Abstract:Distant hybridization is the important pathway of forming hybrid vigor and polyploid individuals. By combined distant hybridization and interploid hybridization, we have obtained different ploid crucian carp. MeCP2 is a kind of DNA methylation binding protein and functions as a crucial gene expression regulator by translating epigenetic DNA methylation to proper cell response. To discover the epigenetic regulation mechanism of the reproductive ability in different ploid crucian carp, this study cloned the MeCP2 gene in different ploid crucian carp based on the transcriptome of testis. Sequence alignments indicated that there are genetic recombination and variation in genome during distant hybridization. A pair of common primers was designed to analyze the spatial and temporal expressional characteristics of MeCP2 gene during the development process of different ploid fish through semi-RT PCR and real-time PCR. The results showed that MeCP2 gene was expressed widely with the highest expression in the brain. Compared the different stages of gonadal development, the expression of MeCP2 gene decreased coupled with gonad maturation. While compared the different ploid crucian carp at the same developmental stages, MeCP2 gene was expressed significantly higher in the ovary of triploid fish than in diploid and tetraploid fish, while increased in the testis coupled with the ploid change. Taken together, the expression of MeCP2 gene is closely related to ovary maturation of different ploid fish, which lays the foundation for studying the mechanism and application of fish infertility in genetic breeding and aquaculture.

Abstract:Olfaction is the important tool of sensing their surroundings to fish and may take part in spawning migration. Fish perform olfactory function by the means of the olfactory receptor proteins that olfactory receptor genes encode. The main olfactory receptor gene is the largest number of olfactory receptor families, which can identify water-soluble odor molecules. To find out the main olfactory receptor genes' differences of C. nasus between settlement population and migratory population, the MOR-2AK2 cDNA sequence of migratory C. nasus using rapid amplification of cDNA ends (RACE) was cloned. The sequencing results showed that the open reading frame was 972 bp in length, encoding 323 amino acid residues. The MOR-2AK2 protein had seven hydrophobic and alpha helix transmembrane structures, belonging to the G-protein-coupled receptor superfamily. Based on qRT-PCR among 10 tissues in migratory C. nasus, the result revealed that MOR-2AK2 was expressed in the olfactory rosettes and sex glands more highly than in others. Further analysis showed, the expression level in olfactory rosettes was 5 to 27 times higher than that in sex glands. In addition, there were differences in tissue expression quantity between male and female. The female olfactory rosette's expression level was 2 times that in male olfactory rosette. But the testis' expression level was 2 times that in ovary. Sequence analysis displayed that the 5 untranslated regions of MOR-2AK2 gene had microsatellite sequence, (GT)₅. The settlement population had a 14 bp more sequence (GTGTGTGTGTGTTT) than the migratory population. And the amino acid sequence similarity was just 84% between them. From the above, MOR-2AK2 gene not only took part in olfactory function, but also participated in gonad development or breeding migratory, and may be involved in the formation of settlement population.

