• ISSN 1000-0615
  • CN 31-1283/S
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Effects of feeding methods of carbon sources on the water treatment of suspended growth reactors in a recirculating aquaculture system
Wenchang LIU, Hongxin TAN, Guozhi LUO, Wei CHEN, Nannan ZHANG, Yongxia YU, Miaolan YAO, Pronapinyakun Pimpron
Accepted Manuscript  doi: 10.11964/jfc.20180411257
[Abstract](19) [FullText HTML](8) [PDF 1083KB](0)
A suspended growth reactor under continuous carbon resource feeding method (SGR-Con) and an another under several steps carbon resource feeding method (SGR-Sev) were conducted in a pilot-scale recirculating aquaculture system to compare their water treatment efficiency. It indicated that the concentrations of dissolved organic carbon in the reaction zone of SGR-Con was at a high and stable level, while that in SGR-Sev firstly increased to the same level of SGR-Con during the 0 h in cycle to the instantaneously carbon feeding time and it subsequently decreased to a lower and stable level after 4 h in cycle. Dissolved oxygen in the reaction zone of SGR-Sev, as well as the settling zone, were significantly higher than that in SGR-Con. The pH in the reaction zone among different reactors had no significant difference, and it was same to settling zone. Several steps carbon resource feeding method significantly increased the nitrogen removal of reactor. Overall the experimental period, the removal rate of nitrate and total nitrogen in SGR-Sev could be 63.91%±14.31% and 64.07%±12.11%, respectively. Besides, alkalinity in the effluent of SGR-Sev was (278.18±80.33) mg/L. SGR-Sev was favorable to achieve good settling performance of bio-flocs. However, the concentrations of total ammonia nitrogen and nitrite in the effluent of SGR-Sev were higher than them of SGR-Con. In summary, it suggested to take several steps carbon resource feeding method to suspended growth reactors.
Cloning and expression of sox9a/b gene in the large yellow croaker (Larimichthys crocea)
Meng ZHANG, Yangyang ZHU, Wanbo LI, Weiliang SHEN, Xiongfei WU, Kun YE, Zhiyong WANG
Accepted Manuscript  doi: 10.11964/jfc.20181111531
[Abstract](16) [FullText HTML](9) [PDF 0KB](0)
To elucidate the role of sox9 in sex determination and gender differentiation of the large yellow croaker (Larimichthys crocea), the full length of sox9a and sox9b were cloned using reverse transcription-polymerase chain reaction (RT-PCR) and rapid amplification of cDNA ends (RACE). The difference of the gene expression in various tissues and development stages was analyzed through quantitative real-time PCR. Expression profiles of sox9a/b after 17β-estradiol or 17α-methyl testosterone treatments were also examined. The full-length cDNA of sox9a gene is 2,442 bp (NCBI: MH996431), including a 476 bp 5′ UTR, a 466 bp 3′ UTR and a 1,500 bp ORF coding a polypeptide of 499 amino-acid. The full-length cDNA of sox9b gene is 2,199 bp (NCBI: MH996432), including a 335 bp 5′ UTR, a 415 bp 3′ UTR and a 1,449 bp ORF coding a polypeptide of 482 amino-acid.Quantitative Real-time PCR results showed that sox9a was primarily expressed in gonad, eye, brain, liver, and the expression level in testis was significantly higher than in ovaries (p<0.05). Sox9b was widely expressed in multiple tissues in large yellow croaker; the expression level was highest in testes but can be barely detected in ovaries. At early developmental stages of fry, sox9a/b expressed in a lower level. Sox9a/b peaked at 84 dph (day post hatch) and 123 dph, then their expression declined and gradually rose again in 10 mph. In addition, 17β-estradiol can significantly down-regulate the expression of sox9a (P<0.05) and sox9b (P<0.01) in testes. 17α-methyl testosterone can significant elevate the expression of sox9a (P<0.05) and sox9b (P<0.05) in gonads. The study demonstrated that sox9a/b may play important roles in sex determination and differentiation in the large yellow croaker. However, the functions of the two genes may be different.
