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Biomacromolecules, including matrix proteins, are important in shell-mineralization processes, such as inducing crystal nucleation, regulating crystal polymorphism and morphology. In this study, we identified the novel matrix protein hc-upsalin from the freshwater pearl mussel Hyriopsis cumingii, associated with its significantly high similarity to the known matrix protein upsalin in the freshwater mussel Unio pictorum. The full-length cDNA of hc-upsalin encodes 119 amino acids, including a potential signal peptide of 17 residues. The mature protein is characterized by high proportions of Gly (13.7%), Cys (11.8%), and Pro (11.8%), with a molecular weight of 11.4 kDa and a theoretical pI of 6.93. Hc-upsalin is specifically expressed in muscular tissues mostly in the mantle, with positive signals detected in dorsal epithelial cells of the mantle by in situ hybridization, and decreased expression of hc-upsalin leaded to random accumulation of tablet, suggesting its involvement in shell nacreous-layer biomineralization. Furthermore, hc-upsalin gene-expression patterns during pearl biomineralization indicated a potential role in the process of pearl nacreous-layer calcification.

In this study, Illumina MiSeq sequencing was used to characterize the bacterial communities in the biofilms that formed on different substratum types including glass, steel, acrylic and hydrophobic glass. The link between bacterial community structure and plantigrade settlement of the mussel Mytilus coruscus was explored. The settlement preference of plantigrades for non-biofilmed substrata was also examined. High-throughput sequencing revealed that the bacterial communities of biofilms varied on different substrata. The overwhelming majority of sequences (>80% of all sequences) belonged to only three bacterial phyla: Proteobacteria, Bacteroidetes and Firmicutes. The phylum Proteobacteria was the dominating group in all biofilm samples. Cluster analysis based on Bray–Curtis distances analysis showed that communities in glass samples differed from those on other samples. The bacterial densities of the biofilms developed on glass were higher than those on other three samples. The present results showed that the percentage of plantigrade settlement was higher in biofilms on glass and steel samples than that in acrylic and hydrophobic glass samples. The variation of bacterial community structure might lead to differences of biofilm activity. There was no selective choice of non-biofilmed substratum for plantigrades. Thus, substratum type affected cell densities of bacteria and bacterial communities of biofilms, which were differentially attractive to plantigrades of M. coruscus.

B cells in fish were recently proven to have potent innate immune activities like macrophages. This inspired us to further explore the innate nature of B cells in fish. Moreover, antimicrobial peptides (AMPs) are representative molecules of innate immunity, and they can modulate the functions of macrophages. These make fish an appropriate model to study the interactions between B cells and AMPs. Interestingly, the results in this study revealed that the IgM+ and IgT+ B cells of rainbow trout could express multiple AMP genes, including four cathelicidin genes and one β-defensin gene. The expression levels of the cathelicidin genes were obviously higher than that of the β-defensin gene. Further studies revealed that intracellular, extracellular, in vitro, and in vivo stimulations could significantly increase the expression of the cathelicidin genes in trout IgM+ and IgT+ B cells but not the expression of the β-defensin gene, indicating that cathelicidin peptides are the main innate immune effectors of trout B cells. More interestingly, we found that cathelicidin peptides could significantly enhance the phagocytic, intracellular bactericidal, and reactive oxygen species (ROS) activities of trout IgM+ and IgT+ B cells, a phenomenon previously reported only in macrophages, and these activities might also be mediated by the P2X7 receptor (P2X7R). These results collectively suggest that B cells play multiple roles in the innate immunity of fish, and they provide new evidence for understanding the close relationship between B cells and macrophages in vertebrates.