Abstract:Fatty acyl-CoA Δ6-b desaturase is a membrane-bound enzyme, and it is the rate-limiting enzyme in the biosynthetic pathway of highly unsaturated fatty acids (HUFA). Fatty acyl-CoA Δ6 desaturase can catalyze the first step of the desaturation in the HUFA pathway, and it can convert linoleic acid(LA, 18:2n-6) to Gamma linolenic acid(GLA, 18:3n-6), convert α-linolenic acid(ALA, 18:3n-3) to Stearidonic acid(SDA, 18:4n-3), and it also can convert Eicosapentaenoic acid (EPA, 20:5n-3) to Docosahexaenoic acid (DHA, 22:6n-3) with other enzymes. In this study, fatty acyl-CoA Δ6-b desaturase gene was cloned from Eriocheir sinensis using reverse transcriptase polymerase chain reaction (RT-PCR) and rapid-amplification of cDNA ends (RACE). The full-length cDNA sequence of fatty acyl-CoA Δ6-b desaturase is 2 310 bp, including a 1 326 bp open reading frame (ORF) and codes a protein of about 442 amino acids. The molecular weight is 50.86 ku, and the isoelectric point is 8.47. GenBank accession number of this gene is KP876058. A homology analysis using BLASTn and BLASTx revealed that the fatty acyl-CoA Δ6-b desaturase gene shared 76% identity with fatty acyl-CoA Δ6 desaturase gene of E. sinensis. The ORF of fatty acyl-CoA Δ6-b desaturase gene was subcloned into the prokaryotic expression vector pCold-TF DNA, to generate recombinant expression vector pColdTF-fad6b, which was then transformed into the expression E. coli BL21(DE3)pLysS. Experiments showed that the fatty acyl-CoA Δ6-b desaturase was successfully expressed in E. coli BL21(DE3)pLysS by the IPTG induction and at the temperature of 15, and the concentration of IPTG was 0.3 mmol/L. The result of SDS-PAGE analysis showed that the recombinant protein had an approximately molecular weight of 105.86 ku which was consistent with the theoretical molecular weight, and the target protein was mainly detected in supernatant. As the purpose protein contains a 6×His-tag, we have chosen His-tag nickel ion affinity chromatography column for recombinant protein purification and anti-6×His-tag antibody for Western-blotting experiments, and results showed that pColdTF-fad6b recombinant protein was successfully expressed in E. coli. The Western-blotting revealed that recombinant protein pColdTF-fad6b had specifically been recognized by the 6×His antibody, indicating that the recombinant protein had antigen activity. Our report provides a new fatty acyl-CoA Δ6 desaturase gene, FAD6b. It may offer a basic method for purification and activity detection of E. sinensis FAD6b, and promote further study of the FADs function.

Abstract:In order to study the effect of individual growth on beak morphology of Dosidicus gigas, the samples of the jumbo flying squid were collected by the Chinese jigging fleet between 2009 and 2014 off the Peruvian Exclusive Economic Zone (79°22'-84°30'W, 10°00'-18°16'S). The beaks were extracted from 1208 samples and 12 morphometric parameters of beaks were measured. Meanwhile, the microstructure of beak was used to estimate the age of D. gigas. The differences of beak morphometry were analyzed which were influenced by body size, age and sexual maturity. The results showed that upper hood length (UHL), upper crest length (UCL), upper lateral wall length (ULWL), lower crest length (LCL), lower rostrum length (LRL) and lower lateral wall length (LLWL) could be used to represent the length features of beak. The ANOVA indicated that morphologic parameters of beak had significant differences between females and males, the average of the beak morphometric parameters of females was greater than that of males. The Least Significant Difference (LSD) analysis showed that the growth of beak between females and males varied according to mantle length, age and sex maturity. The growth had differences among the sections of beak of same sex individuals. However, if the mantle length is greater than 400mm, the age of female is greater than 300 days, the age of male is greater than 250 days or the sex maturity stage is greater than stage 3, then the growth of beak is slow. This study suggested that the morphometry of beak was influenced by body size, age and sex maturity.

Abstract:Chinese shrimp (Fenneropenaeus chinensis) in stocking program was used an example, to illustrate the uncertainty associated with empirically estimated natural mortality on the dynamics and structure of fish populations. Two empirical formulas based on the growth parameters were used to estimate the natural mortality rates which were then compared to the natural mortality estimated based on catch data. The differences in three natural mortalities over time and their impacts on sex ratio were evaluated. Our analyses showed that estimation methods could greatly affect the estimation of natural mortality. Although the mating mortality was included in the natural mortality obtained based on the catch data (Ye et al., 1987), the M of young fish has been underestimated at early releasing stages. The M was overestimated with the empirical formula proposed by Chen & Watanabe (1989), and the sex ratio was 4.44:1 at the end of one-year old China shrimp. The M was underestimated at the early releasing and overestimated at the steadily growth stage with the Gislason's method, and the sex ratio was 2.22:1 at the same time. At the beginning and end of fishing, the amounts of resources and catches based on Ye et al method were over 2.42 and 2.87 times the amounts estimated using the Gislason's empirical formula, and about 76.25 and 102.50 times of the values estimated with Chen and Watanabe's method. These data suggest that the empirical methods selected to estimate the natural mortality need to be based on the most rigorous criterion, and when calculating the natural mortality gender-specifically by empirical methods, sex parameter should be included to ensure that the results are biologically meaningful.

