Tracing the genetic footprints of vertebrate landing in non-teleost ray-finned fishes.

Rich fossil evidence suggests that many traits and functions related to terrestrial evolution were present long before the ancestor of lobe- and ray-finned fishes. Here, we present genome sequences of the bichir, paddlefish, bowfin, and alligator gar, covering all major early divergent lineages of ray-finned fishes. Our analyses show that these species exhibit many mosaic genomic features of lobe- and ray-finned fishes. In particular, many regulatory elements for limb development are present in these fishes, supporting the hypothesis that the relevant ancestral regulation networks emerged before the origin of tetrapods. Transcriptome analyses confirm the homology between the lung and swim bladder and reveal the presence of functional lung-related genes in early ray-finned fishes. Furthermore, we functionally validate the essential role of a jawed vertebrate highly conserved element for cardiovascular development. Our results imply the ancestors of jawed vertebrates already had the potential gene networks for cardio-respiratory systems supporting air breathing.

The American Paddlefish Genome Provides Novel Insights into Chromosomal Evolution and Bone Mineralization in Early Vertebrates.

Sturgeons and paddlefishes (Acipenseriformes) occupy the basal position of ray-finned fishes, although they have cartilaginous skeletons as in Chondrichthyes. This evolutionary status and their morphological specializations make them a research focus, but their complex genomes (polyploidy and the presence of microchromosomes) bring obstacles and challenges to molecular studies. Here, we generated the first high-quality genome assembly of the American paddlefish (Polyodon spathula) at a chromosome level. Comparative genomic analyses revealed a recent species-specific whole-genome duplication event, and extensive chromosomal changes, including head-to-head fusions of pairs of intact, large ancestral chromosomes within the paddlefish. We also provide an overview of the paddlefish SCPP (secretory calcium-binding phosphoprotein) repertoire that is responsible for tissue mineralization, demonstrating that the earliest flourishing of SCPP members occurred at least before the split between Acipenseriformes and teleosts. In summary, this genome assembly provides a genetic resource for understanding chromosomal evolution in polyploid nonteleost fishes and bone mineralization in early vertebrates.

Pathogenicity of Plesiomonas shigelloides and Citrobacter freundii isolated from the endangered Chinese sturgeon (Acipenser sinensis).

Chinese sturgeon (Acipenser sinensis) is an indigenous species of China and is listed as a critically endangered species. Recently, second filial generations of Chinese sturgeon in the Yangtze River Fisheries Research Institute suffered from a severe disease. In this study, two kinds of pathogenic bacteria were isolated from diseased sturgeon and identified as Plesiomonas shigelloides and Citrobacter freundii, based on 16S rDNA gene sequence alignment analysis. Antimicrobial susceptibility testing showed that P. shigelloides was resistant to ampicillin, penicillin, midecamycin, oxacillin, and clindamycin; and sensitive to tocefatriaxone, piperacillin, cefoperazone, cefazolin, and ciprofloxacin. C. freundii was resistant to ampicillin, penicillin, midecamycin, oxacillin, and clindamycin; and sensitive to chloramphenicol, cefuroxime, norfloxacin, ciprofloxacin, and ceftazidime. The median lethal dose (LD50) values of P. shigelloides and C. freundii were 4.50 × 103 colony forming units (CFU)/g and 3.20 × 103 CFU/g, respectively. Clinical symptoms of challenged sturgeons were the same as those of naturally infected sturgeons. Histopathological examination disclosed severe damage in the viscera of P. shigelloides and C. freundii-infected sturgeons. This is the first report suggesting that P. shigelloides infection is associated with mortality of Chinese sturgeon. The results of this study revealed the pathogenesis and severe pathogenicity of P. shigelloides and C. freundii in cultured Chinese sturgeon, and offer insights into the prevention and treatment of bacterial infection caused by P. shigelloides and C. freundii in cultured sturgeons.

Screening and identification of female-specific DNA sequences in octaploid sturgeon using comparative genomics with high-throughput sequencing.

