Shuhuan Zhang

Sequencing and De Novo Assembly of the Gonadal Transcriptome of the Endangered Chinese Sturgeon (Acipenser sinensis)

Background The Chinese sturgeon (Acipenser sinensis) is endangered through anthropogenic activities including over-fishing, damming, shipping, and pollution. Controlled reproduction has been adopted and successfully conducted for conservation. However, little information is available on the reproductive regulation of the species. In this study, we conducted de novo transcriptome assembly of the gonad tissue to create a comprehensive dataset for A. sinensis. Results The Illumina sequencing platform was adopted to obtain 47,333,701 and 47,229,705 high quality reads from testis and ovary cDNA libraries generated from three-year-old A. sinensis. We identified 86,027 unigenes of which 30,268 were annotated in the NCBI non-redundant protein database and 28,281 were annotated in the Swiss-prot database. Among the annotated unigenes, 26,152 and 7,734 unigenes, respectively, were assigned to gene ontology categories and clusters of orthologous groups. In addition, 12,557 unigenes were mapped to 231 pathways in the Kyoto Encyclopedia of Genes and Genomes Pathway database. A total of 1,896 unigenes, potentially differentially expressed between the two gonad types, were found, with 1,894 predicted to be up-regulated in ovary and only two in testis. Fifty-five potential gametogenesis-related genes were screened in the transcriptome and 34 genes with significant matches were found. Besides, more paralogs of 11 genes in three gene families (sox, apolipoprotein and cyclin) were found in A. sinensis compared to their orthologs in the diploid Danio rerio. In addition, 12,151 putative simple sequence repeats (SSRs) were detected. Conclusions This study provides the first de novo transcriptome analysis currently available for A. sinensis. The transcriptomic data represents the fundamental resource for future research on the mechanism of early gametogenesis in sturgeons. The SSRs identified in this work will be valuable for assessment of genetic diversity of wild fish and genealogy management of cultured fish.

Development of 27 novel cross-species microsatellite markers for the endangered Hucho bleekeri using next-generation sequencing technology

In this study, we developed 27 polymorphic microsatellite loci in Hucho bleekeri, a glacial relict and freshwater resident salmonid fish in China. The number of alleles varied from 2 to 8 for each primer set. The observed and expected heterozygosity ranged from 0.250 to 0.906 and 0.508 to 0.845, respectively. The polymorphic information content ranged from 0.371 to 0.808. These microsatellite loci should be useful to study population genetics, paternity identification, speciation and adaptive evolution of this lineage.

Isolation and characterization of 23 microsatellite loci in the Chinese sucker ( Myxocyprinus asiaticus )

Chinese sucker (Myxocyprinus asiaticus) is a second class state-protected animal in China. In this study, we developed twenty-three polymorphic microsatellite loci in Chinese sucker. The observed and expected heterozygosity ranged from 0.292–0.958 to 0.423–0.900, respectively. The polymorphic information content ranged from 0.356 to 0.869, with a mean of 0.710. These microsatellite loci are expected to be useful for further studies of genetic diversity, population genetic structure, and assessments of the artificial propagation release effect of Chinese sucker.

Gender and gonadal maturity stage identification of captive Chinese sturgeon, Acipenser sinensis, using ultrasound imagery and sex steroids

Long lifespan and late maturation make it difficult to establish gamete maturity and breeding age of captive endangered Chinese sturgeon, Acipenser sinensis. This greatly handicaps timely breeding and future conservation stocking efforts. We used ultrasound imagery and sex steroids to determine the gender and gonadal maturity stage of captive Chinese sturgeon (age, 10-17years old). The echogenicity of the reproductive organs and the respective morphology of the gonads were described and two quantitative parameters po (proportion of the ovary to the entire reproductive organs) and d (thickness of the reproductive organs) were measured to characterize sex and maturity stage of Chinese sturgeon. Females were accordingly placed fish into several categories: FII (FII-, FII, FII+), FIII (FIII, FIII+) and FIV (FIV, FIV+) and FVI and males as MII, MIII, MIV, MV and MVI. The accuracy of gender and maturity stage determination provided by ultrasonographic methods was 72.7% for FII- ovary (n=11) and 76.2% for MII testis (n=42). Accuracy of sex and maturity determination using only serum sex steroid of testosterone (T) and estradiol-17β (E2) was low (58-73%, depending on maturity stage). However, when the two methods were used together, accuracy increased sharply, especially for immature (II stage) females. In summary, of 151 Chinese sturgeon, whose sex and maturity stage were independently confirmed, 88.1% (n=133), 62.9% (n=95), and 96.7% (n=146) were successfully sexed and staged using ultrasound, sex steroids, or both methods, respectively. The results provide reliable non-invasive techniques for determining sex and gonadal maturation of captive Chinese sturgeon. These methods can track individual gonad characteristics over multi-year reproductive cycles, which will assist captive broodstock management, artificial reproduction, and future conservation stocking.