Abstract:The aim of this study was to investigate the secretion, structure and function of peritrophic membrane (PM) in digestive tract of Eriocheir sinensis. Histology and electron microscopy techniques were employed to observe the intestinal and PM morphology of E. sinensis. Chitinase dissolution test and SDS-PAGE gel electrophoresis were used to research the composition of the PM and then the function of PM protein was indentified by LC-ESI-MS/MS. Microscopic examination showed the surfaces of gastrointestinal tract of E. sinensis were all covered with PM. Compartmentalization of PM was observed in intestine except midgut. When PM was detached from the epithelial cells, the columnar epithelial cells were becoming irregular round with the nucleus disappeared. A large number of mitochondria and secretory vesicles were observed in cytoplasm under the electron microscope. PM was noticed to be dissolved by chitinase. SDA-PAGE map showed that there were 7 clearer electrophoresis bands and their molecular weight was mainly distributed in more than 40 ku. Results from LC-ESI-MS/MS analysis showed that the PM proteins included sodium-potassium ATPase, ATP synthetase, actin and tubulin, thioredoxin, oxygenase, etc. These results indicated that the PM of E. sinensis is secreted by epithelial cells, improves absorption by forming compartment, and may participate in physiological processes of immune, osmotic regulation and nutrients transportation.

Abstract:In order to study the growth and survival rate of the Chinese sucker (Myxocyprinus asiaticus) under different weaning strategies, two weaning strategies were adopted to feed the M. Asiaticus, which is from larvae (9.50±0.84) mg to 60 days post hatching (dph). (1) Three groups were divided depending on different weaning starting points, 15 dph (W15), 20 dph (W20) and 25 dph (W25), respectively, and each group was performed in triplicate. The results showed the survival rates of all three weaning groups were more than 80%. The W20 group had the highest survival rate 91.21%±1.93%, but there was no significant difference among them. The specific growth rate (SGR) of each group was decreasing. In 35-60 dph period all groups were fed micro-diet until the end of the experiment, the SGR of W20 and W25 groups was significantly higher than that of W15 group. The difference between the two groups was not significant. The W25 group had the highest total length and body weight, which was significantly higher than that of W20 group. The body weight of the W20 group and W25 group didn't show significant between each other. (2) Beginning to wean at 20 dph, we divided the fish into three groups depending on different weaning transition time, 5 d (W20-1), 10d (W20-2) and 15d (W20-3) in W20 group, respectively. Each group was performed in triplicate. The results showed that:the survival rate of W20-2 and W20-3 groups was 95.73%±0.60% and 91.21%±1.93%, respectively, there was no significant difference. The survival rates of this two groups were higher than that of W20-1 group. The SGR of each group was decreasing. In 35-60dph period all groups were fed micro-diet until the end of the experiment, the SGR of W20-2 and W20-3 groups was significantly higher than W20-1 group. The difference between the two groups was not significant. The length and body weight of W20-3 group were significantly higher than those of the other two groups. These results indicated that delaying the starting point and extending the transition time of weaning would promote the SGR after weaning, total length and body weight of larvae Chinese sucker. But we should try our best to shorten the feeding time of organism food prey to reduce the cost as much as possible if it has not significant impact on the growth and survival of the larvae Chinese sucker. In conclusion, starting weaning at 20 dph and a 10-day transition time of weaning are the most appropriate weaning strategy for M. asiaticus in this experiment.

