Yingeng Wang

Transcriptome Analysis and Discovery of Genes Involved in Immune Pathways from Coelomocytes of Sea Cucumber (Apostichopus japonicus) after Vibrio splendidus Challenge.

Vibrio splendidus is identified as one of the major pathogenic factors for the skin ulceration syndrome in sea cucumber (Apostichopus japonicus), which has vastly limited the development of the sea cucumber culture industry. In order to screen the immune genes involving Vibrio splendidus challenge in sea cucumber and explore the molecular mechanism of this process, the related transcriptome and gene expression profiling of resistant and susceptible biotypes of sea cucumber with Vibrio splendidus challenge were collected for analysis. A total of 319,455,942 trimmed reads were obtained, which were assembled into 186,658 contigs. After that, 89,891 representative contigs (without isoform) were clustered. The analysis of the gene expression profiling identified 358 differentially expression genes (DEGs) in the bacterial-resistant group, and 102 DEGs in the bacterial-susceptible group, compared with that in control group. According to the reported references and annotation information from BLAST, GO and KEGG, 30 putative bacterial-resistant genes and 19 putative bacterial-susceptible genes were identified from DEGs. The qRT-PCR results were consistent with the RNA-Seq results. Furthermore, many DGEs were involved in immune signaling related pathways, such as Endocytosis, Lysosome, MAPK, Chemokine and the ERBB signaling pathway.

Development of new microsatellite DNA markers from Apostichopus japonicus and their cross-species application in Parastichopus parvimensis and Pathallus mollis.

Twenty microsatellite DNA markers were developed for sea cucumber and used to investigate polymorphisms of 60 wild Apostichopus japonicus individuals collected from China. It revealed that all the markers were polymorphic. A total of 164 alleles were detected at 20 loci. The number of alleles per locus varied from 3 to 17 with an average of 8.2, and the expected heterozygosities of each locus ranged from 0.03 to 0.89 with an average of 0.64. Cross-species amplification was also conducted in Parastichopus parvimensis collected from the United States and Pathallus mollis collected from Peru. The result showed that 17 loci amplified Parastichopus parvimensis DNAs while only 4 loci could amplify Pathallus mollis DNAs. All of the polymorphic markers would be useful for future genetic breeding and the assessment of genetic variation within sea cucumbers.

Diagnosis and ORF gene sequencing analysis of the nervous necrosis virus (NNV) isolated from cultured pearl gentian grouper, Epinephelus lanceolatus × Epinephelus fuscoguttatus, in China

Pearl gentian grouper, Epinephelus lanceolatus × Epinephelus fuscoguttatus, is a hybrid species with increasing market requirement in China. A disease outbreak occurred in the fish juvenile stages in Shandong Province, P.R. China. The gross signs were characterized by mass mortality, exhibiting dark skin coloration, reduce of feeding and having abnormal swimming behavior. Based on histopathological study, a large number of cellular vacuoles were observed in the diseased fish tissues of brain and retina. In addition, RT-PCR confirmed nervous necrosis virus (NNV) infection, and it was considered as the causative agent of the outbreak. This is, so far, the first report to detect NNV infection in Pearl gentian grouper. Furthermore, the open reading frames (ORF) of RNA dependent-RNA polymerase gene and coat protein gene of NNV also were sequenced after purification and cloning. Nucleotide sequence and the phylogenetic tree analysis of RNA1 and RNA2 showed extra high similarity (98.9%, 99.3% respectively) to red-spotted grouper nervous necrosis virus (RGNNV). These indicated that the NNV belong to RGNNV genotype, which is the only one genotype in P.R. China. Based on molecular study, the Cp genome included the ORF of 1017(6-1022) bases which encode protein of 338 amino-acids and the RdRp genome included the ORF of 2949 (49-2997) bases which encode protein of 982 amino-acids. These results would be helpful to understand NNV infection and provide information for fish health management.

Development of an SYBR Green I real-time PCR assay for detection of Edwardsiella tarda and its application

According to the sequenced gyrB gene sequence of Edwardsiella tarda,a pair of primers was designed for establishing an SYBR Green I real-time fluorescence quantitative PCR method. A 207 bp gene fragment w as amplified from chromosomal DNA of E. tarda from different sources,and no positive reaction w as detected in 9 other bacteria species using conventional PCR,w hich indicated that the primer pair has good inter-species specificity and intra-species commonality. Recombinant plasmid containing gyrB gene of E. tarda w as constructed and used to construct the standard curve. The standard curves w as y =- 3. 32x + 39. 38,the correlation coefficient w as 0. 998 and the amplification efficiency w as 1. 00,w hich indicated that it had a good linear relationship betw een initial templates and C t values. The melting curve has only one specific peak w hen annealing temperature w as 63 ℃. The detection limit of the assay w as 60 copies per reaction. Turbot samples infected by E. tarda artificially w ere detected using the real-time PCR assay. All the three samples w ere positive,w hich had good agreement w ith bacteriological analysis by isolation and culture. The results show ed that the developed SYBR Green I real-time PCR assay had the advantages of specificity, sensitivity,rapidity and quantification,and w ould be helpful for E. tarda diagnosis and epidemiology investigation.