Effects of LED spectrum on feeding, growth and energy distribution of juvenile Dicentrarchus labrax
Jilong REN, Pingping WEI, Fan FEI, Mingyun DAI, He MA, Dongkui GAO, Changbin SONG, Tao CHEN, Ying LIU
Accepted Manuscript  doi: 10.11964/jfc.20181111534
[Abstract](14) [FullText HTML](6) [PDF 660KB](0)
European sea bass is an important economic fish in aquaculture in China. In the process of industrialized recirculating aquaculture, different spectral environments have important implications for the effects of feeding and growth. In this study, five different LED spectral processing groups of white light (λ400-780nm), red light (λ630nm), yellow light (λ595nm), green light (λ530nm) and blue light (λ455nm) were set in the seawater circulating aquaculture experimental system. The effects of five different LED spectra on feeding, growth and energy distribution of juvenile larvae [(29.91±0.39) g, (13.78±0.35 cm)) were studied. The results showed that the effects of different LED spectra on the feeding, growth and energy distribution of juveniles were significantly different. The red light group had the best growth of the juveniles (41.09±5.70)g, and the blue group had the worst growth of the juveniles (36.02±4.18)g; the DGC (Daily growth coefficient) of the blue group Significantly lower than the other groups, the difference between the other groups was not significant; there were significant differences in the feeding rate of the three groups of LED spectrum treatment groups, and the red light group had the highest feeding rate. The results of insulin-like growth factor IGF-1 and IGF-2 showed that the expression of IGF-1 mRNA was highest in the white light group; the mRNA expression level of IGF-2 was highest in the yellow light group. In terms of energy distribution, the red light group has lower fecal energy and metabolizable energy, and has the highest feeding energy and growth energy; the blue light group has the highest fecal energy, lower feeding energy and metabolic energy, and the lowest growth energy. The ratio of RNA/DNA in the muscle and liver of juvenile fish showed that the RNA/DNA ratio of the liver in the blue light group and the juvenile fish was significantly higher than that in the other groups; the RNA/DNA ratio in the muscles of the blue light group and the white light group existed. Significant differences were not significantly different from the other groups. The results showed that the red light Eurasian larvae had better feeding and growth performance, and the blue-spectrum larvae had poor feeding and growth performance, and the red-spectrum larvae had higher feeding energy and growth energy. Therefore, the red spectrum has a better promoting effect on the growth of juvenile larvae, and the blue spectrum has an adverse effect on the feeding growth of juveniles. This study provides a reference for the ground-based industrialized recirculating aquaculture of the European larvae, creating a suitable spectral environment for growth.
Correlation analysis of fish growth performance on serum hormone and digestive enzyme activities of juvenile pearl gentian grouper (Epinephelus lanceolatu♂×E. fuscoguttatus♀) fed with different protein levels diets
Xinwei GUO, Beiping TAN, Shuyan CHI, Xiaohui DONG, Qihui YANG, Hongyu LIU, Shuang ZHANG
Accepted Manuscript  doi: 10.11964/jfc.20180711385
[Abstract](61) [FullText HTML](34) [PDF 816KB](2)
An 8-week feeding trial was conducted to assess the correlation of the dietary protein to growth of juvenile pearl gentian grouper (Epinephelus lanceolatu♂×E. fuscoguttatus♀), serum hormone and digestive enzyme activities, what could to explain the effects of dietary protein levels on healthy growth. Grouper [average body weight (6.50±0.00) g] were randomly divided into 6 groups with 4 replicates in each group. The fish were fed isoenergetic and isolipidic experimental diets with protein levels 35%, 40%, 45%, 50%, 55% and 60%, respectively. The results showed that weight gain ratio and specific growth rate of the fish fed with 50% protein diet were significantly higher than those of fish fed other protein level diets. The concentrations of total protein (TP) in 50% group and 60% group were significantly higher than 35% group. The concentration of growth hormone (GH) and Insulin (INS) in 50% group was significantly lower than than that in other groups. The highest Insulin-like growth factors-Ⅰ (IGF-Ⅰ) concentration was found in 45% group and significantly higher than than that in other groups. Pepsin and intestinal trypsin activities in 50% group was significantly higher than than that in other groups. With increasing dietary protein levels, intestinal amylase activities was decreased, the lowest value was found in 55% group and 60% group and significantly lower than than that in other groups. There was extremely significantly negative correlation between WGR and GH. According to these results, Based on broken-line regression analysis of WGR, a diet containing 51.57% protein is recommended for efficient growth of juvenile pearl gentian grouper.