Abstract:To investigate the effects of hypoxia exposure and subsequent normoxic recovery on growth performance, energy metabolism and oxidative stress of juvenile Mugil cephalus, the dissolved oxygen content (DO, mean±SE) of the aquarium was controlled at hypoxia (1.66±0.41), moderate hypoxia (4.35±0.53), saturation (7.03±0.36) mg/L, respectively, for 10 days and then recovery to normoxic state 7.0 mg/L for 30 days. Specific growth rate, oxygen consumption rate, ammonia excretion rate, O:N ratios and oxidative stress indicators, including the content of T-AOC,SOD, ASOR, MDA, LD,T-GSH, GSSG, and GSH in the plasma, muscle, liver and gill were measured. The results showed that hypoxia exposure for 10 days generally decreased the levels of body weight, specific growth rate, oxygen consumption and ammonia excretion rate of juvenile fish. Fish experienced moderate hypoxia would achieve completely compensatory growth in a short period with normal dissolved oxygen content. But fish under hypoxia conditions had non-compensatory growth. Hypoxia and recovery had a great influence on the oxidative stress indicators. Hypoxia stimulated increases in the activities of lactic acid (LD) levels in blood, activities of superoxide dismutase (SOD) in muscle and liver, activities of anti-superoxide anion (ASOR) in gill, which supported the idea that anticipatory preparation takes place in order to deal with the oxidative stress that will occur during reoxygenation. Meanwhile the SOD activity in blood, muscle, liver and activities of ASOR in muscle, liver increased at different phases during reoxygenation, indicating that the oxidative stress during normoxic recovery was intense. GSH level in liver was elevated under recovery which appears to trigger the protection mechanism. Hypoxia and reoxygenation also significantly increased MDA level in liver and gill, and it seems that hypoxia may cause lipid peroxidation damage in liver and gill.

Abstract:Because of the enhanced of solar UVR and the destruction of Sargassum horneri in Nanji archipelago, it is necessary to study the effect of solar UVR on growth and photophysiology of S. horneri, in order to recovery algae forest by artificial seeding. We carried out experiments using long-term exposure with three radiation treatments, photosynthetically active radiation (PAR, 400-700 nm); photosynthetically active radiation + Ultraviolet A (PA, 320-700 nm) and photosynthctically active radiation+Ultraviolet A+Ultraviolet B (PAB, 280-700 nm) respectively. Big quartz tubes with thalli, which were put into 10℃ thermostatic baths with running tap water, were exposed to three different solar radiations for 14 days acclimation. At the end of experiment, the relative growth rate (RGR) of S. horneri was promoted by 6.7% in UV-A (PA treatment), while was inhibited by 8.98% in UV-B (PAB treatment) compared with P treatment. The results of photosynthetic pigments and chlorophyll fluorescence characteristics revealed that the UV-A significantly raised the concentrations of chlorophyll a, carotenoid and UV-absorbing compounds (UVACs), but UV-B do decrease the pigments. Moreover, UV-A had a significantly positive effects on the maximum relative electron transport rates (rETR_max) and the light utilized efficiency (α). After 14 days acclimated under solar radiation, the thalli were removed indoor to re-exposed to the artificial solar lamp (PAR=213.3WUV-A=14.3WUV-B=6.7 W) in order to check the short-term response to elevated UVR. The results shown the repair rate (r) of D1 protein is faster in UV-A than in both PAR and UV-B significantly. But the damage rate (k) has not difference among three radiation treatments.

