Comparison of Body Characteristics, Carotenoid Composition, and Nutritional Quality of Chinese Mitten Crab (Eriocheir sinensis) with Different Hepatopancreas Redness.

In this study, we investigated the body characteristics, carotenoid composition, and nutritional quality of Eriocheir sinensis with different hepatopancreas redness (a*). We distributed the crabs into two groups based on the hepatopancreas a* values and compared their body characteristics, chroma, carotenoid composition, and protein, lipid, total sugar, amino acid, and fatty acid content via paired t-test. The results revealed that the relationships between hepatopancreas a* values and crab quality are sex specific. In female crabs, the differences in nutritional characteristics were evident mainly in the hepatopancreases and ovaries. In the redder hepatopancreases, the content of zeaxanthin and ß-carotene increased, and the levels of C22:6n3 and C20:5n3 decreased (p < 0.05). In the ovaries, the content of astaxanthin, canthaxanthin, ß-carotene, umami, and sweet amino acids were lower in the redder hepatopancreas crabs (p < 0.05). In male crabs, there were positive relationships between hepatopancreas a* and amino acid and fatty acid content. The content of leucine, arginine, and total umami amino acids in muscles and of unsaturated fatty acids and n-6 polyunsaturated fatty acids in hepatopancreases and testicles increased with increasing hepatopancreas a* values (p < 0.05). Therefore, the redder the hepatopancreas, the higher the nutritional quality of male crabs.

Reproductive performance and semen characteristics of pond-reared and wild-caught large-sized male broodstock of the Chinese mitten crab Eriocheir sinensis.

There has been a recent emphasis on production of large-sized Eriocheir sinensis broodstock. In China, aquaculturists generally prefer wild-caught (WC) crabs from the Yangtze River as broodstock because offspring performance is superior to that of pond-reared (PR) broodstock. Currently, however, there is a ban on fishing in the Yangtze River, and effects on E. sinensis breeding have not been ascertained. There was comparison in the present study of reproductive performance and semen characteristics of male broodstock of PR and WC groups. After copulation, sperm quantity in the vas deferens of crabs in specimens of both groups was large, although there was a consistent decrease in vaso-somatic index. Although sperm density of PR crabs was less, that of WC specimens remained relatively constant. Specimens of neither group, however, had changes in the hepatopancreas index or condition factor, and sperm survival was close to 100%. Although the acrosome reaction was detected in response to cold-temperature induction, there were differences in extent of reaction to cold temperatures. Importantly, in as many as 98% of sperm from female spermathecae, the reaction was completed, which was considerably greater than 15% for sperm of males post-mating. It is concluded there was no difference between PR and WC crabs with respect to reproductive performance or semen characteristics, and, notably, sperm from PR crabs were of sufficient quality for use in E. sinensis aquaculture enterprises. Accordingly, it is predicted the Yangtze River fishing ban would only have a limited effect on supply of male E. sinensis broodstock.

Dissecting the chromosome-level genome of the Asian Clam (Corbicula fluminea).

The Asian Clam (Corbicula fluminea) is a valuable commercial and medicinal bivalve, which is widely distributed in East and Southeast Asia. As a natural nutrient source, the clam is rich in protein, amino acids, and microelements. The genome of C. fluminea has not yet been characterized; therefore, genome-assisted breeding and improvements cannot yet be implemented. In this work, we present a de novo chromosome-scale genome assembly of C. fluminea using PacBio and Hi-C sequencing technologies. The assembled genome comprised 4728 contigs, with a contig N50 of 521.06 Kb, and 1,215 scaffolds with a scaffold N50 of 70.62 Mb. More than 1.51 Gb (99.17%) of genomic sequences were anchored to 18 chromosomes, of which 1.40 Gb (92.81%) of genomic sequences were ordered and oriented. The genome contains 38,841 coding genes, 32,591 (83.91%) of which were annotated in at least one functional database. Compared with related species, C. fluminea had 851 expanded gene families and 191 contracted gene families. The phylogenetic tree showed that C. fluminea diverged from Ruditapes philippinarum, ~ 228.89 million years ago (Mya), and the genomes of C. fluminea and R. philippinarum shared 244 syntenic blocks. Additionally, we identified 2 MITF members and 99 NLRP members in C. fluminea genome. The high-quality and chromosomal Asian Clam genome will be a valuable resource for a range of development and breeding studies of C. fluminea in future research.

