Abstract:In this study, eyestalks of red claw crayfish were dissociated with trypsin to gain single neurons and classified neurons by microscopic inspection. And then, dissociated neurons were cultured into sterile Leibovitz's L-15 medium to screen out proper conditions, etc. The goal of the study is to establish a standard for neurons classification and carry out further research about the endocrine system regulation for crustacean in vitro and in vivo. The results showed that eyestalk XO-SG neurons of red claw crayfish, Cherax quadricarinatus were classified into six different types based on morphological characters with differences on the cell size, and some other differences at microscopic levels. A cell culture approach has been established for XO-SG neurons in vitro, and neurons showed an immediate outgrowth and could regenerate in modified L-15 medium for axon or dendrite. Primary neurons could survive more than 14 d with different growth rate. Some neurons exhibit regeneration of axon or dendrite two days later. Regenerative process can last for 7 days, from then on, the neurons start to shrink and apoptosis. This research provided basis for further study in classifying XO-SG neurons and understanding the endocrine metabolism and regulation at the cell level.

Abstract:Thryssa of the family Engraulidae are widely distributed in the Atlantic Ocean, the Pacific Ocean and the Indian Ocean, with important commercial values in fishery and aquaculture of many countries. In order to achieve unambiguous species recognition and define species status, 652 bp segments of mtDNA cytochrome oxidase subunit I from 62 specimens representing 6 species of genus Thryssa were used as DNA barcoding to examine genetic distances within and among all reported morphological species in coastal waters of China mainland. The results showed that the nucleotide compositions were T: 29.0%, C: 26.3%, A: 25.3%, G: 19.4%, A+T(54.3%) is higher than C+G(45.7%), and the average value of transition/transversion ratios was 3.76. All species were monophyletic and formed into 5 clades with 100% bootstrap supports, of which T.vitirostris and T.mystax were in the same clade, and the average genetic distances within and among clades were 0.2%(0.0%-0.4%), 17.7%(15.7%-19.0%) respectively. Genetic distances among and within T.kammalensis, T.hamiltoni, T.dussumieri and T.setirostris were 15.7%-18.6%, 0.0%-0.3% respectively, i.e., inter-species distances were 10 times greater than intra-species divergence, which confirmed their species status. Although distances within T.mystax and T.vitirostris were both 0.1%, which were close to the 4 congeneric species, and the genetic distance between T.vitirostris and T.mystax was 0.6%, which fell within the scope of most reported intra-species distances, suggesting their closer relationship. Due to some distinguishable morphological and molecular differences, T.mystax and T.vitrirostris should be treated as 2 subspecies or recently diverged species. In conclusion, DNA barcoding based on COⅠ sequences is useful for identifying correctly 4 Thryssa species in coastal waters of China, yet the data also revealed possible cases of unrecognized subspecies or recently diverged species. T.mystax and T.vitrirostris should be managed as 2 significant units, highlighting the importance of synergy between molecular, and biological researches in understanding and documenting Chinese marine fish biodiversity.

Abstract:Keystone species play a decisive role in community structure and stability, and the key species study has both important theoretical and practical significance for the entire ecosystem. In this paper, we tried to screen out the fishery key species from the fish community in the Laizhou Bay based on the bottom trawl survey data in May, 2011. Firstly, we established a relationship network of the 24 fish species according to the predation relationships among fish populations. Then 13 network important indices were calculated using the Ucinet 6 and CoSBiLab Graph1.0 software. A hierarchical clustering was conducted to show the relationships between 13 network indices, and 3 Key Player Problem parameters were calculated using keyplayer1.44 programme. The result showed these 13 indices can be divided into 4 different information groups: a (D, CC, IC, TI¹ and TI⁷), basic information group; b (D_in, H_in and K_t), input information group; c (D_out, H_out and K_b), output information group; d (BC and K), control information group. The species were identified as the key species based on the information of 13 indices and 3 Key Player Problem parameters. Liparis tanakae (D, D_in, BC, CC, IC, H_in, TI¹, TI⁷, K, K_t, F, ^DF and ^DF) and Amblychaeturichthys hexanema (D_out, H_out and K_b) had the highest rank among the 24 study species, and closely tied to others in the fish community, controlled the structure and energy flow of the community, indicating that they were the key species in fish community in the Layzhou Bay. And L. tanakae was the key predator which could control the density of the predators and other competitors in fish community while A. hexanema was the key prey which could restrict the density of other prey species by maintaining the density of the predator.