Signal transducer and activator of transcription 3 (STAT3) binds to Janus kinase 2 (JAK2) to initiate the JAK2/STAT3 signal transduction pathway, which plays an important role in cancer cell proliferation, immune regulation, reproduction, lipid metabolism, and other physiological processes of the organism. In this study, the cDNA sequence of the STAT3 gene from grass carp was cloned using RACE (rapid-amplification of cDNA ends). Twelve characteristics of the STAT3 gene and its encoded protein sequence were predicted and analyzed using bioinformatics methods; these features included the general physical and chemical properties, the hydrophobicity, the secondary structure and the three-dimensional structure of the protein. Quantitative real-time PCR was employed to detect grass carp STAT3 expression pattern in different tissues. The results showed that the full-length STAT3 gene from grass carp is 2739-bp long and contains a 216-bp 5′UTR, a 300-bp 3′UTR, and a 2223-bp open reading frame (ORF) that encodes a 740-amino acid peptide. The deduced protein exhibited 99%∼94% homology to the STAT3 protein of zebrafish (Danio rerio), medaka (Oryzias latipes), turbot (Scophthalmus maximus), white-spotted char (Salvelinus leucomaenis), mandarin fish (Siniperca chuatsi), rainbow trout (Oncorhynchus mykiss), and green pufferfish (Tetraodon fluviatilis). The deduced grass carp STAT3 protein contains a protein interaction domain, an alpha domain, a DNA binding domain, and an SH2 domain. The STAT3 protein of grass carp is a hydrophilic and non-secretory protein, and its molecular mass and isoeletronic point were found to be 98,5412.1 Da and 6.39, respectively. The structural elements of STAT3 included α-helixes, β-sheets, and loops. The grass carp STAT3 is expressed in all of the six tissues tested, which were the liver, spleen, gill, muscle, heart, and brain. The highest expression level was found in the liver (P

In this study, the dicer gene (designated as cidicer) was identified and characterized from grass carp Ctenopharyngodon idella. The complementary DNA (cDNA) of cidicer contained an open reading frame (ORF) of 5646 nucleotides (nts) encoding a putative protein of 1881 amino acids (aa). The deduced Dicer protein contained all known functional domains identified in other organisms. Tissue tropism analysis indicated that cidicer is abundantly expressed in brain, gill, head kidney, liver, spleen, heart, muscle and intestine. In the C. idella kidney (CIK) cells, messenger RNA (mRNA) expression of cidicer was significantly up-regulated at 24 h (6·36-fold, P < 0·01) after grass carp reovirus (GCRV) infection, and its transcriptional expression level was also transiently induced to a high level (6·54-fold, P < 0·01) at 2 h post-stimulation of synthetic double-stranded polyinosinic-polycytidylic potassium salt [poly(I:C)]. In vivo analysis further showed that the expression of cidicer mRNA in the liver was induced to a significantly high level at 12 h (8·46-fold, P < 0·01), and then dropped to normal level at 72 h post-challenge with GCRV. The transcriptional expression pattern of cidicer in the spleen tissue was similar to that of liver tissue upon GCRV challenge. These results collectively implied that the identified cidicer was an inducible gene responding to viral infection both in vitro and in vivo, and the data would shed light on the interaction between RNA interference (RNAi) antiviral pathway and aquareovirus infection.

This study reported the nutritional components and heavy metals of Hijiki seaweed (Hizikia fusiformis) and investigated the efficacy of aqueous extraction method to remove inorganic arsenic from Hijiki seaweed. Hijiki contained 12.2% of crude proteins, 1.8% of total lipids, 14.0% of ashes, 11.3% of total fiber with dry weight, respectively. The content of protein and ashes were slightly lower than other edible seaweeds including Laminaria japonica, Porphyra tenera, Undaria pinnatifida, Palmaria palmate, while the total fiber was much higher compared with those four seaweeds (6.7-7.8%), which showed more benefits to human health especially to intestines. However, Hijiki contained extremely high amount of heavy metal arsenic (total arsenic: 100mg/kg; inorganic arsenic: 44 mg/kg, dry weight), which might be a major safety concern for human consumption. We developed an aqueous removal method to remove inorganic arsenic from Hijiki. The majority of inorganic arsenic (95.9-96.6%) was removed at optimal condition (50°C, pH 4, 8-12h).The inorganic arsenic content of Hijiki ranged from 0.37 mg/kg to 0.46 mg/kg with wet weight after the treatment and met the hygienic standard for marine algae and algae products.