A chromosome-level reference genome of red swamp crayfish Procambarus clarkii provides insights into the gene families regarding growth or development in crustaceans.

Red swamp crayfish Procambarus clarkii is an ecologically and economically important crustacean species. Here, based on a de novo assembly strategy combining PacBio with Hi-C sequencing, we presented a high quality chromosome-level P. clarkii genome. The assembled genome is 2.75 Gb in size with a contig N50 of 216.75 kb. Transposable elements (TEs) make up the largest fraction of the genome (~79.61%), and LINEs comprise the majority of the TEs. Frequent molting and rapid growth of the red swamp crayfish may be explained by the expansion of multiple gene families regarding growth or development. Phylogenetic analysis revealed that P. clarkii diverged from Portunus trituberculatus at 278-407 million years ago (Mya). PSMC analysis identified multiple bottleneck events of the P. clarkii population between 2 kaBP to 14 kaBP. The obtained P. clarkii genome should not only facilitate us understanding the development and evolution of the crayfish species, but also contribute to the genetic improvement in future breeding selections.

Hypoxia-reoxygenation stress modulates the hepatopancreas transcriptome of Chinese mitten crab Eriocheir sinensis.

Hypoxia is a critical, but frequently overlooked problem, which commonly exists in Chinese mitten crab rearing. However, little information is available on the molecular mechanisms of the detrimental effects of hypoxia in this species. In the present study, crabs were subjected to acute hypoxia stress (DO 1.0 mg/L), followed by reoxygenation (DO 6.8 mg/L). Hepatopancreas from five groups of crabs (three to four crabs per group), including normoxia, hypoxia for one and six hours, and reoxygenation for one and 12 h, were used for transcriptome sequencing. The pooled total RNA of all samples were utilized to reconstruct a reference transcriptome with PacBio RS II sequencing, obtaining 49.19 G clean data, with a mean length of 2,180 bp. Seventeen cDNA libraries were constructed and sequenced to identify differentially expressed genes (DEGs) among the different samples (FDR < 0.05 and |log2 fold change| ≥1). A total of 103 and 251 DEGs were identified when exposed to hypoxia for one and six hours, respectively. Totally 462 and 673 DEGs were identified during reoxygenation at 1 and 12 h, respectively. Among these DEGs, two transcripts with complete ORFs were identified to encode hypoxia-inducible factor 1 (Es-Hif-1α/ß), which is a transcriptional activator of various genes correlated to the cellular adaptive responses to hypoxia. Es-Hif-1a/ß expressions were significantly upregulated when exposed to hypoxia treatment, and no significant difference was observed for Es-Hif-1α between hypoxic treatment for 6 h and reoxygenation. The significant KEGG enrichment revealed that the DEGs under hypoxia were mainly enriched in "PPAR signaling pathway", "Gap junction" and "Phototransduction-fly". Compared with crabs in normoxia, even with 12 h of reoxygenation, the hepatopancreas transcriptome under hypoxia remained severely affected, implying its adverse effect on growth and development, or increased susceptibility to diseases.

Genomic organization of the molt-inhibiting hormone gene in the red swamp crayfish Procambarus clarkii and characterization of single-nucleotide polymorphisms associated with growth.

Molt-inhibiting hormone (MIH), a neuropeptide synthesized in the eyestalk in crustaceans, is mainly responsible for the molting by negatively controlling the ecdysteroids secretion. Although there are several reports of the isolation and protein sequencing of MIH in the red swamp crayfish, little is known about the nucleotide sequence and gene organization of this neuropeptide, even less about the association of MIH polymorphisms and growth traits. Here, a 1237 bp full-length MIH cDNA was obtained from the crayfish eyestalk, which encodes a putative protein of 106 amino acids, with a 191 bp 5'-UTR and a 728 bp 3'-UTR. The MIH genomic DNA sequence is 4205 bp in length, which includes three exons interrupted by two introns, and a 929 bp 5'-flanking region. Potential transcription initiation site and transcription factor binding sites were identified in the 5'-flanking region, implying a potential role in transcriptional regulation. Seventeen SNPs in the 5'-flanking region and 3'-UTR were identified, and the associations between these SNPs and growth traits were evaluated with a two-stage design. A SNPs g. -12C > G that showed a significant association with body weight was identified. Individuals with GG genotype had a significantly higher body weight than those with CC genotype (43.98 ±â€¯9.82 g vs. 34.27 ±â€¯6.87 g; P ﹤ 0.001), indicating a beneficial effect of the G allele on the growth of red swamp crayfish. The obtained MIH gene, as well as the identified SNPs, may serve as targets for molecular marker-aided selection in growth improvement of the red swamp crayfish in future studies.