Analysis of melanocortin receptor 1 gene (MC1R) of Amphilophus citrinellus by whole-moant in situ hybridization
Xianhui PAN, Hongmei SONG, Kangqi ZHOU, Xuejie WANG, Yi LIU, Xidong MU, Fandong YU, Yexin YANG, Chao LIU, Shuming ZHENG, Yinchang HU
In Press  doi: 10.11964/jfc.20180511269
[Abstract](435) [FullText HTML](153) [PDF 1123KB](8)
In order to explore the melanocortin receptor 1 gene (MC1R) in Amphilophus citrinellus embryo development and the formation of the body color expression location and function of the test preparation of MC1R gene RNA is antisense probes, T7 has justice direction of transcription of RNA probe concentration is 447.529 ng/uL, SP6 direction of transcription antisense RNA probe concentration is 342.698 ng/uL. After 10 to 20 times dilution probe used for in situ hybridization, express the MC1R gene probe positioning shows that with the A. citrinellus embryo developmental hybridization signal was weakening trend in general, the intestinal phase and bubble phase, yolk and lateral embryonic body parts have a hybridization signal distribution; in the film stage, hybridization signals appeared in the spine, yolk sac and yolk sac contents and pigment cells. There is a signal distribution on the yolk surface. In general, hybridization signals are specifically expressed in chordate, yolk sac and yolk sac contents and pigment cells, and the probe on justice as a negative control group in five both embryonic stage without any signal, sage MC1R express positioning shows that the signal is revealed in the A. citrinellus pigment cells play an important regulating role in differentiation, migration, also associated with nerve, nutrition, immune function, preliminary build fish body color related gene function and orientation of the entire embryo in situ hybridization method.
Ploidy identification for Carassius auratus var. dongting based on microsatellites
Junjie TAN, Kejun LIU, Ligang LÜ, Jianbo YU, Cong ZENG, Tiaoyi XIAO
In Press  doi: 10.11964/jfc.20181111519
[Abstract](518) [FullText HTML](131) [PDF 1133KB](11)
Carassius auratus var. dongting originates from Beimin Lake, Hunan. Compared to other Carassius spp., C. auratus var. dongting had a higher growth ratio and larger body size, and we therefore hypothesise that these growth advantages were related to polyploidization. To test this hypothesis, a 9 microsatellite ploidy identification method was developed to examine the ploidy level of C. auratus var. dongting, and flow cytometry assays and chromosome karyotype assays was used to further confirm the findings. The results showed that 9 microsatellites could completely distinguish 37 ploidy-known specimens. The genotyping results showed that all the 136 ploidy-unknown individuals are triploid, all three genotypes (AAA, AAB/ABB, and ABC) were observed in this population. The mean DNA contents of blood cells in C. auratus var. dongting (159.42±5.64) was 1.5 times with that of blood cells in C. auratus var. red (102.43±3.54), and the results from microsatellite markers are totally concordant with those from flow cytometry analysis. The karyotype analysis revealed that the number of chromosomes is 156± and the karyotypic formula was 3n=39m+36sm+81sta, NF=231. This study not only established a microsatellite marker system for ploidy identification of C. auratus, but also systematically confirmed that C. auratus var. dongting is triploid.
Tempo-spatial distribution characteristics of fish resources in Jiaozhou Bay
Shannan XU, Jianzhong GUO, Zuozhi CHEN, Kui ZHANG, Youwei XU, Yancong CAI, Chunhou LI
In Press  doi: 10.11964/jfc.20180911461
[Abstract](482) [FullText HTML](120) [PDF 1171KB](8)
In order to understand the tempo-spatial distributions of fish resources in Jiaozhou Bay, we conducted four surveys on the fishery resources in Jiaozhou Bay by the underlying single trawl during 2016 and 2017. The surveyed results showed that there existed obvious differences in the seasonal variations in the density of fish resources. Individual density of fish resources was the highest in summer (233 427 ind/km2), followed by those in winter (40 746 ind/km2) and autumn (32 246 ind/km2), while that in spring was the lowest (18 080 ind/km2). Mass density of fish resources in winter was the highest (1 593.16 kg/km2), followed by those in summer (825.55 kg/km2) and spring (681.96 kg/km2), while that in autumn was the lowest (337.91 kg/km2). Among them, the average weight of fish in winter was the highest (39.10 g), followed by those in spring (37.72 g) and autumn (10.48 g), while that in summer (3.54 g) was the lowest. Compared with the historically surveyed data, the number of fish resources in Jiaozhou Bay has shown a significant downward trend during the past 30 years. The fish resource density (1 593.16 kg/km2) was lower than those in 1981—1982 (10 857.00 kg/km2) and 2008—2009 (2 196.00 kg/km2); the average individual mass of fish was 10.60 g, which was lower than those in 1981—1982 (37.70 g). There existed a trend of miniaturization and low-quality in the structure of fish community in Jiaozhou Bay. These changes may have been mainly caused by destruction of fish habitat and the deterioration of the ecological environment of the sea due to anthropogenic activities.