In this study, six candidate female-specific DNA sequences of octaploid Amur sturgeon (Acipenser schrenckii) were identified using comparative genomic approaches with high-throughput sequencing data. Their specificity was confirmed by traditional PCR. Two of these sex-specific sequences were also validated as female-specific in other eight sturgeon species and two hybrid sturgeons. The identified female-specific DNA fragments suggest that the family Acipenseridae has a ZZ/ZW sex-determining system. However, one of the two DNA sequences has been deleted in some sturgeons such as Sterlet sturgeon (Acipenser ruthenus), Beluga (Huso huso) and Kaluga (H. dauricus). The difference of sex-specific sequences among sturgeons indicates that there are different sex-specific regions among species of sturgeon. This study not only provided the sex-specific DNA sequences for management, conservation and studies of sex-determination mechanisms in sturgeons, but also confirmed the capability of the workflow to identify sex-specific DNA sequences in the polyploid species with complex genomes.

Transcriptome analysis of ovarian maturation in a chondrostei Chinese sturgeon Acipenser sinensis.

Chinese sturgeon (Acipenser sinensis) with an evolutionary history of over 200 million years, has a long lifespan, and an extremely late and asynchronous sexual maturation (8-18 years for males and 14-26 years for females), resulting in the difficulty of mature adult culture. However, little is known about the regulatory mechanisms of the transition among ovarian maturation stages in the Chinese sturgeon. We performed de novo transcriptome sequencing of the Chinese sturgeon at different ovarian maturation stages (FII, FIII, and FIV). The number of differentially expressed genes (DEGs) between FII and FIII/FIV (33,517/34,217) was more than that between FIII and FIV (22,123), suggesting that the transition from FII to FIII/FIV is more important than that from FIII to FIV for ovarian maturation. The number of upregulated genes was more than that of the downregulated genes, suggesting that increased gene expression was involved in the transition from FII to FIII/FIV. The representative pathways of DEGs were steroid biosynthesis, fatty acid biosynthesis, fatty acid elongation, glycerolipid metabolism, biosynthesis of unsaturated fatty acid, and α-linolenic acid metabolism. The differential expressions from the transcriptome sequencing were validated with real-time reverse-transcription polymerase chain reaction. We also found 13 genes in sexual development, female sex determination, gonadal development, ovarian maturation, ovarian follicle development, and oocyte development pathways, which were differently expressed among fish at FII, FIII, and FIV. We suggest that enhanced synthesis of steroid, unsaturated fatty acid, and α-linolenic acid may contribute to ovarian maturation of the Chinese sturgeon. These potential determinants provide a glimpse of the molecular architecture of ovary development in sturgeons.

Molecular characterization, expression pattern and evolution of nine suppressors of cytokine signaling (SOCS) gene in the swamp eel (Monopterus albus).

Suppressors of cytokine signaling (SOCS) family members have negative effects on cytokine signaling pathways involved in immunity, growth and development. Owing to their typical feature, they have been extensively studied in mammalians, but they have not offered systematic studies among teleosts. In the present study, nine SOCS family genes were identified in the swamp eel genome and analyzed regulation mechanisms of SOCS family members in swamp eels. The open reading frames of MaSOCS1a, MaSOCS1b, MaSOCS2, MaSOCS3a, MaSOCS3b, MaSOCS4, MaSOCS5, MaSOCS6 and MaSOCS7 were 663 bp, 603 bp, 717 bp, 618 bp, 645 bp, 1188 bp, 1488 bp, 1611 bp and 1998 bp and encoded 220, 238, 200, 205, 214, 395, 496, 536 and 655 amino acids, respectively. All SOCS proteins have no signal peptides. Multiple alignment revealed that MaSOCS family members possessed a typical conserved SOCS box and SH2 region. Phylogenetic analyses showed that all SOCS proteins were divided into two main clusters. Taken together with the similarity and identity of SOCS protein amino acids, these results indicated that MaSOCS family members shared conserved with other homologous genes, in which MaSOCS7 was more conserved. Further syntenic analysis confirmed the phylogenetic analysis results and annotation of SOCS protein, suggesting that MaSOCS5 shared a common ancestor gene with that of fish and humans. MaSOCS family members were constitutively expressed in a wide range of tissues with different levels. In particular, spleen and head kidneys play an important role in immune-related pathways. After Aeromonas veronii and polyinosinic-polycytidylic acid (poly I:C) challenge in the spleen and head kidney, MaSOCS family members exhibit different expression profiles. These expression patterns indicated that MaSOCS family members could make acute responses after pathogen invasion. Taken together, these results indicate that MaSOCS family members participate in the immune response against pathogens and offer a solid foundation for future studies of SOCS function.