Abstract:Aeromonas is regarded as an important pathogen of freshwater animals. However, little is known about genetics of antimicrobial resistance in Aeromonas sp. in Chinese aquaculture. The aim of this study was to investigate the presence of class Ⅰ integron and characterize multidrug-resistant Aermonas sp. isolated from integrated farms. Three hundred and seventeen Aeromonas strains were isolated from pig feces, fish, pond water and sediments. These samples were collected from 5 pig-fish integrated farms in Foshan and Zhaoqing of Guangdong province. All strains were evaluated for resistance to 20 antimicrobials of 7 classes by two fold dilution method. Genomic DNA was extracted with the Omega DNA mini kit. All the isolates were screened for the presence of intI1. All the intI1-positive strains were also amplified sulI, qacE1 fragment and gene cassette region by PCR. Gene cassettes arrays were analyzed by BLAST. Percentages of isolates resistant to 20 antimicrobials were variable. Resistance was most prevalent for ampicillin, sulfamonomethoxine and nalidixic acid. Among 317 Aeromonas isolates, 50 (15.77%) isolates were positive for intI1. The incidence of class 1integron was more prevalent in Aeromonas from fish and pig than that from environmental sources. Comparing the resistance phenotypes represented in integron-positive and integron-negative strains, the results showed that the resistance rates of trimethoprim/sulfamethoxazole, doxycycline, enrofloxacin, neomycin and florfenicol among integron-positive isolates are higher than those from integron-negative strains. All of the integron-positive strains were resistant to more than three classes of agents. There were16 different gene cassettes encoding resistance to aminoglycosides (aadA1, aadA2, aac6-, aacA4), trimethoprim (drfA1, dfrA12, dfrA15, dfrA17, dfrB4), β-lactams(bla_OXA-10, bla_OXA-21), chloramphenicl (catB3, catB8), rifampicin (arr-2, arr-3) and plasmid-mediated quinolone resistance (PMQR) gene aac(6')-Ib-cr. Most of the integron-positive isolates carrying gene cassettes mentioned above corresponded to the resistance profile of antimicrobial agents such as trimethoprim, streptomycin, chloramphenicol. It indicated that there was a close relationship between class Ⅰ integron and multi-drug resistance in Aeromonas isolates. It is concluded that class Ⅰ integron mediated multi-drug resistance was wide spread in Aeromans from different origins of pig-fish integrated farms. Further study on resistance mechanism should be undertaken so as to provide a basis for rational usage of antimicrobial agents in Chinese aquaculture.

Abstract:It has been shown that host protein cyclophilin A (CypA) played a key role in the replication of several viruses. In order to address the function of Mandarin fish CypA (SC-CypA) in infectious spleen and kidney necrosis virus (ISKNV) replication, the full ORF of SC-CypA was first cloned and characterized. The ORF of SCCypA encoded a polypeptide of 164 amino acids with calculated molecular weight of 17.59 ku. The deduced amino acid sequences of the SC-CypA shared highly conserved structures with CypAs from other species, indicating that SC-CypA should be a new member of the CypA family. Cyclosporin A (CsA) is a specific inhibitor of CypA. ISKNV replication was inhibited by the addition of CsA in a manner of dose dependence. CsA could effectively inhibit the replication of ISKNV at different time points after ISKNV infection, and the strongest inhibitory effect was observed at 72 h poi. The addition of CsA could inhibit the mRNA expressions of IL-1β, IL-8, IL-18 and ISG15 in CPB cells after the infection of ISKNV. These suggest that SC-CypA plays a key role in the ISKNV replication, and CsA could effectively inhibit the replication of ISKNV.

Abstract:The diversity of the epiphytic bacteria was studied based on the morphological characteristics and 16S rDNA sequence analysis, which were isolated from Ulva spp. (Enteromorpha spp.) distributed in Zhujiajian Zhoushan, Guohua power plant of Ninghai and Nansha Fenghua in China. The 65 isolated strains were grouped into 26 morphotypes, which had high similarities with strains of 23 generas such as Acinetobacter sp., Alteromonas sp., Vibrio sp., Pseudoalteromonas sp., Exiguobacterium sp., Bacillus sp., Erythrobacter sp., Micrococcus sp., Pseudomonas sp., Idiomarina sp., Phaeobacter sp., Roseivirga sp., Silicibacter sp. and so on. The results suggested that:(1) The diversity of Ulva spp. (Enteromorpha spp.) epiphytic bacteria was most abundant in Guohua power plant of Ninghai, and the Shannon-Wiener index was shown at 93.98%. (2) The Flora of Ulva spp. (Enteromorpha spp.) epiphytic bacteria was relevant with its growth and living environment. Although the community structure and dominated bacterica from different origins were various, the main species of bacteria belonged to the phylum of Proteobacteria. There were still some unidentified strains in this study, which need further investigation.