The complete mitochondrial genome of the Endangered Hucho bleekeri (Salmonidae: huchen).

Abstract Hucho bleekeri, a glacial relict and freshwater resident salmonid fish, is endemic to the Yangtze River drainage in China. This species has important significances for studies of biogeography, evolution, phylogeny, ecology, reproduction and development. The complete mitochondrial genome of H. bleekeri was sequenced and characterized. The genome is 16,837 bp in length and contains 13 protein-coding genes, 2 ribosomal RNAs, 22 transfer RNAs, and a non-coding control region. The gene arrangement and nucleotide composition of the mitochondrial genome are similar to those of H. taimen. An 82 bp tandem repeat was identified in the control region. We conclude that the control region is variable in length and copy number of repeat between and within species. The complete mitochondrial DNA of H. bleekeri should be useful to study population genetics, biogeography, speciation and adaptive evolution of this lineage.

Molecular characterization and expression analysis of TLR1 and TLR4 from the endangered fish Dabry's sturgeon ( Acipenser dabryanus )

Abstract Toll‐like receptors (TLRs) are important pattern recognition receptors (PRRs) of teleost innate immune system. However, information about TLRs is absent in Dabrys sturgeon (Acipenser dabryanus), one of the most primitive actinopterygii species. In the present study, the full lengths of adaTLR1 and adaTLR4 were cloned from Dabrys sturgeon using RT‐PCR and RACE‐PCR. The obtained adaTLR1 was 2957 bp in length, encoding a putative protein of 767 amino acids and adaTLR4 cDNA was 2902 bp in length, encoding a putative protein of 830 aa. Both adaTLR1 and adaTLR4 possessed several typical TLRs motifs, including signal peptides, leucine‐rich repeat (LRR) motifs, a transmembrane domain and a TIR motifs. In addition, adaTLR4 contained three conserved boxes in its TIR motif, involving in TLRs signal transduction. A proline, important for LPS recognition of mammalian TLR4, was also found in adaTLR4. Physicochemical features of adaTLR1 and adaTRL4 were also analyzed. Quantitative realtime PCR showed that both transcripts were ubiquitously expressed in all 11 normal tissues selected, but they exhibited different expression patterns, with adaTLR1 highly expression in heart and adaTLR4 highly in skin. Further, adaTLR1 and adaTLR4 were up‐regulated in the primary head‐kidney leucocytes following LPS and polyI:C stimulation, indicating that both genes involved in the sturgeon immune response to LPS and polyI:C. To our best knowledge, this was the first report of these genes in sturgeon and these results provided the basis for further elucidating the ligand specificity and signaling pathway of fish TLRs. HighlightsTLR1 and TLR4 were firstly identified from the endangered fish Dabrys sturgeon (Acipenser dabryanus).Sequence features of adaTLR1 and adaTLR4 were analyzed.Expression patterns of adaTLR1 and adaTLR4 in normal tissues of sturgeon were analyzed.Expression patterns of adaTLR1 and adaTLR4 in the primary HK leucocytes following PAMPs stimulation were analyzed.