Based on the cDNA sequence of GPX in Hyriopsis cumingii, the complete genomic DNA of GPX gene and it's 5'-flanking region were identified from H. cumingii using PCR and genome walking technique. The length of the complete genomic sequence was 6 708 bp including the 5'-flanking region, two exons, and one intron. Sequence analysis of the 992 bp 5'-flanking region revealed that it contained a core promoter element (TATA-box) and other transcription regulation elements such as AP1, C/EBP, and CdxA. The sequence lengths of the two exons were 273 bp and 991 bp, respectively, and the intron was 4 491 bp in length. Sixteen single nucleotide polymorphisms (SNPs) were detected in the GPX gene from resistant stock (RS) and susceptible stock (SS) of H. cumingii. These polymorphisms were analyzed with regard to resistance to Aeromonas hydrophila. Among them, three SNPs including A-99G, A-86C, and A-49C in GPX promoter and five SNPs including A2841T, C2847T, G3146C, A3150G, and G4645T in GPX introns were associated with resistance/susceptibility of H. cumingii to A. hydrophila, both in genotype and allele frequency. Linkage disequilibrium analysis revealed that A-86C, A-49C, C2847T, A3150G, G4645T, A2841T, and G3146C were in high linkage disequilibrium, and haplotype analysis revealed that the frequency of two major predominant haplotypes (ACTGT and TG) in the resistant group was significantly higher than that in the susceptible group. The results suggest that the polymorphic loci in the GPX gene could be potential genetic markers for future molecular selection of strains resistant to diseases.

The means of survival of genomic dsRNA of reoviruses from dsRNA-triggered and Dicer-initiated RNAi pathway remains to be defined. The present study aimed to investigate the effect of Grass carp reovirus (GCRV) replication on the RNAi pathway of grass carp kidney cells (CIK). The dsRNA-triggered RNAi pathway was demonstrated unimpaired in CIK cells through RNAi assay. GCRV-specific siRNA was generated in CIK cells transfected with purified GCRV genomic dsRNA in Northern blot analysis; while in GCRV-infected CIK cells, no GCRV-specific siRNA could be detected. Infection and transfection experiments further indicated that replication of GCRV correlated with the increased transcription level of the Dicer gene and functional inhibition of in vitro synthesized egfp-siRNA in silencing the EGFP reporter gene. These data demonstrated that although only the genomic dsRNA of GCRV was sensitive to the cellular RNAi pathway, unidentified RNAi suppressor protein(s) might contribute to the survival of the viral genome and efficient viral replication.

Antarctic krill (Euphausia superb) is a species of krill (shrimp-like crustaceans) found in the Antarctic waters of the Southern Ocean. It is the most abundant species of krill, which catchable stock is believed to amount up to 10 million tons per year, and may be the most potential marine resource for utilization as food in the world. This paper compared the nutritional components and heavy metals of edible portions of Antarctic krill, greasy-back shrimp (Metapenaeus ensis), Chinese white prawn (Exopalaemon modestus), and oriental river prawn (Macrobrachium nipponense). Antarctic krill meat contained 76.39% of water, 17.22% of crude proteins, 2.66% of crude lipids, and 1.43% of ashes, respectively. At dry basis, Antarctic krill had relatively lower content of crude protein (72.92%) than oriental river prawn (85.35%), greasy-back shrimp (81.12%), or Chinese white prawn (78.18%). However, it had significantly higher lipid content (11.25%) than the three species of shrimps (4.89%~6.65%). And the total amino acids in dry samples of Antarctic krill meat was 74.46g/100g, which was lower than the shrimps, but the essential amino acid content (45.90g/100g protein) was higher than others. Regarding to minerals, no significant difference was found in Antarctic krill and the shrimps, with exceptions that Antarctic krill contained two to three times higher content of magnesium (458.28mg/100g) and copper (4.96mg/100g) than shrimps. In addition, the heavy metals including lead (Pb), mercury (Hg), arsenic (As), chromium (Cr) in Antarctic krill meat met the limit standard of contaminants in aquatic products. But fluorine content of Antarctic krill meat surpassed the safety limit (2.0mg/kg) and might be a safety concern.