Molecular cloning and functional characterization of a short peptidoglycan recognition protein from triangle-shell pearl mussel (Hyriopsis cumingii).

Peptidoglycan (PGN) is an important target of recognition in invertebrate innate immunity. PGN recognition proteins (PGRPs) are responsible for PGN recognition. In this study, we cloned and functionally analyzed a short PGRP (HcPGRP2) from the triangle-shell pearl mussel Hyriopsis cumingii. The full-length cDNA sequence of HcPGRP2 gene was 1185 bp containing an open reading frame of 882 bp encoding a 293 amino acid protein. HcPGRP2 was predicted to have two SH3b domains and a conserved C-terminal PGRP domain. Quantitative real-time RT-PCR showed that HcPGRP2 was expressed in all examined tissues and its expression was induced most significantly by Staphylococcus aureus and Vibrio parahaemolyticus in the hepatopancreas and gills. RNA interference by siRNA results revealed that HcPGRP2 was involved in the regulation of whey acidic protein, theromacin, and defensin expression. As a pattern-recognition receptor, recombinant HcPGRP2 (rHcPGRP2) protein can bind and agglutinate (Ca2+ dependent) all tested bacteria. rHcPGRP2 exhibited specific binding to PGN but not to lipopolysaccharide. Moreover, rHcPGRP2 inhibited the growth activities of S. aureus and V. parahaemolyticus in vitro and accelerated the clearance of V. parahaemolyticus in vivo. Overall, our results indicated that HcPGRP2 may play an important role in the antibacterial immune mechanisms of H. cumingii.

Genome-wide identification, phylogeny and expressional profile of the Sox gene family in channel catfish (Ictalurus punctatus).

The Sox gene family has been systematically characterized in some fish species but not in catfish Ictalurus punctatus. In this study, 25 Sox genes were identified in the channel catfish genome and classified into seven families based on their conserved domains as follows: eight genes in SoxB group (six in SoxB1 subgroup and two in SoxB2 subgroup); five genes in SoxC group; three genes in SoxD and SoxF groups; four genes in SoxE group; and one gene in SoxH and SoxK groups. The mammalian Sox groups SoxA, G, I, and J were not present in catfish. The number of introns in channel catfish Sox genes varied from zero to 13. Sox genes were distributed unevenly across 17 chromosomes. Five members of the ancestral vertebrate Sox genes (Sox1, Sox4, Sox9, Sox11 and Sox19) experienced teleost-specific whole genome duplication during evolution, and now have two copies on different chromosomes. Expression profiles analyses indicated that the accumulation of Sox genes was associated with different tissues, and the expression pattern also differed among each Sox gene group and duplicated gene. This study constitutes a comprehensive overview of the Sox gene family in channel catfish and provides new insights into the evolution of this gene family.

Eriocheir sinensis microRNA-7 targets crab Myd88 to enhance white spot syndrome virus replication.

MicroRNAs (miRNAs) are non-coding RNAs that regulate gene expression at the posttranscriptional level. In this study, the function of microRNA-7 (miR-7) in host-virus interaction was investigated. Replication of White spot syndrome virus (WSSV) was enhanced with the overexpression of miR-7 and inhibited with the downregulation of miR-7 by using anti-miRNA oligonucleotide AMO-miR-7. The target gene of miR-7 was predicted using bioinformatics methods. Results showed that crab myeloid differentiation factor 88 (Myd88) could be targeted by miR-7. When the expression of Myd88 was knocked down by sequence-specific siRNA, WSSV copies in crabs were significantly increased. Further findings revealed that knockdown of Myd88, Tube, or Pelle inhibited the expressions of interleukin enhancer-binding factor 2 homolog (ILF2) and interleukin-16-like gene (IL-16L). While ILF2 was silenced, IL-16L expression was inhibited. The overexpression of miR-7 inhibited the expressions of ILF2 and IL-16L. Moreover, when ILF2 or IL-16L was silenced, WSSV copies in crabs were increased. Thus, the upregulated expression of miR-7 during WSSV challenge suppressed the host Myd88-ILF2-(IL-16L) signaling pathway in crabs and enhanced WSSV replication. Our study indicated that WSSV utilized crab miR-7 to enhance virus replication during infection.