Abstract:In this study, full-length cDNA sequence of caspase2 gene from Fenneropenaeus chinensis was first cloned using RACE method. The full-length cDNA sequence of caspase2 was 1517 bp, which contains a 78 bp 5'-UTR, 515 bp 3'-UTR and 924 bp open reading frame that encoded 307 amino acid residues, which had the isoelectric point(PI) of 7.62 and molecular mass of 34.21 ku. Homology analysis revealed that the amnio acid sequences of caspase2 highly identified with caspase family of other species, for example, it had 88% identity with caspase2 of Litopenaeus vannamei, and had 80% identity with caspase of Penaeus monodon. The phylogenetic analysis showed that Fenneropenaeus chinensis caspase2 was in the same class with other arthropods caspase. The expression levels of caspase2 gene in different tissues were analyzed by quantitative real-time PCR. The results showed that the highest level of caspase2 gene was in hepatopancreas. Real-time PCR analysis showed that the expression level of caspase2 was up-regulated distinctly in muscle, hepatopancreas and gill after stimulation with WSSV infection, and caspase2 showed different expression profiles. The results implied that caspase2 might play an important role in WSSV-challenge response of F. chinensis.

Abstract:The aim of this study is to prepare the ScFv antibody (single chain variable fragment antibody) against viral hemorrhagic septicemia virus (VHSV) and analyze the biological properties. The variable heavy (VH) and the variable light (VL)chain gene fragments were derived from mAb 1G5 hybridoma cells against VHSV. The VH and the VL DNA fragments were assembled through a flexible linker DNA to generate ScFv DNA that was cloned subsequently in the pET28a vector to express ScFv protein in E.coli cells. The expressed ScFv protein, with a relative molecular mass of about 28 ku, existed in a form of soluble expression in cytoplasma. The ScFv protein could specifically identify VHSV glycoprotein (G), and neutralize viral virulence of VHSV in vitro. The ScFv protein showed good affinity for VHSV glycoprotein (G) antigen, as indicated by KD values of 1.4×10^-8 M. ScFv protein preparation has laid a foundation for further study of VHSV therapeutic antibodies as well as rapid diagnostic reagent.

Abstract:Test of statistical significance uses samples to infer the statistic property of population from which the samples are taken. Two types of error, Type I (i.e., reject a true null hypothesis) and Type II errors (i.e., accept a false null hypothesis) can be made in a test of significance. Significance tests are commonly used in fisheries to identify statistical differences. Of the 6 major domestic fisheries journals, significance test methods were used in 2235 research articles within the recent 5 years (from January 2011 to April 2015), which accounted for 58.4% of all the research articles that were published in these 6 journals during the corresponding period. However, attention was only paid to the Type I errors in all these studies, and the Type II error was totally neglected. However, Type II error is not unimportant. The probability of having Type II error is often higher than that of Type I error, and sometimes the consequences are more serious for committing Type II errors. Neglecting Type II errors in fisheries is likely to result in fisheries mismanagement, consequently leading to overexploitation of fishery resources. In this study, the analyses of the Type II errors in fisheries researches were reviewed, and the potential consequence and implication of committing Type II errors in domestic fisheries researches were discussed; and the calculation method of the probability of having Type II error β (or statistical power 1-β) was described using fisheries examples. The relationships between β and several main influence factors were analyzed. We strongly suggest that Type II errors be considered and evaluated in the future fisheries research to reduce the likelihood of making serious mistakes in fisheries management.