Abstract:Neosalanx taihuensis is a small-sized freshwater fish from the Salangidae family, and is found mainly in the middle and lower reaches of the Yangtze River, including its tributaries and affiliated lakes. N. taihuensis has a high commercial value, and was therefore introduced to lakes and reservoirs in many Chinese provinces during the 1980s. It very rapidly adapted to the new conditions present in these lakes and reservoirs, quickly becoming the dominant species of fish. This caused a population decline in several native species, which led to notable changes in the community structure of fish and even local extinctions. It is currently understood that the level of genetic variation present in a population influences its ability to adapt to changing conditions. Therefore, in order to better understand the genetic mechanisms that allow N. taihuensis to successfully invade and adapt to new ecosystems, 10 microsatellite loci were performed to comparatively analyze population genetic structure of N. taihuensis between 5 invasive areas and 2 original areas. Genetic diversity parameters indicated that invasive populations from Dianchi Lake, Qionghai Lake, Fuxianhu Lake and Three Gorges reservoir had higher levels of genetic diversity than those living in original areas of Taihu Lake and Chaohu Lake, and invasive population from Erhai Lake had the lower level of genetic diversity than that from Taihu Lake, but higher level of genetic diversity than that from Chaohu Lake, whilst original area of Chaohu Lake had the lowest level of genetic diversity. The value of pairwise genetic distance was from 0.0062 to 0.1013. The genetic distance between Qionghai and Chaohu populations was the greatest, whilst the genetic distance between Taihu Lake and Fuxianhu Lake populations was the smallest. Both genetic distance and UPGMA clustering results showed that the Qionghai population had the farthest genetic relation with the other populations, whilst relationship between Taihu Lake and Fuxianhu Lake populations was the nearest. ANOVA analysis revealed most of the total variation occurred within populations (95.78%) and a small amount of differentiation among populations (4.22%). F_st value (0.0422) was significant, which exhibited a low and significant population differentiation. Out of 21 pairwise comparisons of populations, significant genetic divergence was detected in 18 pairs (85.7%) of populations, which also confirms the significant population differentiation. The value of F_st between Qionghai Lake and Chaohu Lake populations was maximum among all pairwise comparisons. Significant genetic differentiation was not found in three pairs of populations: Taihu Lake vs. Fuxianhu Lake, Taihu Lake vs. Erhai Lake and Fuxianhu Lake vs. Erhai Lake. MVSP principal component analysis demonstated the populations from Qionghai Lake, Three Gorges reservoir, Chaohu Lake and Dianchi Lake displayed a clear differentiation. It could conclude that high level of genetic diversity and the significant difference of genetic structure might be the important reasons for successful invasion of N. taihuensis.

Abstract:For analyzing the cost of cultivation and discussing the economic benefit of scallop at different cultivation density, a field experiment of five density groups (10N/layer, 15N/layer, 20N/layer, 25N/layer and 30N/layer) was carried out from May 2013 to April 2014 in the period when scallop grew from 1 year old to the 2 year old,. The results of scallop height on different cultivation denstiy showed that the average shell height of low density cultivation (10N/layer, 15N/layer) was greater than the average shell height of high denstiy cultivation (20N/layer, 25N/layer, 30N/layer), and the cumulative mortility of low denstiy cultivation was lower than the cumulative mortility of high denstiy cultivation (10N/layer<15N/layer, 20N/layer < 25N/layer < 30N/layer). The low denstiy cultivation groups had outstanding advantage on the shell height and reducing mortility of scallop that grew from one year old to two years old. The result of economic benefit showed that in the two economic patterns of fixed number of scallop and fixed number of cultural raft, the economic benefit of 10 N/layer denstiy group was best; the result of experiment showed that low denstiy cultivation of raft cultural scallop had obvious advantage on shell height character and economic benefit. The concept that the higher cultivation denstiy can bring more economic benenfits should be changed, then the mode of ecologic cultivation would be established.