Spatiotemporal distribution and the estimated abundance indices of small yellow croaker in winter in the Yellow Sea based on Geostatisticaldelta-generalized linear mixed models
Qingpeng HAN, Xiujuan SHAN, Rong WAN, lisha GUAN, Xianshi JIN, Yunlong CHEN, Qiang Wu
In Press  doi: 10.11964/jfc.20180911448
[Abstract](464) [FullText HTML](108) [PDF 2192KB](8)
Larimichthys polyactis is an important economic bottom fish in China. Understanding the spatial and temporal distribution and abundance index of small yellow croakers (Larimichthys polyactis) contributes to the scientific management of small yellow croaker resources. In this study, the Geostatistical delta-generalized linear mixed models (geostatistical delta-GLMM) was used to analyze the spatial distribution of the wintering population of the Yellow sea and Bohai sea small yellow croakers from 2001 to 2011 and from 2015 to 2017 in the central and southern yellow sea. The biomass index of small yellow croakers was estimated by geostatistical delta-GLMM, ordinary Kriging interpolation-based method and design-based swept area method, respectively. The results showed that there were mainly three high-density areas of biomass areas in the overwintering ground of the Yellow Sea in 2001 and 2002, namely, the north (36°00′N-37°37.5′N, 123°15′E-124°15′E), the central (33°75′N-36°00′N, 123°15′E-124°75′E) and the southeast (32°00′N-33°75′N, 124°00′E-125°15′E), and the central region had the highest density. Since 2003, the biomass density of small yellow croaker has begun to decline, and the density core area in the north and southeast has declined more than the central density core area. In winter, the total biomass index of small yellow croaker is contrary to the change trend of the annual yield of small yellow croaker and fishing boat power, showing a downward trend. In most years, the number of stations is more than 37, and the range of stations covers the study area. The factors of sampling stations can be excluded, which indicates that the resources of small yellow croaker are still facing overfishing and the population is in a declining state. By 2016-2017, the density core area became less obvious. The geostatistical delta-GLMM estimated the total biomass index of the yellow croaker in the winter of 2001-2017 is more accurate than that of the swept area method and ordinary Kriging interpolation-based method.
Molecular cloning, prokaryotic expression and positioning analysis of C-type lectin 3 (MnLec3) cDNA from oriental river pawn(Macrobrachium nipponense)
Shengming SUN, Hongtuo FU, Fujun XUAN, Xianping GE, Jian ZHU, Xugan WU
In Press  doi: 10.11964/jfc.20180611327
[Abstract](435) [FullText HTML](128) [PDF 1221KB](4)
C-type lectins are a large family of proteins that exist in all deuterostomia. C-type lectins can bind to carbohydrate moieties normally in a calcium-dependent manner and play important roles in immune defense. This study aims to explore the expression patterns of C-type lectins gene in different tissues, celluar localization and becteria challenge in Macrobrachium nipponense. The cDNA sequence of M. nipponense (MnLec3) was obtained using rapid amplification of cDNA ends method (RACE) and RT-PCR. The expression levels of MnLec3 in different tissues and different time of artificially challenged with Aeromonas hydrophilia were analyzed by qRT-PCR. The full-length cDNA sequence of MnLec3 was 1 357 bp, which contained a 5′ untranslated region of 125 bp, a 3′ untranslated region of 206 bp, a 1 026 bp open reading frame (ORF) encoding 341 amino acids. The deduced amino acid sequence of MnLec3 had a signal peptide containing 17 amino acid residues and a carbohydrate recognition domain (CRD). Phylogenetic tree analysis stated that oriental river prawn has the closest relationship with other crustacean. The expressed recombinant MnLec3 protein and polyclonal antibody were obtained in present study using a conventional method. Furthermore, immunofluorescent staining technique was used to determine and cellular localization of MnLec3 in hepatopancreas of prawns. Quantitative real-time RT-PCR analysis showed that the MnLec3 gene was expressed in haemocytes, hepatopancreas, muscles, gill, testis, ovary and intestines with the highest level of expression in the hepatopancreas. Real-time PCR analysis indicated that MnLec3 transcripts level showed significant change in hepatopancreas after the prawns was artificially challenged with A. hydrophilia, followed return to control levels at post-injection 96 h, which were similar with MnLec3 protein expression abundance using western blot. The results suggested that MnLec3 might be involved in the immune response against bacterial.