Molecular cloning, characterization and expression analysis of two juvenile hormone esterase-like carboxylesterase cDNAs in Chinese mitten crab, Eriocheir sinensis.

Precise regulation of methyl farnesoate (MF) titer is of prime importance throughout the crustacean life-cycle. Although the synthetic pathway of MF is well-documented, little is known about its degradation and recycling in crustaceans. Juvenile hormone esterase-like (JHE-like) carboxylesterase (CXE) is a key enzyme in MF degradation, thus playing a significant role in regulating the MF titer. We identified and characterized two cDNAs, Es-CXE1 and Es-CXE2, encoding JHE-like CXEs in Chinese mitten crab. Full-length cDNAs of Es-CXE1 and Es-CXE2 encode proteins composed of 584 and 597 amino acids, respectively, both of which contain a typical carboxylesterase domain. Alignment and phylogenetic analyses revealed that the Es-CXEs are highly similar to those of other crustaceans. To further validate their functions, we evaluated the mRNA expression patterns of the Es-CXEs in various tissues and in different physiological conditions. Tissue-specific expression analysis showed that the two Es-CXEs were predominantly expressed in the hepatopancreas and ovaries, which are the major tissues for MF metabolism. Es-CXE2 expression levels in the hepatopancreas and ovaries were about 100 and 25-fold higher, than the respective Es-CXE1 expressions. During ovarian rapid development stage, the global expressions of Es-CXEs were up-regulated in the hepatopancreas and down-regulated in the ovaries. After eyestalk ablation (ESA), the mRNA expressions of the two Es-CXEs were up-regulated in the hepatopancreas, further indicating their potential in degrading MF. Taken together, our results suggest that Es-CXEs, the key component of the juvenile hormone degradation pathway, may play vital roles in the development and reproduction of the Chinese mitten crab.

A new microsporidium, Potaspora macrobrachium n.sp. infecting the musculature of pond-reared oriental river prawn Macrobrachium nipponense (Decapoda: Palaemonidae).

This paper described a novel microsporidian infection in the pond-reared oriental river prawn Macrobrachium nipponense. A conspicuous symptom of the infection was progressive white opacity associated with the musculature. Although neither bacteria nor viruses were detected in routine diagnostic tests, apparently degenerated microsporidian cells or spores were frequently observed in wet smears of the musculature from diseased prawns. Histological observations also revealed characteristics typical of microsporidian infection throughout the host. Transmission electron microscopy revealed multiple life stages of a microsporidian parasite within the cytoplasm of host muscle cells. In addition, partial small subunit ribosomal RNA (SSU rRNA) gene was obtained by a nested PCR using microsporidian specific primers. A consensus sequence was then deposited in GenBank (accession no. KU307278) and subjected to a general BLASTn search that yielded hits only for microsporidian sequence records. Phylogenetic analysis showed that the isolate was most similar to the fish microsporidian clade containing the genera Kabatana, Microgemma, Potaspora, Spraguea, and Teramicra. The highest sequence identity, 87%, was with Potaspora spp. Based on histological, ultrastructure and molecular phylogenetic data, we erected a new species, Potaspora macrobrachium for the novel microsporidium. The description of microsporidium in this important commercial host was fundamental for future consideration of factors affecting stock health and sustainability.

Transcriptome profiling of the eyestalk of precocious juvenile Chinese mitten crab reveals putative neuropeptides and differentially expressed genes.