Abstract:The yellow catfish (Pelteobagrus fulvidraco) is extensively distributed throughout rivers and lakes of China. With overexploitation and water eco-environment deterioration, the resources of P. fulvidracao are severely affected. The genetic diversity and population structure of the commercial fish were examined by using 413 bp of the first hypervariable region of mitochondrial DNA sequences in 258 specimens collected from 9 localities in China. Only 18 polymorphism sites were detected and 22 haplotypes were defined. All of the 9 populations exhibited middle-low haplotype diversity and low nucleotide diversity (h=0.336±0.095~0.700±0.078; π=0.087±0.096%~0.258±0.208%). Low genetic differentiation was estimated within Yellow River deme and Yangtze River deme and significant level of genetic structure was detected between two drainages via AMOVA and pairwise F_ST. The uncertainty of genetic structure in Sheyang River and Hongze Lake populations may be connected with the extensive gene flow between the two populations and the two drainages especially during 1128-1855 AD. Neutrality tests, analysis of mismatch distribution and Bayesian skyline analysis suggested that all populations and Yangtze River deme experienced historical sudden and special population expansion. The climatic changes in the Quaternary may have had an important influence on P. fulvidraco and climate warming during the last interglacial age may have played an important role in population expansion. Information on genetic diversity and genetic structure will have implications for the management of fisheries and conservation efforts.

Abstract:Tannic acid and phytic acid are two important anti-nutritional factors that limit the utilization of plant ingredients in aqua-feed industry. Enrichment-culture technique was used to isolate, screen and identify the efficient microorganisms from soil which could degrade both tannic acid and phytic acid. In this study, 109 microorganisms were isolated, of which 27 isolates could degrade phytic acid and 14 isolates exhibited tannic acid degradation potential. Among these isolates, fungi M-6, M-3 and M-1 degraded both tannic acid and phytic acid, of which fungus M-6 possessed higher degradative capacity than fungi M-3 and M-1. With the increase of fermentative temperature (20℃~35℃), the activities of tannase and phylase produced by fungus M-6 in submerged fermentation both showed a firstly-increase-and-then-decrease trend, with the highest activities observed at 30℃ (P<0.05). With the increase of fermentative pH (4~7), the activities of tannase and phylase of fungus M-6 in submerged fermentation both firstly increased and then decreased (P<0.05). The activity of tannase from pH 5 treatment was significantly higher than other groups (P<0.05), the activity of phylase from pH 5 group was significantly higher than pH 4 and 7 groups (P<0.05), but the difference was not significant compared with pH 6 (P<0.05). The isolate fungus M-6 was characterized as Aspergillus niger, supported by the morphologic taxonomy as well as the sequences of the ITS1-5.8S-ITS2 ribosomal DNA region. Therefore, fungi M-6, M-3 and M-1 could degrade both tannic acid and phytic acid. The optimum fermentative temperature for fungus M-6 was 30℃ and the optimum fermentative pH was 5, under submerged fermentation.

