breeding of sea cucumbers is difficult because the sexes cannot be identified by appearance. Therefore, studies on sex-related genes are helpful in revealing the mechanisms of sex determination and differentiation in sea cucumbers. P-element induced wimpy testis (piwi) is a germ cell marker involved in gametogenesis in vertebrates; however, the expression pattern and function during gametogenesis remain unclear in sea cucumbers. In this study, we identified a piwi homolog gene in A. japonicus (Ajpiwi1) and investigated its expression pattern, and function. Ajpiwi1 is a maternal factor and is ubiquitously expressed in adult tissues, including the ovary and testis. Ajpiwi1 expression is strong in early oocytes, spermatocytes, and spermatogonia; weak in mature oocytes; and undetected in spermatids and intra-gonadal somatic cells. The knockdown of Ajpiwi1 by RNA interference (RNAi) led to the downregulation of other conserved sex-related genes such as dmrt1, foxl2, and germ cell-less. Therefore, Ajpiwi1 might play a critical role during gametogenesis in A. japonicus. This study creates new possibilities for studying sex-related gene functions in the sea cucumber and builds a gene function research platform based on RNAi for the first time.

[Dynamic changes of volatile components of Qiai from different harvest time based on GC-MS and chemometrics analysis].

Artemisiae Argyi Folium, the dried leaves of Artemisia argyi, has been widely used in traditional Chinese and folk medicines for a long time. Qiai is one of the top-geoherb of Artemisiae Argyi Folium. Qiai contains various bioactive constituents, such as volatile oils, phenolic acids, flavonoids and terpenoids. Phytochemical studies demonstrated that volatile compounds are the main bioactive constituents in Qiai. Try to investigate dynamic changes of volatile components of Qiai from different harvest time and explore the optimum harvest time of Qiai, in this study, the contents of total volatile oils in Qiai collected from five different harvest time were analyzed by steam distillation method. The results showed that the contents of volatile oils of Qiai were higher in the third harvest time(around the Dragon Boat Festival), which is basically consistent with the traditional harvest time. Furthermore, a sensitive method based on gas chromatography-mass spectrometry(GC-MS) was established for qualitative analysis of volatile compounds in Qiai, and a total of thirty volatile compounds were identified. Chemometrics methods including principal component analysis(PCA) and orthogonal partial least-squares discriminate analysis(OPLS-DA) were applied to explore chemical markers and dynamic changes of volatile components in Qiai from different harvest time, and the results indicated that there were obvious differences in the relative contents of volatile compounds of Qiai samples from different harvest time. Eight volatile compounds, including α-terpinene, γ-terpinene, D-camphor, trans-carveol, α-copaene, isobornylisobutyrate, humulene, and caryophyllene oxide were selected as potential chemical markers. Among the eight chemical markers, the relative contents of α-terpinene, γ-terpinene, α-copaene and caryophyllene oxide were higher in the third harvest period(around the Dragon Boat Festival), which is consistent with the contents of total volatile oils. The present study could provide the basis for investigating the optimum harvest time of Qiai, and might be useful for the quality control of this herbal medicine.

Molecular Cloning and Sexually Dimorphic Expression Analysis of nanos2 in the Sea Urchin, Mesocentrotus nudus.

Sea urchin (Mesocentrotus nudus) is an economically important mariculture species in China and the gonads are the solely edible parts to human. The molecular mechanisms of gonad development have attracted increasing attention in recent years. Although the nanos2 gene has been identified as a germ cell marker in several invertebrates, little is known about nanos2 in adult sea urchins. Hereinto, we report the characterization of Mnnano2, an M. nudus nanos2 homology gene. Mnnanos2 is a maternal factor and can be detected continuously during embryogenesis and early ontogeny. Real-time quantitative PCR (RT-qPCR) and section in situ hybridization (ISH) analysis revealed a dynamic and sexually dimorphic expression pattern of Mnnano2 in the gonads. Its expression reached the maximal level at Stage 2 along with the gonad development in both ovary and testis. In the ovary, Mnnanos2 is specifically expressed in germ cells. In contrast, Mnnanos2 is expressed in both nutritive phagocytes (NP) cells and male germ cells in testis. Moreover, knocking down of Mnnanos2 by means of RNA interference (RNAi) reduced nanos2 and boule expression but conversely increased the expression of foxl2. Therefore, our data suggest that Mnnanos2 may serve as a female germ cell marker during gametogenesis and provide chances to uncover its function in adult sea urchin.