The polyphenol and flavonoid contents and antioxidation capabilities, including total reducing power, DPPH radical scavenging capability and lipid peroxidation inhibition, of ethanol extracts of water hyacinth (Eichhomia crassipes) were investigated and compared with those extracted from red tide seaweed (Enteromorpha prolifera) and from the most common seaweed (Porphyra haitanensis). Results showed that E. crassipes contained higher amounts of polyphenols [6.68 mg gallic acid equivalents (GAE)/g] in the leaf than in the stem (4.37 mg GAE/g) or in P. haitanensis (4.27 mg GAE/g). The E. crassipes also contained higher contents of flavonoids [1524 and 453 mg rutin equivalents (RE)/g in leaf and stem, respectively] than did E. prolifera (233 mg RE/g). Accordingly, both the leaf and stem of E. crassipes had relatively higher reducing power [118.35 and 47.21 100μg ascorbic acid equivalents (AscAE)/g, respectively] and DPPH radical scavenging capability (74.6% for leaf and 62.7% for stem) when compared with E. prolifera (reducing power of 16.5 and DPPH radical scavenging capability of 42.96%). Statistical analysis showed that the antioxidation capabilities, especially reducing power, of extracts of the three aquatic plants were positively correlated with their total polyphenol contents, but not with the contents of flavonoids. The correlation coefficient (r) of the content of polyphenols and reducing power of those extracts was r=0.9028, that for DPPH scavenging and lipid peroxidation inhibition was r=0.9311 and 0.9099, respectively. These results indicated that phenolic compounds are the main compounds contributing to the antioxidation activities of seaweed extracts. E. crassipes can be a source for extracting natural antioxidants.

Collagen is regarded as one of the most useful biomaterials. Collagen was extracted from wild Chinese soft-shelled turtles (Pelodiscus sinensis) and its molucular patterns and antioxidant activities were investigated. The SDS-PAGE analysis revealed that the collagen had three α bands with an approximate molecular weight of 400-410 kDa, which was similar to type V collagen with a molecular pattern of α1(V)α2(V)α3(V) or α1(V)[α2(V)]2. The antioxidant activity of collagen were examined by the radical-scavenging activity (RSA) assay and reducing power (RP) methods respectively. In the RSA assay, the DPPH free radical scavenging capability of the sample collagen was positively correlated with its concentration with a IC50 of 1.95mg/ml. The same trend was found in the RP assay. The reducing power of the sample collagen was also dependent with its dose employed.

The complete mitochondrial genome of Cristaria plicata was obtained using long amplification polymerase chain reaction (LA-PCR). Analysis showed that the total length of sequence was 15 712 bp, including 13 protein-coding genes, 2 ribosomal RNA genes, 22 transfer RNA genes, and 26 non-coding regions ranged from 2 bp to 328 bp in size. The nucleotide composition of A, T, C, G were 36.54% 27.22% 23.22% and 13.02%, respectively. Most genes were encoded on the L strand, While ND3-ND5, ND4L, COI-COIII, ATP6, ATP8, tRNAAsp, and tRNAHis were encoded on the H strand. The arrangement of genes in C. plicata was identical to that of Lampsilis ornata, but was different from that of Hyriopsis cum-ingii between COII and 12S rRNA in the same family. Thirteen protein genes contained 3 initiation codons, i.e., I (AUU, AUC), V (GUG), and M (AUA, AUG), all of which had complete stop codons (UAA or UAG), except for the stop codon in ND2 that had an incomplete T. Fifteen tRNAs had typical cloverleaf structure, except for tRNAThr, tRNALys, tRNASer(UCN), tRNAAsp, tRNAArg, tRNATyr, and tRNAMet. Like other freshwater bivalvia, C. plicata contained ATP8 gene, which might be related to the balance of osmotic pressure in the cytoplasm.