Abstract:Biological sex differentiation and hormonal regulation are related with the expression of CYP19 gene in teleost, and so it can be used to explore the relationship between environmental hormone pollution and gene expression. The Gambusia affinis CYP19a cDNA of full sequence was cloned and analyzed for the first time. This would provide comprehensive experimental data for the study of the CYP19 gene as a biomarker for monitoring environmental hormones. Primers were designed according to the conserved region of CYP19a cDNA, and the conserved region was amplified and sequenced. RACE method was used to amplify the G. affinis CYP19a cDNA of full sequence and its protein sequence homology analysis, and the RT-PCR method was used to detect the transcription level of CYP19a mRNA in the sequence. The full length cDNA sequence of G. affinis CYP19a type was cloned. This sequence contains 2020 bp nucleotides and codes 518 amino acids with an open reading frame (ORF) from 238 bp to 1791 bp. We made an analysis of the signal peptide, transmembrane helices, hydrophilic/hydrophobic, primary structure, secondary structure and tertiary structure. When making the homology comparison with CYP19a gene in gonads of other teleost, the gene fragment similarity of mosquitofish were 93%, 84%, 84%, 71%, 71% and 66% with Fundulus heteroclitus, Oryzias laticeps, Rhabdosargus sarba, Carassius auratus, Cyprinus carpio and Danio rerio respectively. This showed that the cloned gene was G. affinis CYP19a gene. Phylogenetic analysis of the CYP19 gene by using MEGA4.0 indicates that CYP19a gene is highly conserved when clustered with other 19 species ovary-derived P450arom gene. We identified the CYP19a cDNA of full sequence is gonadal aromatase gene, and the proof of G. affinis aromatase is by two genes of CYP19a and CYP19b encoding. G. affinis CYP19a has 3 highly conserved fragments, and has catalytic activity.

Abstract:Ranaviruses not only infect frogs but can also infect many other aquaculture animals, such as fish, amphibians and reptiles. Here, three aquatic animal cell lines, including Chinese giant salamander thymus cell line (GSTC), Epithelioma papulosum cyprini cell line (EPC) and Xenopus kidney cell line (A6), were infected with two raninvirus strains, Rana grylio virus (RGV) and Andriasda davidianus ranavirus (ADRV), respectively. After the ranavirus infection, the morphological changes in these three cell lines were observed by light microscope. The pathological changes and virus titer were recorded and compared at different time post infection. Microscopic observation showed that A6 cells and EPC cells swelled and ruptured. GSTC cells shrank and gathered together to multilayer. The virus titers of two ranaviruses varied significantly in different cells lines. For RGV, 10^3.6, 10^5.9 and 10^6.6TCID₅₀/mL in A6, EPC and GSTC cells respectively. For ADRV, 10^4.3, 10^5.4 and 10^6.1 TCID₅₀/mL in A6, EPC and GSTC cells, respectively. These results showed that the susceptibilities of the same aquatic animal cell lines to different ranavirues were different. This suggested that GSTC is the most sensitive cell line to the ranaviruses. This study provides valuable information and materials for the further study of the pathogenesis of ranaviruses.

Abstract:Nile tilapia (Oreochromis niloticus) is a tropical species that prefers to live in shallow waters, and it has the merits of fast growth and high reprodu-ctive rate, also has the characteristics of less bone spurs, thick fleshy, which has been widely cultured in southern China, and China is by far the largest producer of farmed Nile tilapia. In order to establish a two-dimensional electrophoresis (2-DE) system for proteomic analysis of Nile tilapia muscle, protein samples of Nile tilapia muscle were separated using 2-DE after extracting. By optimizing different preparation methods, different pH of immobilized pH gradients(IPG) gel strips and their lengths, as well as different concentration of SDS-PAGE gel and staining methods (silver staining and Coomassie blue staining), we obtained the best 2-DE experiment conditions. The results show that the optimum method for protein samples preparation of Nile tilapia muscle is combination of liquid nitrogen grinding and acetone precipitation. For 2-DE assay, using 24 cm pH 4-7 IPG gel strips for isoelectric focusing(IEF) and 12.5% gel for the second SDS-PAGE. Besides, dyeing the gels by silver staining could get the optimized 2-DE map of Nile tilapia muscle protein with excellent degree of separation, clear protein points, high resolution and less cross band. The 2-DE technical system was successfully established in this study, which could be used for the separation of proteins in the future Nile tilapia muscle proteomics research.