Chinese mitten crabs that reach maturity 1 year earlier than normal crabs are known as precocious juvenile crabs. The molecular mechanisms underlying the precocity of the Chinese mitten crab are poorly understood. To identify the genes that may be involved in the control of precocity in Chinese mitten crab, we measured the expression profile of eyestalk genes in precocious and normally developed juvenile crabs using high-throughput sequencing on an Illumina HiSeq 2500 platform. We obtained 56,446,284 raw reads from the precocious crabs and 58,029,476 raw reads from the normally developed juvenile crabs. Reads from the two libraries were combined into a single data set. De novo assembly of the combined read set yielded 78,777 unigenes with an average length of 1563 bp. A total of 41,405 unigenes with predicted ORFs were selected for functional annotation. Among these genes, we identified three neuropeptide genes belonging to the crustacean hyperglycemic hormone family and two neuropeptide genes encoding the chromatophorotropic hormones. Transcriptome comparison between the two libraries revealed 42 genes that exhibited significant differential expression, of which 29 genes were up-regulated and 13 genes were down-regulated in the precocious crabs. To confirm the sequencing data, six differentially expressed genes with functional annotations were selected and validated by qRT-PCR. In conclusion, we obtained the comprehensive transcriptome of the eyestalk tissues of precocious juvenile crabs. The sequencing results may provide new insights into the biomolecular basis of precocity in the Chinese mitten crab.

The first detection of white spot syndrome virus in naturally infected cultured Chinese mitten crabs, Eriocheir sinensis in China.

An epidemic with a high mortality rate (80-100%) recently occurred in the cultured Chinese mitten crab, Eriocheir sinensis, which is a very important economic crustacean species in China. Using negative stain, histopathology and nested PCR supplemented by sequencing we identified white spot syndrome virus (WSSV) in these crabs. Challenge experiments revealed that the disease was caused by WSSV and confirmed the crab's susceptibility to this virus, which was consistent with previous laboratory-based studies. A cumulative mortality of 100% was observed within 10 days post WSSV injection. This is the first report of WSSV-associated disease outbreaks in the Chinese mitten crab, which is normally reported as an important penaeid-shrimp viral pathogen. Furthermore, this is only the second report to describe a significant pathogen in pond-cultured E. sinensis. These results will enhance the early diagnosis of WSSV in the crab farms and help in monitoring efforts directed at determining the prevalence of the virus in E. sinensis.

Generation of myostatin B knockout yellow catfish (Tachysurus fulvidraco) using transcription activator-like effector nucleases.

Myostatin (Mstn), a member of the transforming growth factor ß superfamily, plays an inhibiting role in mammalian muscle growth. Mammals like human, cattle, mouse, sheep, and dog carrying null alleles of Mstn display a double-muscle phenotype. Mstn is conserved in fish; however, little is known whether the fish with mutated mstn display a similar phenotype to mammals because of the lack of mutant fish with mstn null alleles. Previously, we knocked out one of the duplicated copies of myostatin gene (mstna) in yellow catfish using zinc-finger nucleases. In this study, we report the identification of the second myostatin gene (mstnb) and knockout of mstnb in yellow catfish. The gene comprises three exons. It is predicted to encode 373 amino acid residues. The predicted protein exhibits 59.3% identity with yellow catfish Mstna and 57.3% identity with human MSTN. Employing TALEN (transcription activator-like effector nucleases) technology, we obtained two founders (from four randomly selected founders) of yellow catfish carrying the mutated mstnb gene in their germ cells. Totally, six mutated alleles of mstnb were obtained from the founders. Among the six alleles, four are nonframeshift and two are frameshift mutation. The frameshift mutated alleles include mstnb(nju22), an 8 bp deletion, and mstnb(nju24), a complex type of mutation comprising a 7 bp deletion and a 12 bp insertion. They are predicted to encode function null Mstnb. Our results will help to understand the roles of mstn genes in fish growth.

Genetic diversity and population structure of yellow catfish Pelteobagrus fulvidraco from five lakes in the middle and lower reaches of the Yangtze River, China, based on mitochondrial DNA control region.