Reproductive strategy is a central feature of the ecology of invasive species as it determines the potential for population increase and range expansion. The red swamp crayfish, Procambarus clarkii, has invaded many countries and caused serious problems in freshwater ecosystems. However, little is known about the effects of environmental conditions on crayfish paternity and offspring traits in the wild. We studied these reproductive characteristics of P. clarkii in wild populations from two different habitats (ponds and ditches) in three locations with different environmental conditions in China. Genotyping of 1,436 offspring and 30 mothers of 30 broods was conducted by using four microsatellites. An analysis of genotyping results revealed that gravid females were the exclusive mother of the progeny they tended. Twenty-nine of 30 mothers had mated with multiple (2-4) males, each of which contributed differently to the number of offspring in a brood. The average number of fathers per brood and the number of offspring per brood were similar (P > 0.05) among six sampling sites, indicating that in P. clarkii multiple paternity and offspring number per brood are independent of environmental conditions studied. Indirect benefits from increasing the genetic diversity of broods, male and sperm competition, and cryptic female choice are a possible explanation for the high level multiple paternity and different contribution of fathers to offspring in this species.

An Ecopath model was constructed to describe the ecosystem of Lake Qiandaohu, a stock-enhanced large deep Chinese reservoir with silver carp (Hypophthalmichthys molitrix) and bighead carp (Aristichthys nobilis) dominated in its pelagic community. The food web structure and ecosystem property of the reservoir were analyzed and evaluated. The results showed that there were seven trophic levels (TLs) in the system, with the trophic flows primarily occurring through the first four TLs. The food web structure of this ecosystem was characterized with a bulged intermediate trophic level, which was contrary to the wasp-waist food web structure occurred in most natural aquatic ecosystems. The corresponding trophic flow pattern showing by transfer efficiencies (TEs) between TLs indicated that the trophic flows primarily went through from TL I to II with a high TE (of over 50%) and through a flow loop or short cut between detritus and TL II but greatly reduced from TL II to III with a lowest TE of 2.5% due to the bulged biomass at TL II. The trophic flow loop greatly increased the throughput recycled, which, together with high connectance index (CI), system omnivory index (SOI), Finn's cycled index (FCI) and Finn's mean path length (FML), might be beneficial to the maintaining of ecosystem stability. Finally, ecosystem property indices showed that this reservoir had a high value of Pp/R and Pp/B, indicating this ecosystem of short history was immature, but highly productive. This silver carp and bighead carp dominated deep reservoir ecosystem had both the characteristics of high productivity of an immature ecosystem and the feature of high stability of a mature ecosystem.

Hyriopsis cumingii is the most important species for freshwater pearl culture in China. Seventeen polymorphic microsatellite repeat markers were identified from mantle expressed sequence tags of Hyriopsis cumingii. The number of alleles per locus ranged from two to 22, the observed and expected heterozygosities ranged from 0.042 to 1.000 and from 0.061 to 0.962, respectively. These markers should be useful for population genetics studies, parentage and genome mapping in this species.

The yellow catfish Pelteobagrus fulvidraco is a freshwater fish species. Due to overfishing and pollution of freshwater ecosystems, the wild stocks of this fish reduced substantially. We isolated and characterized 12 polymorphic microsatellites of this species. The number of alleles at the 12 microsatellite loci ranged from four to eight, with an average of 6.6/locus. The average observed heterozygosity was 0.72, whereas the expected heterozygosity ranged from 0.60 to 0.86 (average: 0.80). All 12 microsatellites conformed to Hardy–Weinberg Equilibrium and were in linkage equilibrium. These 12 novel microsatellites could facilitate studies of genetic diversity and population structure of the yellow catfish to supply necessary information of conservation of the yellow catfish.

Here we report the isolation and characterization of 17 polymorphic loci isolated from a partial genomic DNA library of grass carp ( Ctenopharyngodon idellus ) enriched for CA repeats. We tested variability of these microsatellites on 24 unrelated individuals collected in China. All microsatellites were polymorphic. The average allele number was 7.9 per locus, ranging from four to 13. The observed heterozygosity was from 0.46 to 0.88 with an average of 0.71, whereas the average expected heterozygosity was 0.78. Sixteen of the 17 microsatellites conformed to Hardy–Weinberg equilibrium, and inherited independently. These microsatellites can be used to study genetic diversity and population structure of wild populations, and facilitate selective breeding of cultured broodstocks.