Study on interaction of autophagosome and microtubule during autophagy process in Cherax quadricarinatus
Fanjuan MENG, Guangcheng ZHANG, Wenjuan YIN, Liyan WANG, Jinsheng SUN
In Press  doi: 10.11964/jfc.20180811406
[Abstract](407) [FullText HTML](110) [PDF 775KB](8)
Insect research suggests that autophagy plays an important role in virus infection and proliferation, while shrimp autophagy research reports are rare. Understanding the autophagy of shrimp cells will open up new ideas for shrimp disease immunity research. Autophagy related protein LC3 is a marker protein of autophagy, existing in the membrane of autophagosome during the whole autophagy process. Our bioinformatics analysis found that Cherax quadricarinatus LC3 (CqLC3) and α-tubulin (Cqα-tubulin) has the interaction relations. To further reveal transport way of autophagosome during autophagy process, in this study, recombinant plasmids pET-HIS-CqLC3 and pET-GST-Cqα-tubulin were constructed and induced for protein expression in vitro. Then HIS and GST tagged fusion proteins HIS-CqLC3 and GST-Cqα-tubulin were purified by using affinity chromatography respectively. Recombinant proteins HIS-CqLC3 and GST-Cqα-tubulin were found binding each other by the GST pull down assay. In order to further reveal the role of microtubules for autophagosome transport, C. quadricarinatus cell microtubules were depolymerized by vincristine which was one microtubule inhibitor, then the autophagosomes were detected by MDC staining. The results showed that autophagic reaction was incomplete, and autophagosomes increased after microtubule depolymerization. Therefore, the autophagosomes in C. quadricarinatus cells can interact with microtubules through CqLC3, and microtubules play an important role in autophagosome transport during autophagy process. This paper firstly revealed the transport pathway of autophagosomes in shrimp autophagy, and the results laid the foundation for the study of autophagy in shrimp cells.
Review on methods for migration and distribution of aquatic animals
Jin MA, Siquan TIAN, Xinjun CHEN
In Press  doi: 10.11964/jfc.20181011504
[Abstract](514) [FullText HTML](97) [PDF 815KB](14)
Migration and distribution of aquatic animals has been always considered as the research hotspots of aquatic ecology, and the aim of it is to understand the pattern of migration and the relationship between migration and environmental factors. This kind of research plays an important role in the protection and management strategy of aquatic animals. Monitor by Tagging, prediction by species distribution models, speculate based on analysis of microchemistry and stable isotope recorded in organism tissues are the main three methods and have been widely used on the research aspects including species distribution under climate influence. The three methods consist of many technological tools, models and test contents in its own method system respectively, but little study has focused on the summary of these methods, and collaborative usage of the three methods has not been paid high attention. So the three main methods for understanding migration and distribution of aquatic animals were reviewed. Meanwhile, literature research on migration and distribution of aquatic animals using related methods were introduced in some extent. The three methods were recognized as effective tool for understanding migration and distribution of aquatic animals, and present study has focused on the compare among the different means which was used in each of the three methods, but little research has played high value on collaborative usage of the three methods. In addition, Statistical analysis of catch can be used to know the migration pattern of fishes and other fishery resources in fishery. The suggestions listed in this paper can provide good reference to the research on migration and distribution of aquatic animals.