Analysis of the expression patterns of the cytokine receptor family B (CRFB) and interferon gamma receptor (IFNGR) in Dabry's sturgeon (Acipenser dabryanus)

&NA; Teleost fish have more complex interferon receptor systems than mammals. In the present study, genes encoding four cytokine receptor family B (CRFBs) and two interferon gamma receptors (IFNGRs) in Dabrys sturgeon (Acipenser dabryanus) were identified by RNA‐sequencing. Sequence analysis revealed that the Dabrys sturgeon CRFBs and IFNGRs contained several conserved characteristics features, including signal peptides and a transmembrane domain. Phylogenetic analysis suggested that they belong to the CRFB3, CRFB5, and IFNGR protein families, and were named CRFB3a, CRFB3b, CRFB5a, CRFB5b, IFNGR1, and IFNGR2. The expression patterns of the CRFB and IFNGR genes were investigated in Dabrys sturgeon. The expression levels of CRFB5a, CRFB5b, and IFNGR1 showed no significant changes, suggesting that those genes do not mediate embryonic development. By contrast, the high expression levels of CRFB3a, CRFB3b, and IFNGR2 in the fertilized egg, 16‐cell phase, and initial blastula stage implied the existence of maternally expression in the oocyte and association with embryonic development. Tissue distribution analysis revealed that the CRFB and IFNGR proteins have potential functions in immune and non‐immune tissue compartments. Comprehensive analysis in Dabrys sturgeon revealed that the expression fold changes of CRFB3a, CRFB3b, CRFB5a, and CRFB5b in Dabrys sturgeon stimulated with poly I:C were higher than those in fish administrated with lipopolysaccharide (LPS). Conversely, the fold changes IFNGRs mRNA levels stimulated with LPS were higher than those in fish administrated with poly I: C. CRFB5a and IFNGR2 genes showed the earliest responses to the poly I: C, and the CRFB5a and IFNGR1 genes showed the earliest responses to LPS. These results implied that CRFB5a has important role in the IFN immune response. Our findings indicated that the Dabrys sturgeon CRFB and IFNGR genes have important functions in antiviral and antibacterial immune responses. The differential responses of these genes to poly I: C and LPS implied differences in the defense mechanisms against viruses and bacteria.

Sequence analysis and characterization of type I interferon and type II interferon from the critically endangered sturgeon species, A. dabryanus and A. sinensis

In the present study, we identify three type I interferon (IFN) genes (Ad/AsIFNe1-3) and a type II IFN gene (Ad/AsIFNγ) from the Dabrys sturgeon (Acipenser dabryanus) and the Chinese sturgeon (Acipenser sinensis). Sequence analysis revealed that Ad/AsIFNe1-3 and Ad/AsIFNγ contain several conserved characteristics, including signal peptides, interferon alpha, beta, and delta (IFabd) domains, and N-glycosylation sites. Ad/AsIFNe1-3 belongs to the type I IFN group I subgroup, possessing two conserved cysteines residues (C1 and C3), and Ad/AsIFNγ contained a conserved nuclear localization sequence (NLS) motif. Ad/AsIFNe1-3 and Ad/AsIFNγ contain signature motifs indicative of their corresponding IFN group. The Ad/AsIFNe1-3 and Ad/AsIFNγ genes were found to consist of 5 exons/4 introns and 4 exons/3 introns, respectively. These IFNs were separated by four phase 0 introns (type I IFN) and three phase 0 introns (type II IFN). The sequences of IFNe1-3 and IFNγ from the Dabrys sturgeon and the Chinese sturgeon were closely aligned, suggested that these two species are closely related. Phylogenetic analysis revealed that Ad/AsIFNe1-3 and Ad/AsIFNγ clustered together with the corresponding homologous proteins from other fish species. AdIFNe1-3 were found to be high expressed in early embryonic development, suggesting that AdIFNe1-3 might indicate maternal transmission, while AdIFNγ may not mediate embryonic development. Tissue distribution analysis revealed that AdIFNe1-3 and AdIFNγ carry out biological functions in immune and non-immune tissues compartments. AdIFNe1-3 and AdIFNγ can be stimulated by polyinosinic-polycytidylic acid (poly I:C) and lipopolysaccharides (LPS). AdIFNe1-3 have stronger antiviral activity than AdIFNγ, and AdIFNγ has a stronger antibacterial activity than AdIFNe1-3. The differential responses of these genes to poly I:C and LPS suggest differences in the mechanisms of defense against viruses and bacteria.