Integrated metabolomic and transcriptomic analyses suggest that high dietary lipid levels facilitate ovary development through the enhanced arachidonic acid metabolism, cholesterol biosynthesis and steroid hormone synthesis in Chinese sturgeon (Acipenser sinensis).

Chinese sturgeon (Acipenser sinensis) is an endangered species, listed as a grade I protected animal in China. The females rarely successfully develop their gonads from stage II to III in captivity, which handicaps the propagation of cultured Chinese sturgeon. The present study aimed to understand the effects of dietary lipid level on the ovarian development and the related regulation mechanism in female Chinese sturgeon. A 24-month feeding trial was conducted with 10-year-old Chinese sturgeons with ovaries at the developmental stage II, with three experimental diets containing 10, 14 and 18 % lipids. Ovary, muscle and serum samples were collected at four time points (6, 12, 18 and 24 months) for further analyses. Serum metabolomics and ovary transcriptomics analyses were conducted at 18 months. Results showed that only the 18 % lipid diet promoted ovary development to stage IV. Oocytes at stage II in this group also exhibited higher diameter and more lipid droplets. Serum TAG content in the 18 % group was significantly higher than in 10 and 14 % groups (both at 12 and 18 months). Oestradiol content in the 14 % group was significantly higher than in 10 and 18 % groups, except at 24 months. Metabolomic and transcriptomic results indirectly indicated that 14 % of dietary lipids benefited steroid hormone synthesis, while 18 % lipid facilitated arachidonic acid metabolism, cholesterol biosynthesis and vitellogenesis, although serum cholesterol content did not vary with dietary lipid level. In conclusion, 18 % dietary lipid is the optimal level for improving gonad development of female Chinese sturgeon.

Evaluation of the potential probiotic Bacillus subtilis isolated from two ancient sturgeons on growth performance, serum immunity and disease resistance of Acipenser dabryanus.

In the present study, we aimed to screen the potential probiotic Bacillus subtilis isolated from the gut of healthy fish using in vitro assays and to evaluate its effect on Dabry's sturgeon (Acipenser dabryanus) using in vivo feeding experiments. Among the isolates, B. subtilis BSth-5 and BSth-19 exhibited antimicrobial effect against four sturgeon-pathogenic bacteria, including Aeromonas hydrophila, A. veronii, A. media, and Streptococcus iniae. The cell number of B. subtilis BSth-5 and BSth-19 changed little after 2 h of exposure to pH 3.0 or fresh Dabry's sturgeon bile at 2.5% and 5.0%. Meanwhile, B. subtilis BSth-5 and BSth-19 produced extracellular protease, cellulose, and lipase. And it was proved that B. subtilis BSth-5 and BSth-19 were harmless after injection of Dabry's sturgeon. One group of Dabry's sturgeon was fed a control diet and two groups were fed experimental diets containing 2.0 × 108 CFU/g BSth-5 (T1 group) or BSth-19 (T2 group) for 8 weeks. No significant differences in final weight, weight gain rate, and special growth rate were observed in the T1 and T2 groups compared to the control group (P > 0.05), but a significant improvement in survival rate was detected after 4 and 8 weeks of feeding (P < 0.05). After 8 weeks, serum total antioxidant capacity, total superoxide dismutase activity, and IgM levels were significantly higher in the T1 and T2 groups compared to the control group (P < 0.05). Moreover, serum lysozyme activity was significantly higher in the T1 group relative to the control group during the whole experiment period (P < 0.05); however, the differences were not significant between the T2 and control groups (P > 0.05). Serum malondialdehyde levels in the T1 and T2 groups were significantly lower than those in the control group after 4 weeks (P < 0.05). Sturgeons in the T1 and T2 groups showed a higher survival rate after Aeromonas hydrophila infection. To summarize, dietary supplementation with BSth-5 and BSth-19 could enhance the survival rate, antioxidant activity, serum immunity, and disease resistance in A. dabryanus.