Abstract:In order to study the role of another Cathepsin L (CtsL) isoform from Hyriopsis schlegelii in immune responses, we isolated and characterized a novel CtsL gene from the freshwater pearl mussel Hyriopsis schlegelii using cDNA library screening and rapid amplification of cDNA ends (RACE), and it was named HsCtsL1-like (GenBank accession No. KF015273). The HsCtsL1-like cDNA was 1280 bp long, and consisted of a 5'-untranslated region (UTR) of 31 bp, a 3'-UTR of 256 bp with a polyadenylation signals (AATAAA) at 11 nucleotides upstream of the poly(A) tail, and an open reading frame (ORF) of 993 bp encoding a polypeptide of 330 amino acids with 36.86 ku predicted molecular weight. The theoretical isoelectric point was 6.23. Sequence analysis showed that the putative HsCtsL1-like protein sequence sharing similar structural features with other reported CtsLs containing a typical signal peptide sequence with 16 amino acids (Met¹~Ser¹⁶), a propeptide inhibitor domain(Try²⁵~Phe⁸⁴) and a mature domain(Leu¹¹⁴~Val³³⁰). And some signature sequence tags were identified for CtsL family proteins in other species including the oxyanion hole (Gln), the active triad formed by Cys, His and Asn, and the conserved ERF/WNIN, GNFD, GCXGG and OCHN motifs can all be found in the HsCtsL1-like protein sequence. Moreover, homology analysis revealed that HsCtsL1-like shared the highest identity (67%) with CtsL1 (AGL33704.1) from the razor clam, Sinonovacula constricta, while the similarities of HsCtsL1-like with the reported Hyriopsis cumingii (ADV03094) and H. schlegelii CtsL (AEX88474) were both 52.91%. The phylogenetic tree revealed that the HsCtsL1-like clustered with the invertebrate CtsL cysteine proteases and was also closely related to S. constricta, Crassostrea gigas and Pinctada fucata CtsL1. Real-time quantitative reverse transcription PCR (qRT-PCR) showed that HsCtsL1-like is expressed in a wide range of tissues including the liver, gills, foot, heart, ovary, testis, kidney, blood, intestines and mantle, with the highest level of transcripts in liver, followed by ovary, testis, intestines and blood, while there is rare expression in other tissues. After Vibrio anguillarum stimulation, the expression level of HsCtsL1-like mRNA was significantly up-regulated at 2 h and 8 h in liver and blood. These results suggested that HsCtsL1-like belongs to subtype 1 of the CtsL subfamily, and it might play an important role in the freshwater mussel, H. schlegelii immune response.

Abstract:To understand the potential mechanisms of the Wnt4 gene at different development stages and adult tissues of the mussel Mytilus coruscus, the Wnt4 gene was cloned by RACE technique. The full length of Wnt4 gene was 3 342 bp with 1 074 bp of open reading frame (ORF) encoding 357 amino acids. Homologous analysis of the amino acids of Wnt4 gene in M. coruscus shared high similarity with Homo sapiens (61%), Mus musculus (61%), Paracentrotus lividus (61%), Chlamys farreri (71%) and Crassostrea gigas (76%). qRT-PCR showed that Wnt4 mRNA expression was detected in all tissues (mantle, adductor muscle, gill, foot, digestive gland, male and female gonad) and the highest expression was found in mantle, indicating that Wnt4 gene may play an important role in shell formation. Relative gene expression of Wnt4 during larval development stages including trochophore, D-shaped, umbo, pediveliger and the juvenile stage was also detected. The Wnt4 mRNA was highest expressed in umbo stage larvae, suggesting that Wnt4 gene may participate in the process of shell morphological structure change and certain organ formation and development. Taken together, the finding provides a new insight into investigating the function of Wnt gene family.

Abstract:Catalase (CAT) is a main member of the plant cell anti-oxidant enzymes, which plays an important role in clearing reactive oxygen species (ROS) under the stressful conditions. In this study, the full-length sequence of CAT gene of Pyropia haitanensis was obtained by rapid amplification of cDNA ends (RACE), which was named PhCAT (GenBank accession: KM655303). Full-length of the gene is 1983 bp which could code a 542 amino acid polypeptide. The sequence similarity was 83.08% when compared with the CAT gene from P. yezoensis. Multiple sequence alignment and phylogenetic tree analysis results confirmed that PhCAT belongs to the CAT gene family. PhCAT gene quantitative analysis found the gene expression levels rose significantly when water loss equal or greater than 60%. The CAT expressions rose under high temperature stress (29℃) regardless of the treatment time. However, the responses of CAT activity showed different trends: CAT activities were significantly decreased by all levels of water loss; but only significantly enhanced under 24 h of high temperature treatment, while at the rest of the time point, enzyme activity level decreased significantly. All of these results suggested that the CAT enzyme plays an important role in responses of P. haitanensis to high temperature stress, but did not exhibit significant effects under the water loss stress.

