Shaoxiong Ding

Transcriptome analysis revealed changes of multiple genes involved in immunity in Cynoglossus semilaevis during Vibrio anguillarum infection

Half-smooth tongue sole (Cynoglossus semilaevis) is one of the most valuable marine aquatic species in Northern China. Given to the rapid development of aquaculture industry, the C. semilaevis was subjected to disease-causing bacteria Vibrio anguillarum. It therefore is indispensable and urgent to understand the mechanism of C. semilaevis host defense against V. anguillarum infection. In the present study, the extensively analysis at the transcriptome level for V. Anguillarum disease in tongue sole was carried out. In total, 94,716 high quality contigs were generated from 75,884,572 clean reads in three libraries (HOSG, NOSG, and CG). 22,746 unigenes were identified when compared with SwissProt, an NR protein database and NT nucleotide database. 954 genes exhibiting the differentially expression at least one pair of comparison in all three libraries were identified. GO enrichment for these genes revealed gene response to biotic stimulus, immune system regulation, and immune response and cytokine production. Further, the pathways such as complement and coagulation cascades and Vibrio cholerae infection pathways were enriched in defensing of pathogen. Besides, 13,428 SSRs and 118,239 SNPs were detected in tongue sole, providing further support for genetic variation and marker-assisted selection in future. In summary, this study identifies several putative immune pathways and candidate genes deserving further investigation in the context of development of therapeutic regimens and lays the foundation for selecting resistant lines of C. semilaevis against V. anguillarum.

A Comprehensive Description and Evolutionary Analysis of 22 Grouper (Perciformes, Epinephelidae) Mitochondrial Genomes with Emphasis on Two Novel Genome Organizations

Groupers of the family Epinephelidae are a diverse and economically valuable group of reef fishes. To investigate the evolution of their mitochondrial genomes we characterized and compared these genomes among 22 species, 17 newly sequenced. Among these fishes we identified three distinct genome organizations, two of them never previously reported in vertebrates. In 19 of these species, mitochondrial genomes followed the typical vertebrate canonical organization with 13 protein-coding genes, 22 tRNAs, two rRNAs, and a non-coding control region. Differing from this, members of genus Variola have an extra tRNA-Ile between tRNA-Val and 16S rRNA. Evidence suggests that this evolved from tRNA-Val via a duplication event due to slipped strand mispairing during replication. Additionally, Cephalopholis argus has an extra tRNA-Asp in the midst of the control region, likely resulting from long-range duplication of the canonical tRNA-Asp through illicit priming of mitochondrial replication by tRNAs. Along with their gene contents, we characterized the regulatory elements of these mitochondrial genomes’ control regions, including putative termination-associated sequences and conserved sequence blocks. Looking at the mitochondrial genomic constituents, rRNA and tRNA are the most conserved, followed by protein-coding genes, and non-coding regions are the most divergent. Divergence rates vary among the protein-coding genes, and the three cytochrome oxidase subunits (COI, II, III) are the most conserved, while NADH dehydrogenase subunit 6 (ND6) and the ATP synthase subunit 8 (ATP8) are the most divergent. We then tested the phylogenetic utility of this new mt genome data using 12 protein-coding genes of 48 species from the suborder Percoidei. From this, we provide further support for the elevation of the subfamily Epinephelinae to family Epinephelidae, the resurrection of the genus Hyporthodus, and the combination of the monotypic genera Anyperodon and Cromileptes to genus Epinephelus , and Aethaloperca to genus Cephalopholis .

Transcriptome analysis of the Octopus vulgaris central nervous system.

Background Cephalopoda are a class of Mollusca species found in all the worlds oceans. They are an important model organism in neurobiology. Unfortunately, the lack of neuronal molecular sequences, such as ESTs, transcriptomic or genomic information, has limited the development of molecular neurobiology research in this unique model organism. Results With high-throughput Illumina Solexa sequencing technology, we have generated 59,859 high quality sequences from 12,918,391 paired-end reads. Using BLASTx/BLASTn, 12,227 contigs have blast hits in the Swissprot, NR protein database and NT nucleotide database with E-value cutoff 1e−5. The comparison between the Octopus vulgaris central nervous system (CNS) library and the Aplysia californica/Lymnaea stagnalis CNS ESTs library yielded 5.93%/13.45% of O. vulgaris sequences with significant matches (1e−5) using BLASTn/tBLASTx. Meanwhile the hit percentage of the recently published Schistocerca gregaria, Tilapia or Hirudo medicinalis CNS library to the O. vulgaris CNS library is 21.03%–46.19%. We constructed the Phylogenetic tree using two genes related to CNS function, Synaptotagmin-7 and Synaptophysin. Lastly, we demonstrated that O. vulgaris may have a vertebrate-like Blood-Brain Barrier based on bioinformatic analysis. Conclusion This study provides a mass of molecular information that will contribute to further molecular biology research on O. vulgaris. In our presentation of the first CNS transcriptome analysis of O. vulgaris, we hope to accelerate the study of functional molecular neurobiology and comparative evolutionary biology.

Cloning and expression characterization of the serum transferrin gene in the Chinese black sleeper (Bostrichthys sinensis)

In this study, the gene encoding serum transferrin (STF) in the Chinese black sleeper (Bostrichthys sinensis) was cloned and sequenced. The full BsSTF cDNA is 2324 bp in length with an open reading frame (ORF) of 2067 bp encoding 688 amino acids and has 47.1%-65.7% identity with other fish homologues. The BsSTF genomic DNA is composed of 17 exons and 16 introns and is well conserved with respect to other vertebrate species in the number of exons and in the position of introns. The expression of the BsSTF mRNA was examined in all tissues tested including liver, blood, muscle, spleen, stomach, skin, intestine, brain, head kidney, fin, gill and gonad, with the highest expression levels found in the liver. Temporal transcriptional changes of BsSTF during embryonic development of the Chinese black sleeper were detected, and the results showed a substantial decrease in the early stages, from the cleavage stage to the blastula stage, reaching minimum levels at the blastula stage and increasing gradually until the pre-hatching stage, indicating that BsSTF is likely an important factor in early embryonic development. After infection with Vibrio harveyi, BsSTF expression was found to increase mainly in the liver and stomach, acting as a positive acute protein, suggesting that BsSTF is involved in the immune response.