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 Dabry's 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 Dabry's 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.

Are river protected areas sufficient for fish conservation? Implications from large-scale hydroacoustic surveys in the middle reach of the Yangtze River.

BACKGROUND: The Yangtze River is the third largest river in the world and suffers from extensive anthropogenic impacts. The fishes in the Yangtze River are essential for the sustainable development of freshwater fisheries and the conservation of aquatic biodiversity in China. However, the fishery resources in the Yangtze River Basin have shown rapid decline due to various human activities. In recent years, nature reserves and germplasm resource reserves have become important means to protect fishes in the Yangtze River. However, nature reserves and germplasm resource reserves that regard freshwater fishes as the main object of protection are not common and have been rarely studied in China. In this paper, a hydroacoustic method and systematic conservation planning tool (Marxan) were combined to evaluate the effectiveness of reserves based on the spatial and temporal patterns of mature fishes in the middle reach of the Yangtze River (MRYR) from 2010 to 2017. RESULTS: The hydroacoustic survey results indicated that in the longitudinal direction, low densities of mature fish species were observed in the Jingzhou (S2) and Jianli (S4, S5, S6) sections, whereas high densities of fish were observed in other sections, such as the Yichang (S1), Chenglingji to Huangsangkou (S7-S12), and Hukou (S15) sections. Among the regions preferred by fish, S7, S10 and S12 were non-reserves. No significant difference in mature fish density was observed between the non-reserves and nature reserves, and a similar result was obtained between the non-reserves and germplasm resource reserves. In Marxan, the optimal conservation sites selected for habitat restoration, such as the Chenglingji, Dengjiakou, Zhuankou, Hankou, Yangluo, and Huangsangkou sections, which are located in non-reserves, were identified in the MRYR. CONCLUSIONS: The Chenglingji, Dengjiakou, Zhuankou, Hankou, Yangluo, and Huangsangkou sections, which are located in non-reserves, play equally important roles in the conservation of fish populations in the MRYR. Our results indicated that further optimization is urgently needed for the currently protected areas in this region. These areas should be designated as reserves, and classification protection mechanisms should be adopted to strengthen the effectiveness of fish conservation in the MRYR.

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

Dabry's 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 Dabry's 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. Dabry's 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 Dabry's sturgeon host defense and contributes to a better understanding of the immune system and defense mechanisms of Dabry's 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.

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

Dabry's 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 Dabry's 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 Darby's 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.

Evolution, expression, and characterisation of liver-expressed antimicrobial peptide genes in ancient chondrostean sturgeons.

Liver-expressed antimicrobial peptide 2 (leap-2) is an evolutionarily ancient molecule that acts as the key component in vertebrate innate immunity against invading pathogens. Leap-2 has been identified and characterised in several teleosts, but not yet in chondrosteans. Herein, the complete coding sequences of leap-2b and leap-2c were identified from expressed sequence tags (ESTs) isolated from Dabry's sturgeon (Acipenser dabryanus) and Chinese sturgeon (A. sinensis), designated as adleap-2b, adleap-2c, asleap-2b, and asleap-2c, respectively. Adleap-2b and adleap-2c sequences share 98% and 100% sequence identity with asleap-2b, and asleap-2c, respectively. Sequence alignment revealed that all four genes contain four cysteine residues, conserved in all fish leap-2 homologs, that form two disulfide bonds. Comparative analysis of the exon-intron structure revealed a three exon/two intron structure for that leap-2 genes in animals, but intron 1 is much longer in sturgeons than in other species. The adleap-2c gene was expressed mainly in the liver of Dabry's sturgeon, and transcription of adleap-2c was significantly up-regulated (p < 0.05) in the liver and midkidney in response to Aeromonas hydrophila challenge. These results suggest adleap-2c may contribute to the defence against pathogenic bacterial invasion. The findings further our understanding of the function of adleap-2c and the molecular mechanism of innate immunity in chondrosteans.

Characterization and expression analysis of g- and c-type lysozymes in Dabry's sturgeon (Acipenser dabryanus).