Morphological variation analysis of Octopus minor along thecoastal waters of China
Xiaolei GAO, Ran XU, Zhixin ZHANG, Xiaodong ZHENG
In Press  doi: 10.11964/jfc.20181111521
[Abstract](567) [FullText HTML](126) [PDF 1198KB](12)
The wide coverage of the north and south seas of China makes the ecological environment geologically different. Octopus minor habiting in different sea areas thus formed corresponding apparent differences in morphology. A multi-variant analysis method including the principal component analysis, discriminant analysis, one-way ANOVA and stepwise discriminant analysis was used to comprehensively analyze the 14 morphological indexes of 11 natural O. minor populations along the coasts of China and a population discriminant function was established. The results showed that the arm formula of O. minor was 1>2>3>4 and the web formula was A>B>C>D>E. Principal component analysis showed two principal components were constructed with a cumulative contribution rate of 66.067%. The first principal component included the MW/TL, HW/TL and FFL/TL, with their contribution rate of 36.979%, while the second principal component contained the Twt/TL, ML/TL and FL/TL with the contributory ratio being 29.088%. Importantly, the Penghu group can be clearly distinguished based on the scattered plots of principal component analysis. The integrative discrimination accuracy was 60.400% based on the discriminant functions. One-way analysis of variance (ANOVA) implied that the number of suckers in the mating wirst were significant difference between Penghu and the other population. The cluster analysis revealed that five northern populations and the Yilan population grouped together at first, then clustered with Shengsi, Nanjidao and Lijiang, then finally clustered with Penghu, while Quangang formed into a separated group. All of those indicated that five northern populations showed high morphological similarity while it was not the case for the southern populations; There are significant variations between the northern and southern populations, especially Penghu and Quangang populations exhibited relatively large differences with the others. In summary, eleven O. minor populations in the coastal of Chinese sea exhibited significant morphological divergence, and the morphological diversity was relatively high.
Effects of tea polyphenols on growth, antioxidant capacity and lipid metabolism related genes expression of turbot (Scophthalmus maximus)
Yicong LI, Kai LIAO, Renlei JI, Wei FANG, Dan XU, Kangsen MAI, Qinghu AI
In Press  doi: 10.11964/jfc.20180311205
[Abstract](579) [FullText HTML](341) [PDF 549KB](7)
In aquaculture, high fat diets are widely used for its protein sparing effect which can improve protein utilization efficiency, promote growth and protect environment pollution. Hoevever, it can also lead to excessive lipid deposition of liver and lower disease resistance. In order to solve this problem, additives should be explored. Tea polyphenols are natural plant extracts which have been proved to have the effects of of promoting growth, lipid reduction and immunity improving in many animals. Therefore, the purpose of the experiment is to investigate the effects of dietary tea polyphenols (TP) level on growth, antioxidant capacity and lipid metabolism of turbot. Four diets with graded levels of TP (0.00%, 0.01%, 0.02% and 0.05%. dry weight 0, 100, 200 and 500 mg/kg) was formulated in this experiment. Fish were cultured in indoor aquaculture system, and each diet was randomly assigned to three replicate groups of juvenile fish [initial average weight of (13.51+0.31) g] and fed twice daily (8:00, 18:00).The feeding trial lasted 70 d. The results showed as follows: 1) Compared with the control group, fish fed the diet with 0.01%–0.02% TP had significantly higher weight gain rate (WGR). Feed efficiency (FE) was increased with the increasing TP level and no significant differences were detected among dietary treatments. Compared with the control group, Hepato-somatic index (HSI) was significantly increased by the supplement with TP. 2) The lipid content of whole fish and liver were decreased with increasing TP level and significantly lower in 0.02%–0.05% group compared with the control group. 3) Compared with the control group, the activities of total antioxidant capacity (T-AOC) and glutathione peroxidase (GSH-PX) were significantly higher in fish fed the diet with TP, and no significant differences were detected among fish fed diets with TP. The activity of superoxide dismutase (SOD) was first increased and then decreased with the increasing TP level, and 0.02% supplement level was the highest. The content of malondialdehyde (MDA) was decreased with increasing TP level, and significantly higher in 0.02% group compared with the control group. 4) The expression of sterol regulatory element binding protein 1(SREBP-1) was decreased with increasing TP level and significantly higher in 0.02%–0.05% group compared with the control group. The expression of fatty acid synthetase (FAS) was first decreased and then increased with increasing TP level and significantly lower in 0.02% group compared with the control group. The expression of peroxisome proliferator-activated receptor α (PPAR α) was first increased and then decreased with increasing TP level and significantly higher in 0.02% group compared with the control group. The expression of carnitine palmitoyl transferase 1(CPT 1) was increased increasing TP level and significantly higher compared with the control group. This research indicated that high fat diets supplemented with TP could promote growth, reduce liver fat deposition, and increase antioxidant activity. The optimum supplemental level of TP in diets for turbot is approximatly 0.02%.