Grass carp reovirus (GCRV) is one of the most serious pathogens threatening grass carp (Ctenopharyngodon idella) production in China. Through sequence analysis, the co-existence of two genetically distant grass carp reoviruses, named GCRV-JX01 and GCRV-JX02, was revealed in the same diseased grass carp sample collected in 2011. GCRV-JX01 and GCRV-JX02 shared high levels of homology with GCRV-873 and GCRV-GD108, respectively. In contrast to GCRV-JX01, GCRV-JX02 induced no cytopathic effect in infected cells. A quantitative real-time PCR assay was employed to monitor the replication efficiency of both virus strains in either Ctenopharyngodon idella kidney (CIK) cells or infected cell supernatant. The results demonstrated that, although GCRV-JX02 did reduce the cellular replication level of GCRV-JX01 up to 10-fold during co-infection, there was no significant impact on the productive virus progeny level in supernatant compared to that of cells infected by GCRV-JX01 alone. To validate the hypothesis that both viruses might co-infect grass carp without significant interference in the field, we collected clinical samples from two different fish farms in 2012 and monitored virus loads for each fish. The data showed that 55 % of the collected fish samples were co-infected by GCRV-JX01 and GCRV-JX02, and the single virus infection rate was 10 % for GCRV-JX01 and 20 % for GCRV-JX02. For both viruses, the in vivo viral loads under co-infection and single viral infection were similar. No serological cross-reaction or cross-protection occurred between GCRV-JX01 and JX02 in our immunization and challenge tests. This new information on co-infection by two genetically distant virus strains should be helpful for designing vaccines targeting the causative agents of grass carp haemorrhagic disease.

17 half-sib families and 51 full-sib families of Hyriopsis cumingii were constructed by nested design.40 individuals were randomly selected from each of these full-sib families when the individuals in the cages reached 1 cm,and the number was 2 040 in all.Shell length,shell height,shell width and body weight were measured of these individuals,and these data were used for genetic analysis.The heritability of these four traits was 0.356±0.047,0.488±0.060,0.453±0.055 and 0.518±0.050 respectively.The phenotype correlation and genetic correlation was between 0.476-0.709 and 0.574-0.868.There was enough genetic variance for selective breeding and when we have selective breeding program on weight,the other three traits would have some genetic improvement at the same time.

To examine the effect of different two-carp ratios between silver carp Hypophthalmichthys molitrix and bighead carp Aristichthys nobilis cocultured in the mussel pond on growth of freshwater pearl mussel Hyriopsis cumingii and water quality,an enclosure experiment was carried out in Tangxi of Zhejiang Province from April 23 to September 21,2008.Two-carp ratio was set at six levels:0/0(control group),100/0,70/30,50/50,30/70 and 0/100.Shell length,shell width and wet weight of pearl mussels were measured at the beginning and the end of the experiment,respectively;and the water quality parameters including NO3-N,NO2-N,NH3-N,TN,TP,PO4-P,COD were determined twice a month during the experiment.The results showed that the relative growth rate of mussel shell length in level 100/0(silver carp vs.bighead carp ratio)were significantly lower than that of levels 0/0,50/50 and 0/100(P0.05),and no significant differences were found among survival rate,growth rate of shell width and wet weight of mussels at various polyculturing two-carp ratio levels(P0.05).Besides,enclosures at level of 30/70 had better water quality in which TP was significantly lower than 100/0(P0.05),COD was significantly lower than 100/0 and 70/30(P0.05),NH3-N was significantly lower than 100/0(P0.05)and PO4-P was significantly lower than 70/30(P0.05).So the ratio of 30/70 of silver carp vs.the bighead carp was the best polyculture ratio for mussel culture in terms of the growth and water quality parameters.