Genetic diversity and population structure of yellow catfish Pelteobagrus fulvidraco were examined by using mitochondrial DNA control region sequences in 143 specimens sampled from five lakes in the middle and lower reaches of the Yangtze River, China; 151 polymorphic sites defined 72 distinct haplotypes. Haplotype diversity indices (0.903-0.953) and nucleotide diversity indices (0.00378-0.00970) demonstrated low genetic diversity of the yellow catfish populations in the five lakes. The analysis of molecular variance and the fixation index (F(st) = 0.0896) revealed insignificant genetic difference between samples from different lakes. In addition, neutral tests and analysis of mismatch distribution suggested that yellow catfish might have undergone a population expansion. Neighbor-joining tree indicated a correlation between these population genetic differences and geographic distance. This study revealed the extant population genetic diversity and structure of the yellow catfish and was in favor of the related fishery management issues including fishery stock identification, conservation, and artificial breeding.

Identification and characterization of microRNAs in channel catfish (Ictalurus punctatus) by using Solexa sequencing technology.

Channel catfish (Ictalurus spp.) is an economically important species in freshwater aquaculture around the world and occupies a prominent position in the aquaculture industry of the United States. MicroRNAs (miRNAs) play important roles in the regulation of almost every biological process in eukaryotes; however, there is little information available concerning miRNAs in channel catfish. In this study, a small-RNA cDNA library was constructed from 10 tissues of channel catfish, and Solexa sequencing technology was used to perform high-throughput sequencing of the library. A total of 14,919,026 raw reads, representing 161,288 unique sequences, were obtained from the small-cDNA library. After comparing the small RNA sequences with the RFam database, 4,542,396 reads that represent 25,538 unique sequences were mapped to the genome sequence of zebrafish to perform distribution analysis and to screen for candidate miRNA genes. Subsequent bioinformatic analysis identified 237 conserved miRNAs and 45 novel miRNAs in the channel catfish. Stem-loop RT-PCR was applied to validate and profile the expression of the novel miRNAs in 10 tissues. Some novel miRNAs, such as ipu-miR-129b, ipu-miR-7562 and ipu-miR-7553, were expressed in all tissues examined. However, some novel miRNAs appear to be tissue specific. Ipu-miR-7575 is predominantly expressed in stomach. Ipu-miR-7147 and ipu-miR-203c are highly expressed in heart, but are relatively weakly expressed in other tissues. Based on sequence complementarity between miRNAs and mRNA targets, potential target sequences for the 45 novel miRNAs were identified by searching for antisense hits in the reference RNA sequences of the channel catfish. These potential target sequences are involved in immune regulation, transcriptional regulation, metabolism and many other biological functions. The discovery of miRNAs in the channel catfish genome by this study contributes to a better understanding of the role miRNAs play in regulating diverse biological processes in fish and vertebrates.

Bioinformatic identification and validation of conservative microRNAs in Ictalurus punctatus.

Catfish (Ictalurus spp.) is an important aquaculture species around the world, accounting for over 60 % of the domestic aquaculture output in the United States. However, little information is available about I. punctatus miRNAs which play an important role in the regulation of almost every biological process. In the present studies, we applied a bioinformatic strategy to identify 16 miRNAs which represent 12 miRNA families in I. punctatus by searching both expressed sequence tags and genome survey sequences databases. The A + U contents of the candidate pre-miRNA sequence range from 51 to 63 %, and the pre-miRNA sequences vary from 55 to 63 bp in length. To verify the predicted miRNAs, real-time PCR was used to profile the expression of 16 miRNAs with different tissues of I. punctatus. All the miRNA candidates were detectable in five tissues except for ipu-miR-9-3p. Based on sequence complementarity between miRNAs and their mRNA targets, potential targets for I. punctatus miRNAs were predicted. Due to the limited information for the I. punctatus transcripts, only one sequence targeted by ipu-miR-135 was identified to be an I. punctatus EB1 mRNA. Bioinformatic analyses indicated that the 3' untranslated region (3'-UTR) of EB1 mRNA contains an ipu-miR-135 target site, which are perfectly complementary to the seed region (positions 2-8) of the mature ipu-miR-135. I. punctatus miRNAs characterized in this study may provide useful information for the miRNAs research in I. punctatus and other aquaculture species.

Isolation of yellow catfish ß-actin promoter and generation of transgenic yellow catfish expressing enhanced yellow fluorescent protein.