Transcriptome analysis of the critically endangered Dabry's sturgeon (Acipenser dabryanus) head kidney response to Aeromonas hydrophila

Abstract Dabrys sturgeon (Acipenser dabryanus), as a living fossil, is considered a critically endangered aquatic animal in China. To date, the immune system of this species remains largely unknown, with limited available sequence information. In addition, increasing incidence of bacterial pathogenic diseases has been reported. Hence, the present study aimed to characterize comprehensively transcriptome profile of the head kidney from Dabrys sturgeon infected with Aeromonas hydrophila using Illumina platform. Over 42 million high‐quality reads were obtained and de novo assembled into a final set of 195240 unique transcript fragments (unigenes), with an average length of 564 bp. Approximately 41702 unigenes were annotated in the NR NCBI database. Dabrys sturgeon unigenes had the highest number of hits with 14365 (34.45%) to Lepisosteus oculatus. The 195240 unigenes were assigned to three Gene Ontology (GO) categories: biological process, cellular component, and molecular function. Among them, 27770 unigenes were clustered into 26 Eukaryotic Orthologous Group (KOG) functional categories, and 36031 unigenes were mapped to 335 known Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways. After A. hydrophila administration, 1728 differentially expressed unigenes were identified, including 980 upregulated and 748 downregulated unigenes. Further KEGG enrichment analysis of these unigenes identified 16 immune‐related pathways, including the Toll‐like receptor signaling pathway, chemokine signaling pathway, complement and coagulation pathway, RIG‐I‐like receptor signaling pathway, and NOD‐like receptor signaling pathway. 20 DEGs were selected and their expression patterns are largely consistent with the transcriptome profile analysis, which clearly validated the reliability of the DEGs in transcriptome analysis. This work revealed novel gene expression patterns of Dabrys sturgeon host defense and contributes to a better understanding of the immune system and defense mechanisms of Dabrys sturgeon in response to bacterial infection. The results provide valuable references for studies in sturgeons that lack complete genomic sequences, and could also be helpful for the analyzing evolution among cartilaginous and teleost fish. HighlightsThe head kidneys transcriptome of Dabrys sturgeon were constructed using Illumina XTen platform.Totally, 195240 unigenes were obtained from the transcriptome.Toll‐like signaling pathway, Chemokine signaling pathway, RIG‐I‐like and NOD‐like receptor signaling pathways were analyzed.A total of 1728 differentially expressed unigenes were identified, including 980 upregulated and 748 downregulated unigenes.

Molecular characterization of three toll-like receptors (TLR21, TLR22, and TLR25) from a primitive ray-finned fish Dabry's sturgeon (Acipenser dabryanus)

Abstract Dabrys sturgeon (Acipenser dabryanus) is a useful model for the study of fish evolution, as it is one of the most primitive actinopterygian species. However, studies of the immune system of this fish are limited. Here, we identified three toll‐like receptors (adaTLR21, adaTLR22, and adaTLR25) from Dabrys sturgeon. The three sturgeon TLRs had characteristic TLR features, including a signal peptide, several leucine rich repeat (LRR) domains, a transmembrane domain, and a Toll/interleukin‐1 receptor (TIR) domain. Although the predicted amino acid sequences encoded by the sturgeon adaTLR21, adaTLR22, and adaTLR25 had somewhat low levels of sequence identity and similarity with TLRs from other fish species, the three sturgeon TLRs fell in well‐supported clades with other teleost TLRs in our neighbor‐joining phylogenetic tree. Real‐time quantitative PCR showed that the three sturgeon TLRs were ubiquitously expressed in all examined tissues from healthy adult sturgeon, but that their expression patterns varied greatly among the different tissues. The three sturgeon TLRs were also expressed across all embryonic developmental stages that were examined, but their expression levels differed between developmental stages. All three TLRs were upregulated in head–kidney primary leucocytes following lipopolysaccharide (LPS) and polyinosinic: polycytidylic acid (polyI:C) stimulation. To the best of our knowledge, this is the first characterization of these three TLRs in Darbys sturgeon. Our results provide a framework for further studies of TLR ligand specificity and signaling pathways in sturgeon, and increase our understanding of the functional evolution of TLRs in vertebrates. HighlightsTLR21, TLR22 and TLR25 were identified from the endangered fish cipenser dabryanus.Sequence features of TLR21, TLR22 and TLR25 were analyzed.Expression patterns of TLR21, TLR22 and TLR25 in normal tissues were analyzed.Expression patterns of TLR21, TLR22 and TLR25 in embryonic stages were analyzed.Expression patterns of TLR21, TLR22 and TLR25 in the HK leucocytes were analyzed.