Gonadal transcriptomic analysis and identification of candidate sex-related genes in Mesocentrotus nudus.

The edible sea urchin, Mesocentrotus nudus, is both an economically important mariculture species and an excellent model for studying reproductive development. The gonads are the only edible parts of sea urchins and increasing market demand for high-quality gonads has prompted increasing amounts of research into the molecular mechanisms of reproduction. Using a high-throughput sequencing technology, we performed transcriptome sequencing on the gonads of females and males, sampled before the spawning season, to identify genes involved in sex determination, sex differentiation and gametogenesis. Through a de novo transcriptome assembly approach, we obtained 104,039 unigenes, of which 40,471 (38.90%) showed homologies with known proteins in public databases. By comparing the expression levels of these unigenes in females and males, 15,368 differentially-expressed unigenes (DEGs) were identified. Compared with males, 9473 were up-regulated and 5895 were down-regulated in females. Multiple candidate genes were identified that may play important roles in spermatogenesis, oogenesis, and germ cell development. Furthermore, we identified and characterized several genes involved in sex determination and sex differentiation, such as dmrt1 and foxl2. The current study provides valuable molecular resources for studying the underlying mechanisms of reproduction in sea urchins.

Molecular characterization and expression of NLRP10 in the antibacterial host defense of the sea cucumber (Apostichopus japonicus).

The nucleotide-binding oligomerization domain-like receptors (NOD-like receptors, NLRs) can regulate the innate immune process and is an important part of inflammatory body. In this study, we use transcriptome sequencing and the rapid amplification of cDNA ends approach to identify a novel NLRP gene in Apostichopus japonicus. We designated the gene as AjNLRP10. The full-length of AjNLRP10 is 4509 bp. The putative open reading frame comprising 3489 bp encodes a polypeptide with 1162 amino acid residues. The predicted molecular mass of AjNLRP10 is 132.87 kDa and its theoretical pI is 5.60. AjNLRP10 comprises a signal peptide with two Ig superfamily (IgSF) domains and a NACHT [NAIP (neuronal apoptosis inhibitory protein), CIITA (MHC class II transcription activator), HET-E (incompatibility locus protein from Podospora anserina) and TP1 (telomerase-associated protein)] domain. Spatial distribution expression analysis detected AjNLRP10 in all of the tissues tested, but with higher expression in the coelomocytes, medium expression in the intestine and respiratory tree, and slightly weaker expression in the body wall, tube feet, and longitudinal muscle. The expression levels of AjNLRP10 in the respiratory tree and intestines of sea cucumbers with skin ulceration syndrome were increased by 4-fold and 2.7-fold compared with those in healthy sea cucumbers, respectively. We investigated expression profiles of AjCasepase-1 (Cysteinyl aspartate specific proteinase-1) and AjMMP37 (mitochondrial protein-37) after AjNLRP10 knock-down and discovered that AjCasepase-1 was raised by 2.60-fold and AjMMP37 was raised by 3.84-fold. The study showed that AjNLRP10 has inhibitory effect in the immune process. In conclusion, this study showed that the AjNLRP10 protein found in the sea cucumber involved with the innate immune responses against bacterial infection. It has a similar structure and biological function to that in other organisms, where it appears to be involved with these results provide insights into the innate immune mechanism in the sea cucumber as well as suggesting new strategies for disease prevention, molecular therapy, and the development of novel drugs for sea cucumbers.

Establishment of lysozyme gene RNA interference systemand its involvement in salinity tolerance in sea cucumber (Apostichopus japonicus).