Selection and evaluation of new reference genes for RT-qPCR analysis in Epinephelus akaara based on transcriptome data

Groupers are an economically important fish species in world fishery markets. Because many studies using RT-qPCR have addressed gene expression in groupers, appropriate reference genes are required to obtain reliable and accurate results. In this study, the most suitable reference genes were identified from eleven candidate genes of one of the most valuable species, Epinephelus akaara, in a range of different experimental conditions. Using the software packages geNorm, NormFinder, BestKeeper and refFinder, three traditionally used reference genes, β-actin (β-ACT), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and beta-2-microglobulin (B2M), were identified as not suitable for E. akaara gene expression studies, whereas two newly identified reference genes, conserved oligomeric Golgi complex subunit 5 (Cog5) and brefeldin a-inhibited guanine nucleotide-exchange protein 1 (ARFGEF1), could be universally applied under all the tested conditions. These data provide the foundation for more precise results in RT-qPCR studies of gene expression in E. akaara and other Epinephelus species.

Development and Characterization of 20 Microsatellite Markers for Chinese Black Sleeper, Bostrychus sinensis

Twenty microsatellite markers were isolated and characterized from the Chinese black sleeper, Bostrychus sinensis. Loci were screened in 30 individuals from Taiwan. For each locus, the number of alleles varied from 4 to 22 with mean expected and observed heterozygosity of 0.79 and 0.66, respectively. One locus significantly deviated from Hardy-Weinberg equilibrium after Bonferroni correction and no significant linkage disequilibrium was detected. This set of microsatellites will provide a suitable tool for population genetic studies of Chinese black sleeper.

Differentiation of coral trout (Plectropomus leopardus) based on an analysis of morphology and complete mitochondrial DNA: Are cryptic species present?

Two morphotypes of Plectropomus leopardus have been identified; morphometric and meristic analyses show that there is no diagnostic difference between them. A difference in color pattern was the most appropriate phenotypic character with which to distinguish between the two morphotypes. Complete mitochondrialDNA sequencing, however, indicated a clear difference between the two morphotypes. Barcoding analysis revealed no significant difference (P>0.05) in CO1 or ND2 divergence among intramorphotypic individuals, even between geographically separated populations, whereas the intermorphotypic CO1 and ND2 divergences were large enough (averaging 0.95% for CO1 and 1.37% for ND2) to clearly discriminate between the two morphotypes. The color pattern difference, geographical distribution, together with the mtDNA and barcode sequencing data, suggest that the two morphotypes should be of two subspecies or even two species.

Transcriptome Changes during the Life Cycle of the Red Sponge, Mycale phyllophila (Porifera, Demospongiae, Poecilosclerida).

Sponges are an ancient metazoan group with broad ecological, evolutionary, and biotechnological importance. As in other marine invertebrates with a biphasic life cycle, the developing sponge undergoes a significant morphological, physiological, and ecological transformation during settlement and metamorphosis. In this study, we compare new transcriptome datasets for three life cycle stages of the red sponge (Mycale phyllophila) to test whether gene expression (as in the model poriferan, Amphimedon queenslandica) also varies more after settlement and metamorphosis. In contrast to A. queenslandica, we find that the transcriptome of M. phyllophila changes more during the earlier pre-competent larva/post-larva transition that spans these defining events. We also find that this transition is marked by a greater frequency of significantly up-regulated Gene Ontology terms including those for morphogenesis, differentiation, and development and that the transcriptomes of its pre-competent larvae and adult are distinct. The life cycle transcriptome variation between M. phyllophila and A. queenslandica may be due to their long separate evolutionary histories and corresponding differences in developmental rates and timing. This study now calls for new transcriptome datasets of M. phyllophila and other sponges, which will allow for tests of the generality of our life cycle expression differences and for the greater exploitation of poriferans in both basic and applied research.

Complete mitochondrial genome of yellow grouper Epinephelus awoara (Perciformes, Epinephelidae)

In this paper, the complete mitochondrial (mt) genome of Epinephelus awoara has been determined. The mt genome (16,802 bp) had the canonical mt gene content and arrangement, including 13 protein-coding genes, 2 rRNA genes, 22 tRNA genes, and 2 non-coding regions. The overall base composition of the Heavy-strand is 28.47% A, 27.29% T, 16.54% G, and 27.70% C, with an AT content of 56.76%. It shared 92.6%, 85.5%, 86.1%, 84.9%, and 85.7% identities with that of Epinephelus akaara, Epinephelus bruneus, Epinephelus coioides, Epinephelus lanceolatus and Epinephelus moara, respectively.

Complete mitochondrial genome of black porgy Acanthopagrus schlegelii (Perciformes, Sparidae)

Black porgy, Acanthopagrus schlegelii, is a marine protandrous hermaphrodite and belongs to one of the most important species commercialized for food in various areas of Asia. In this study, the complete mitochondrial genome of A. schlegelii has been determined. The mitogenome was 16,649 bp in length and contained 13 protein-coding genes, 2 rRNA genes, 22 tRNA genes and 2 non-coding regions. It shared 90.2%, 82.3%, and 82.1% mitogenome sequence with Acanthopagrus latus, Parargyrops edita, and Pagrus major, respectively.