Dabry's sturgeon (Acipenser dabryanus) is mainly distributed in the upper Yangtze River. Although extensively farmed, little information is available on its innate immune system. In this study, we conducted de novo transcriptome assembly of the head kidney to create a comprehensive dataset for A. dabryanus. A total of 51,324,686 high quality reads were obtained from head kidney cDNA library by the Illumina sequencing platform and 131,261 unigenes were determined to contain complete ORFs. The complete coding sequences of g- and c-type lysozymes were identified from unigenes, and designated as ADLysG and ADLysC. Aeromonas hydrophila infection of Dabry's sturgeon caused a significant increase (P < 0.05) in blood for both lysozyme types, confirming their active defensive role against bacterial infections. This research provides the first characterization of these enzymes in an ancestral chondrostean. These data suggest that ADLysG and ADLysC have the potential for immune defense system against bacterial infection.

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-17ß (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.

Identification and sexually dimorphic expression of vasa isoforms in Dabry's sturgeon (Acipenser dabryanus), and functional analysis of vasa 3'-untranslated region.

Germ cells are set aside from somatic cells early in embryogenesis, and are responsible for transmitting genetic information through generations. Vasa is a highly conserved germ cell marker across animal phyla, and widely used to label primordial germ cells. Dabry's sturgeon is a rare and endangered species distributed solely in the Yangtze River basin. Here, seven vasa isoforms, named Advasa1-7, were isolated and characterized in Dabry's sturgeon. RT-PCR and western blot analyses revealed that vasa mRNA and protein were mainly restricted to the testis and ovary, but exhibited sexually dimorphic expression. Cellular and subcellular localization uncovered that Advasa mRNA and protein displayed mitotic and meiotic expression in females, and mainly showed mitotic expression in males; surprisingly, they exhibited both cytoplasmic and nuclear expression in the ovarian germ cells, while showing exclusively cytoplasmic expression in the testicular germ cells. By microinjecting chimeric RNA consisting of the red fluorescent protein coding region and the Advasa 3'-untranslated region into embryos of Dabry's sturgeon, zebrafish and medaka, we demonstrated that it had the ability to visualize primordial germ cells (PGCs) in Dabry's sturgeon and zebrafish but not in medaka. It seemed that the machinery of vasa 3'UTR RNA localization was conserved between Dabry's sturgeon and ostariophysan, while possibly changed during the divergence of euteleosts and ostariophysan. Finally, Dabry's sturgeon PGCs moved on the yolk ball, and migrated toward the genital ridge via mesenchyme. Taken together, these results provide new information for vasa expression pattern and function, and lay a foundation for PGC cryopreservation and conservation of Dabry's sturgeon.

Differential expression of fertility genes boule and dazl in Chinese sturgeon (Acipenser sinensis), a basal fish.

The gene family DAZ (deleted in Azoospermia), including boule, dazl and DAZ, performs highly conserved functions in germ cell development and fertility across animal phyla. Differential expression patterns have been demonstrated for the family members in invertebrates and vertebrates including fish. Here, we report the identification of boule and dazl and their expression at both RNA and protein levels in developing and mature gonads of Chinese sturgeon (Acipenser sinensis). Firstly, the isolation of the boule and dazl genes in Chinese sturgeon and the observation of the two genes in coelacanth suggest that dazl originated after the divergence of bony fish from cartilaginous fish but before the emergence of the Actinistia. Quantitative real-time PCR and western blot analyses reveal that boule and dazl RNA and proteins are restricted to the testis and ovary. In situ hybridization and fluorescent immunohistochemistry show that the bisexual mitotic and meiotic germ cell expression of dazl RNA and protein is conserved in vertebrates, while Chinese sturgeon boule RNA and protein exhibit mitotic and meiotic expression in the testis, and also likely display mitotic and meiotic expression in female. Moreover, we directly demonstrate for the first time that sturgeon Balbiani body/mitochondrial cloud disperses in the cytoplasm of early developing oocytes and co-localizes with Dazl to some extent. Finally, urbilaterian boule may also have an ancestral function in oogenesis. Taken together, these results provide useful information on the evolution of DAZ family genes, expression patterns and functions in animal reproduction.