The lysozyme gene was silenced using RNA interference (RNAi) to analyze the function of lysozyme in sea cucumber under salt stress. The interfering efficiency of four lysozyme RNAi oligos ranged from 0.55 to 0.70. From the four oligos, p-miR-L245 was used for further interfering experiments because it had the best silencing efficiency. Peristomial film injection of p-miR-L245 (10 µg) was used for further interfering experiments. The lowest gene expression, determined by RT-PCR assay, in muscle, coelomic fluid, and parapodium occurred 48 h after p-miR-L245 injection, while that of body wall and tube foot was 96 h and 24 h, respectively. Lysozyme activity in muscle and body wall was significantly lower than the controls. The lowest lysozyme activity in muscle, body wall and parapodium, was found at 48, 72, and 48 h, respectively, which was consistent with the transcription expression of lysozyme. The lowest point of lysozyme activity was at 96 h in coelomic fluid and tube foot, which was laid behind lysozyme expression in transcription level. The expression profile of the lysozyme transcription level and lysozyme activity in the body wall and tube foot increased at 12 h after p-miR-L245 injection before the interference effect appeared. NKA gene expression was expressed at a high level in the positive control (PC) and negative control (NC) groups at 12, 24, and 48 h, while NKA was expressed at low levels in the lysozyme RNAi injection group at 12 and 24 h. The level of NKA gene expression recovered to the level of the PC and NC group at 48, 72, and 96 h after the lysozyme RNAi injection. NKCC1 gene expression was high in the PC and NC groups at 96 h, while the NKCC1 was expressed at a low level 96 h after lysozyme RNAi injection. The results suggest that lysozyme decay involves NKA and NKCC1 gene expression under salinity 18 psµ. The K+ and Cl- concentration after lysozyme RNAi injection was lower than in the PC and NC group.

The complete mitochondrial genome of Neptunea arthritica cumingii Crosse, (Gastropoda: Buccinidae).

The complete mitochondrial (mt) genome of the Neptune whelk, Neptunea arthritica cumingii, was determined using genome walking techniques in this study. The total length of the mt genome sequence of N. arthritica cumingii was 15 256 bp, including 13 protein-coding genes, 21 transfer RNA genes and 2 ribosomal RNA genes. The overall composition of the mitogenome was estimated to be 30.85% for A, 38.59% for T, 15.15% for C and 15.40% for G, indicating that an A + T (69.44%)-rich feature occurs in the N. arthritica cumingii mitogenome. The phylogenetic relationships of 11 mollusc species were constructed based on the complete mtDNA sequences by the neighbour-joining method using MEGA 5.0 software (MEGA Inc., Englewood, NJ).

Expression of c-type lysozyme gene in sea cucumber (Apostichopus japonicus) is highly regulated and time dependent after salt stress.

Lysozymes have been confirmed to possess varieties of functions in a range of organisms. In the present study, we cloned and sequenced c-type lysozyme cDNAs, constructed the recombinant protein over-expression of c-type lysozyme and analyzed the expression of transcription level in various tissues. The c-type lysozyme cDNA contained an open reading frame of 759 bp encoding a polypeptide of 252 amino acids. The molecular weight of the deduced amino acid of AjcLYZ is 26.7 kDa with an estimated pI of 4.66. Multiple sequence alignments revealed that AjcLYZ had two highly conserved active sites (Glu147 and Asp159) and eight typical Cys residues. The tertiary structure and modeled AjcLYZ showed structural similarity to Meretrix lusoria LYZ. The results of mRNA transcripts showed that the highest expression was found in the tube foot, followed by the muscle, body wall, and coelomic fluid. In contrast, the intestine, tentacle and respiratory tree exhibited very low expression levels. Under salinity stress, significant down-regulation of AjcLYZ was observed in response to salinity stress in the intestine and coelomic fluid. Significant up-regulation and down-regulation of AjcLYZ were observed in response to salinity stress in body wall and respiratory tree. The purified recombinant protein was analyzed by SDS-PAGE and a single band with a molecular mass of 45.09 kDa, which was in agreement with the theoretical size (26.7 kDa for AjcLYZ and 18.39 kDa Trx-His-S tags) of the recombinant protein. Radial diffusion assay was employed to determine the antimicrobial spectrum of recombinant AjcLYZ against three Gram-positive and Gram-negative bacteria, and three sea cucumber pathogenic Vibrio species. From the radius of the antimicrobial zone, it was found that recombinant AjcLYZ harbored remarkable in vitro inhibitive effect on tested Gram-positive bacteria, while lytic activity against Gram-negative bacteria was relatively weak. The results will provide new clues about the molecular mechanisms that regulate the salinity adaption system.