Effects of probiotic on microfloral structure of live feed used in larval breeding of turbot Scophthalmus maximus

The effects of an exogenous probiotic (Bacillus amyloliquefaciens) on microbial community structure of Branchionus plicatils and Artemia sinica were evaluated in this study during turbot (Scophthalmus maximus) larval breeding. The analysis and comparison of the microfloral composition of live feed with probiotic was conducted using the Illumina HiSeq PE250. The abundance of microbial species and diversity of microflora in live feed with B. amyloliquefaciens were higher than those in the control. The microfloral composition was similar among the three replicate experimental groups of B. plicatils compared with the control after enrichment. Lactococcus, Pseudoalteromonas, and Alteromonas were always dominant. Additionally, some other bacterial species became dominant during the enrichment process. The microbial community during nutrient enrichment of A. sinica was rather similar among the three control replicates. Relative abundance of Cobetia sp., the most dominant species, was 54%–65.2%. Similarity in the microbial community was still high after adding B. amyloliquefaciens. Furthermore, Pseudoalteromonas and Alteromonas replaced Cobetia as the dominant species, and the abundance of Cobetia decreased to 4.3%–25.3%. Mean common ratios at the operational taxonomic unit level were 50%–60% between the two B. plicatils and A. sinica treatments. Therefore, the microbial community structure changed after adding B. amyloliquefaciens during nutrient enrichment of B. plicatils or A. sinica and tended to stabilize. Additionally, the abundance of Vibrio in any kind of live feed was not significantly different from that in the control. These results will help improve the microflora of B. plicatils and A. sinica and can be used to understand the multiple-level transfer role of probiotic species among probiotic products, microflora of live feed, and fish larvae.

Optimization of protectant, salinity and freezing condition for freeze-drying preservation of Edwardsiella tarda

Novel preservation condition without ultra-low temperature is needed for the study of pathogen in marine fishes. Freeze-drying is such a method usually used for preservation of terrigenous bacteria. However, studies using freeze-drying method to preserving marine microorganisms remain very limited. In this study, we optimized the composition of protectants during the freeze-drying of Edwardsiella tarda, a fish pathogen that causes systemic infection in marine fishes. We found that the optimal composition of protectant mixture contained trehalose (8.0%), skim milk (12.0%), sodium citrate (2.0%), serum (12.0%) and PVP (2.0%). Orthogonal and interaction analyses demonstrated the interaction between serum and skim milk or sodium citrate. The highest survival rate of E. tarda was observed when the concentration of NaCl was 10.0, 30.0 and between 5.0 and 10.0 g L−1 for preparing TSB medium, E. tarda suspension and protectant mixture, respectively. When E. tarda was frozen at −80°C or −40°C for 6 h, its survival rate was higher than that under other tested conditions. Under the optimized conditions, when the protectant mixture was used during freeze-drying process, the survival rate (79.63%–82.30%) of E. tarda was significantly higher than that obtained using single protectant. Scanning electron microscopy (SEM) image indicated that E. tarda was embedded in thick matrix with detectable aggregation. In sum, the protectant mixture may be used as a novel cryoprotective additive for E. tarda.

Effects of different microbes on fermenting feed for sea cucumber (Apostichopus japonicus)

The effects of different microbes on fermenting feed for sea cucumber (Apostichopus japonicus) were compared to select the optimal fermentation strain in this study. Saccharomgces cerevisae, Candida utilis, Bacillus subtilis and Geotrichum candidum were independently added into the experimental compound feed, while only saline was mixed with the control feed. The fermentation treatments were inoculated with 10% seed solution under the condition of 25°C and 70% water content, which lasted for 5 days to elucidate the optimal microbe strain for fermenting effect. Physicochemical indexes and sensorial characteristics were measured per day during the fermentation. The indexes included dry matter recovery (DMR), crude protein (CP), the percentage of amino acid nitrogen to total nitrogen (AA-N/tN), the percentage of ammonia nitrogen to total nitrogen (NH3-N/tN), and the ratio of fermentation strains and vibrios to the total microbes, color, smell and viscosity. The results showed that DMR, CP and AA-N/tN of the S. cerevisae group reached the highest level on day 3, but the ratio of fermentation strain was second to C. utilis group. In addition, its NH3-N/tN and the ratio of vibrios were maintained at low levels, and the sensory evaluation score including smell, color and viscosity was the highest in S. cerevisae group on day 3. Therefore, S. cerevisae could be the optimal strain for the feed fermentation for sea cucumber. This research developed a new production method of fermentation feed for sea cucumber.

Isolation, identification and pathogenicity of Listonella anguillarum from diseased cultured Sebastes schlegelii

Sebastes schlegelii is an important economic fish cultured in North China.In recent years,S.schlegelii aquaculture has developed rapidly along the northern coast of China.The expansion and intensification of S.schlegelii farming has led to the occurrence of some diseases.In August 2011,an epizootic occurred among cultured S.schlegelii in a cage culture farm in Qingdao,which caused cumulative mortality rate 30%.To make certain the causative pathogen,we studied the etiology and virulence of S.schlegelii so as to provide basic data for the farming industry of S.schlegelii.In this study,we used the morphological,physiological and biochemical methods to identify the isolated bacterium.Meanwhile,to ensure the results accurately,the phylogenetic analyses were also used.A dominant bacterium strain SS-1 was isolated from the skin,gill,liver,spleen,kidney of the diseased S.schlegelii.Artificial infection test with intramuscular injection method proved that the median lethal concentration(LC50) value of bacterium strain SS-1 in S.schlegelii was 9.6×106CFU/mL.This bacterium could cause severe infections in several tissues and organs of the fish.The results of morphological,physiological biochemical characteristics tests showed that strain SS-1 was gran-negative,rod bacteria,polar flagellum,and the results of oxidase,contact enzyme,nitrate reduction,producing indol,V-P test and arginine digydrolase test were positive;producing H2S test was negative.The morphological,physiological biochemical characteristics indicated that SS-1 could be identified as Listonella anguillarum.The sequence analysis of 16S rRNA gene of strain SS-1 was identical with L.anguillarum and the homogeneity is 99%.This paper reported for the first time that L.anguillarum caused the disease of S.schlegelii in China,which will provide reference in fish health management and disease control.