Yellow catfish (Pelteobagrus fulvidraco Richardson) is one of the most important freshwater farmed species in China. However, its small size and slow growth rate limit its commercial value. Because genetic engineering has been a powerful tool to develop and improve fish traits for aquaculture, we performed transgenic research on yellow catfish in order to increase its size and growth rate. Performing PCR with degenerate primers, we cloned a genomic fragment comprising 5'-flanking sequence upstream of the initiation codon of ß-actin gene in yellow catfish. The sequence is 1,017 bp long, containing the core sequence of proximal promoter including CAAT box, CArG motif and TATA box. Microinjecting the transgene construct Tg(beta-actin:eYFP) of the proximal promoter fused to enhanced yellow fluorescent protein (eYFP) reporter gene into zebrafish and yellow catfish embryos, we found the promoter could drive the reporter to express transiently in both embryos at early development. Screening the offspring of five transgenic zebrafish founders developed from the embryos microinjected with Tg(ycbeta-actin:mCherry) or 19 yellow catfish founders developed from the embryos microinjected with Tg(beta-actin:eYFP), we obtained three lines of transgenic zebrafish and one transgenic yellow catfish, respectively. Analyzing the expression patterns of the reporter genes in transgenic zebrafish (Tg(ycbeta-actin:mCherry)nju8/+) and transgenic yellow catfish (Tg(beta-actin:eYFP)nju11/+), we found the reporters were broadly expressed in both animals. In summary, we have established a platform to make transgenic yellow catfish using the proximal promoter of its own ß-actin gene. The results will help us to create transgenic yellow catfish using "all yellow catfish" transgene constructs.

Heritable targeted inactivation of myostatin gene in yellow catfish (Pelteobagrus fulvidraco) using engineered zinc finger nucleases.

Yellow catfish (Pelteobagrus fulvidraco) is one of the most important freshwater aquaculture species in China. However, its small size and lower meat yield limit its edible value. Myostatin (MSTN) is a negative regulator of mammalian muscle growth. But, the function of Mstn in fish remains elusive. To explore roles of mstn gene in fish growth and create a strain of yellow catfish with high amount of muscle mass, we performed targeted disruption of mstn in yellow catfish using engineered zinc-finger nucleases (ZFNs). Employing zebrafish embryos as a screening system to identify ZFN activity, we obtained one pair of ZFNs that can edit mstn in yellow catfish genome. Using the ZFNs, we successfully obtained two founders (Founder July29-7 and Founder July29-8) carrying mutated mstn gene in their germ cells. The mutated mstn allele inherited from Founder July29-7 was a null allele (mstn(nju6)) containing a 4 bp insertion, predicted to encode function null Mstn. The mutated mstn inherited from Founder July29-8 was a complex type of mutation (mstn(nju7)), predicted to encode a protein lacking two amino acids in the N-terminal secretory signal of Mstn. Totally, we obtained 6 mstn(nju6/+) and 14 mstn(nju7/+) yellow catfish. To our best knowledge, this is the first endogenous gene knockout in aquaculture fish. Our result will help in understanding the roles of mstn gene in fish.

Antitumor activity of the water-soluble polysaccharide from Hyriopsis cumingii in vitro.

As a freshwater pearl mussel, Hyriopsis cumingii is widely cultured in China to produce pearls. This study was made to explore the antitumor activity of water-soluble polysaccharide (WSP) from mantles of H. cumingii. Cell viability of human hepatocarcinoma HepG2 cells was estimated by MTT method. Cell cycle analysis was determined by flow cytometry. Apoptosis was observed under fluorescence microscopy and confirmed by DNA fragment assay. Reverse transcriptase-polymerized chain reaction (RT-PCR) and immunocytochemistry were carried to evaluate c-myc, bcl-2 and cyclinD1 gene expressions in HepG2 cells. Furthermore, intracellular reactive oxygen species (ROS) production was assessed by flow cytometry. After WSP treatment, the growth of HepG2 cells was inhibited and most of cells arrested in G(0)/G(1) phage (p < .01); apoptotic changes in nucleus and cytoplasm were also observed in WSP-treated cells; percentage of apoptotic rate significantly increased in a dose-dependent manner (p < 0.001); DNA fragmentation was detected in treated HepG2 cells; The mRNA level and protein level of c-myc, bcl-2 and cyclinD1 were decreased in the treated HepG2 cells. ROS was significantly increased in a dose- and time-dependent manner as well. The results suggested that WSP has potent antitumor activity.