Effect of intestinal autochthonous probiotics isolated from the gut of sea cucumber (Apostichopus japonicus) on immune response and growth of A. japonicus.

The study isolated 224 bacteria from the intestine of Apostichopus japonicus, then selected and identified three of the bacteria (HS1, HS7, and HS10) which demonstrated amylase, lipase, and protease production capacity as candidate probiotics for sea cucumbers. The three potential probiotics showed no pathogenicity both in hemolytic assays on sheep blood agar plates and after immersing sea cucumbers in a suspension of the bacteria. To reveal the effects of these three potential probiotics on the innate immunity of sea cucumbers, total coelomocyte counts, respiratory burst activity, superoxide dismutase activity, lysozyme activity, acid phosphatase activity, and phagocytic activity by coelomocytes were examined after feeding with four different diets for up to 28 days. Also the specific growth rate and survival rate were investigated after a 60-day feeding trial. Sea cucumbers were fed with 4 diets: one control, three diets supplemented with 1 × 10(9) cell g(-1) of HS1, HS7, and HS10 for 28-60 days. Results showed that sea cucumbers fed diets containing HS1, HS7, and HS10 had led to an enhanced cellular and humoral immune response, notably higher total coelomocytes counts, respiratory burst activity, lysozyme activity, acid phosphatase activity, and phagocytic activity, as recorded during the four weeks of probiotics administration. On the other hand, the survival rate among dietary treatments ranged from 90.71 to 97.97% with significant improvement (P < 0.05) compared to that of the control; and the growth rate observed in the sea cucumbers fed HS1 and HS7 showed sharp increases after 60 days feeding. The present study confirmed the potential beneficial effects of Pseudoalteromonas elyakovii HS1, Shewanella japonica HS7, and Vibrio tasmaniensis HS10 as dietary probiotics in A. japonicus.

[Bacterial diversity in the mantle of Patinopecten yessoensis revealed by 454 pyrosequencing].

Pyrosequencing-technique was used to analyze and compare bacterial diversity in the mantle of healthy and incised symptoms of Patinopecten yessoensis, respectively. For the two samples; 20872 and 16333 effective sequences were selected and utilized to perform bacterial diversity analysis, respectively. Bacterial diversity and richness in the incised samples were higher than that in the healthy samples. The effective sequences acquired from both samples included eight known phyla bacteria, i. e., Proteobacteria, Firmicutes, Actinobacteria, Bacteroidetes, Cyanobacteria, Planctomycetes, Spirochaetes and Tenericutes, among which the front seven phyla were present in the two samples. Proteobacteria was absolutely dominant in the mantle of healthy P. yessoensis accounting for 97.7% of the entire bacterial community, and Firmicutes was the sub-dominant group accounting for 0.8%. In contrast, the most abundant bacterial group in the mantle of incised P. yessoensis was Firmicutes, accounting for 52.2% of the entire bacterial community, and the sub- dominant was Proteobacteria accounting for 47.7%.

Effects of dietary supplementation of probiotic Shewanella colwelliana WA64, Shewanella olleyana WA65 on the innate immunity and disease resistance of abalone, Haliotis discus hannai Ino.

The effects of dietary administration of two probiotics, Shewanella colwelliana WA64 and Shewanella olleyana WA65, on the innate immunity of abalone (Haliotis discus hannai Ino), and survival of juvenile abalone challenged with Vibrio harveyi have been studied. Two groups of abalone were fed with three different diets: one control, and two diets supplemented with 10(9) cell g(-1) of probiotic WA64 (WA64 diet) and WA65 (WA65 diet) for up to four weeks. Results showed that abalone fed diets containing S. colwelliana WA64 and S. olleyana WA65 had led to an enhanced cellular and humoral immune response, notably higher haemocytes, respiratory burst activity, serum lysozyme activity and total protein levels were recorded after one week of probiotic administration. On the other hand, mortality after the challenges with V. harveyi in the group fed with control diet ranged from 77 to 80%, while mortality rates observed in the groups fed with diets supplemented with WA64 and WA65 ranged from 27 to 50% and 30-43%, respectively. The results demonstrated potential for S. colwelliana WA64 and S. olleyana WA65 to improve innate immunity and disease resistance in H. discus hannai.