One-step detection of Bean pod mottle virus in soybean seeds by the reverse-transcription loop-mediated isothermal amplification.

BACKGROUND: Bean pod mottle virus (BPMV) is a wide-spread and destructive virus that causes huge economic losses in many countries every year. A sensitive, reliable and specific method for rapid surveillance is urgently needed to prevent further spread of BPMV. METHODS: A degenerate reverse-transcription loop-mediated isothermal amplification (RT-LAMP) primer set was designed on the conserved region of BPMV CP gene. The reaction conditions of RT-LAMP were optimized and the feasibility, specificity and sensitivity of this method to detect BPMV were evaluated using the crude RNA rapidly extracted from soybean seeds. RESULTS: The optimized RT-LAMP parameters including 6 mM MgCl2, 0.8 M betaine and temperature at 62.5-65°C could successfully amplify the ladder-like bands from BPMV infected soybean seeds. The amplification was very specific to BPMV that no cross-reaction was observed with other soybean viruses. Inclusion of a fluorescent dye makes it easily be detected in-tube by naked eye. The sensitivity of RT-LAMP assay is higher than the conventional RT-PCR under the conditions tested, and the conventional RT-PCR couldn't be used for detection of BPMV using crude RNA extract from soybean seeds. CONCLUSION: A highly efficient and practical method was developed for the detection of BPMV in soybean seeds by the combination of rapid RNA extraction and RT-LAMP. This RT-LAMP method has great potential for rapid BPMV surveillance and will assist in preventing further spread of this devastating virus.

EST dataset of pituitary and identification of somatolactin and novel genes in Chinese sturgeon, Acipenser sinensis.

Chinese sturgeon (Acipenser sinensis) is a rare and endangered species and also an important resource for the sturgeon aquaculture industry, however, a few genes have been identified in this species. We report here construction of a pituitary cDNA library from a 24 years old female Chinese sturgeon just after its spawning, and obtained 2,025 ESTs from the library. 885 unique sequences were identified, which were categorized into 12 functional groups. More than half of the unique sequences (57%) do not match with annotated sequences in the public databases. Three of these novel genes were further identified. Notably, a full-length of cDNA (1,143 bp) encoding somatolactin of 232 amino acids was identified. Phylogenetic analysis showed 97% amino acid identity with White sturgeon somatolactin. RT-PCR analysis indicated that the somatolactin mRNA was only detected in pituitary. Pituitary-specific expression of the somatolactin suggested that the protein may play important physiological functions in pituitary-endocrine system of the Chinese sturgeon.

Malformations of the endangered Chinese sturgeon, Acipenser sinensis, and its causal agent.

The anadromous Chinese sturgeon (Acipenser sinensis) is endangered and listed among the first class of protected animals in China. The possible causes for the decline of this species are the effects of synthetic chemicals, and loss of critical habitat. Chinese sturgeon in the Yangtze River have accumulated triphenyltin (TPT) to 31-128 ng/g wet weigh (ww) in liver, which is greater than the concentrations of tributyltin (<1.0 ng/g ww). Maternal transfer of TPT has resulted in concentrations of 25.5 +/- 13.0 ng/g ww in eggs of wild Chinese sturgeon, which poses a significant risk to the larvae naturally fertilized or hatched in the Yangtze River. The incidence of deformities in fry was 7.5%, with 1.2% of individuals exhibiting ocular abnormal development, and 6.3% exhibited skeletal/morphological deformations. The incidences of both ocular and skeletal/morphological deformations were directly proportional to the TPT concentration in the eggs of both the Chinese sturgeon and the Siberian sturgeon (Acipenser baerii) in controlled laboratory studies. The rates of deformities in the controlled studies were consistent with the rates caused at the similar concentrations in eggs collected from the field. Thus, TPT is the causal agent to induce the malformation of larvae of Chinese sturgeon. The incidence of deformed larvae of Chinese sturgeon is an indicator of overall population-level effects of TPT on Chinese sturgeon, because TPT at environmentally relevant concentrations can result in significantly decrease both quality and quantity of eggs and spawning frequency of fish.