Abstract:In order to evaluate the antiviral activity against IHNV of rainbow trout IFN-γ2, the target gene (471 bp) was successfully amplified from the cultured primary head kidney leucocyte cultures which had been stimulated by PHA using the primers which were designed based on the NCBI reference sequence. Then, the gene was inserted into the pET32a vector and was expressed in E.coli Rosetta. The result of SDS-PAGE showed that the recombinant protein was successfully expressed in the inclusion bodies, which was about 38.4 ku. The recombinant protein could be simply purified by just washing two times. The antiviral ability of the refolding protein was evaluated in CHSE-214 cells. The activity of rtIFN-γ2 against IHNV was 6.63×10⁶ U/mg. Based on this, the rainbow trout were injected with rtIFN-γ2. The results of real-time PCR indicated that rtIFN-γ2 was potent to induce the comparable levels of IRF-1, IRF-2, IFN-I, IFN-γ and Mx transcription in head kidney, spleen and liver. Generally, the antiviral state on day 1 post immunization was stronger than that on day 2 post immunization. In addition, its protection against IHNV was 40% and 80% when the challenge was on day 1 and day 2 post immunization, which was closely correlated with the kinetic profile of the antiviral state. In conclusion, our study here showed that the rtIFN-γ2 of rainbow trout with the activity was successfully produced in the prokaryotic expression system. Furthermore, the rtIFN-γ2 could elicit the ideal antiviral state and provide the subsequent protection against IHNV.

Abstract:The mature peptide domain of melanin concentration hormone1 (MCH1) from Cynoglossus semilaevis Günther was amplified with specific primers based on their cDNA sequences from NCBI. Then the matured peptide fragments were subcloned into the prokaryotic expression vector pET-32a to successfully construct MCH1/pET-32a recombinant plasmid. The obtained recombinant MCH1 protein expressed in form of inclusion bodies with molecular weight of 29.9 ku and maximally accounted for 50% of the whole bacterial protein post 6-hour induction with 0.2 mmol/L IPTG at 32℃. Western blotting analysis indicated that the obtained recombinant MCH1 protein had the antigenicity to His 6 antibody.The obtained target MCH1 protein was purified using Ni²⁺-NTA affinity chromatography method. The bioactivity of obtained target MCH1 protein was tested by the method of in vitro incubation of pituitary gland. The results showed that MCH1 recombinant protein can effectively stimulate or inhibit the secretion of pituitary MCH and MSH peptides. Wherein, the MCH peptide level increased with the MCH1 protein concentration and peaked at 100 nmol/L, however, it dramatically decreased at 1000nmol/L; the MSH peptide level showed a significant rise at 100nmol/L group, and peaked at 1000nmol/L of MCH1 protein. As for gene expression patterns, the MCH1 and MCH2 mRNAs levels showed similar variation trend, wherein MCH1 mRNA showed upregulated expression with the recombinant MCH1 protein increasing, and peaked at 10 nmol/L group, and MCH2 mRNA levels peaked at 100 nmol/L group. The pituitary PACAP and POMC-a mRNAs both exhibited down-regulated expression trends, and POMC-b, MCHR1, MCHR2, MITF mRNAs expression levels were not significantly affected by exogenous recombinant MCH1. In summary, the recombinant MCH1 protein could adjust the physiological functions of pituitary hormone secretion and gene expression. The present results could provide basic knowledge and technical support for development of high-efficient and specific additives which could be applied to aquaculture of C. semilaevis.