Phenotypic variability and correlation analysis of lipid traits in grass carp (Ctenopharyngodon idella)
Peng JIANG, Xue LU, Shengjie LI, Xiaoli KE, Dongmei MA, Jiajia FAN, Junjie Wang
In Press  doi: 10.11964/jfc.20180611324
[Abstract](456) [FullText HTML](125) [PDF 726KB](1)
Grass carp (Ctenopharyngodon idella) is an important economic freshwater fish in China. Under intensive aquaculture conditions, the farmed grass carps often appear excess fat accumulation, such as fatty liver, rotund shape and increased body lipid content. To evaluate the possibility of genetic improvement, this study estimated the phenotypic variability and correlation of lipid traits in C. idella. A total of 15 quantitative traits of 17 month-old C. idella among 296 individuals were analyzed by mathematical statistical methods. The results showed that intraperitioneal fat index IPF, hepatopancreas lipid content HLC and muscle lipid content MLC had high variation in C. idella, and the phenotypic variation coefficients ranged from 21.30 % to 47.04 %. Correlation analysis showed that there was a significant positive correlation among the three lipid traits, suggesting that the process of lipid deposition has a certain synchronization in different tissues of C. idella. Furthermore, factor and cluster analysis revealed that the observed 15 quantitative traits were roughly divided into 4 categories: body shape factor, body size factor, organ index factor, and body lipid content factor. IPF belonged to organ index factor, while HLC and MLC formed body fat content factor together. In addition, multivariate statistical analysis showed that the explanation degree of morphological traits on the variation of the three lipid traits ranged from 7.45% to 24.83%. By contrast, sagittal shape SS can, to some extent, predict body lipid traits. In summary, the lipid traits of C. idella have great potential for genetic improvement, and IPF would be attractive character as breeding target. This study provided important reference for genetic improvement of lipid traits in C. idella.
Transcriptome analysis of spotted steed (Hemibarbus maculatus Bleeker) to identify genes related to ovary development
Huifen LIU, Liangsheng LU, Jing WANG, Chuanjiang ZHOU, Qianhong Gu, Xiao Ma, Junchang FENG, Guoxing NIE, Xuejun LI
Accepted Manuscript  doi: 10.11964/jfc.20180811419
[Abstract](93) [FullText HTML](58) [PDF 833KB](3)
To identify candidate genes involved in ovary development, Illumina Hiseq technology was used to define the transcriptome of spotted steed brain, ovary and liver. A total of 49 484 132, 47 540 538 and 50 622 304 clean reads were generated from brain, ovary and liver, and then assembled into 99 878 Unigenes with an average length of 1 430 bp. DEseq analysis revealed that 2 305 were found only in the expression of brain vs. ovary group, 839 were found only in the expression of brain vs. liver group, 1 474 were found only in the expression of ovary vs. liver group, and 860 genes were found to be co-expressed in the three compare groups. Gene ontology analysis indicated that many of these genes encoded proteins involved in primary metabolic process、single-organism process、organic substance metabolic process. Based on KEGG Pathway analysis, putative genes are novel candidate regulators of oocyte development and meiosis, such as GnRH signaling pathway, Steroid hormone biosynthesis, TGF-beta signaling pathway and Oocyte meiosis. The spotted steed transcriptome data reported here will provide valuable new data to increase genomic resources of this species. The data will also provide a valuable basis for further reproductive molecular biology studies of this commercially species.
Target strength measurements of tethered live Purpleback flying squid (Sthenoteuthis oualaniensis) at sea
Huanhuan WANG, Jun ZHANG, Zuozhi CHEN, Yong TANG, Mingshuai SUN, Jiaxing HUANG, Yongsong QIU
Accepted Manuscript  doi: 10.11964/jfc.20180611318
[Abstract](85) [FullText HTML](55) [PDF 1075KB](1)
Knowledge of target strength (TS, dB) is essential for accurate assessment of fish abundance using acoustic methods. In order to investigate acoustic scattering characteristic of purpleback flying squid Sthenoteuthis oualaniensis, 25 squid specimens were collected by jig in the open sea in the northern South China Sea in September 2017 near geographical position 18°42.9′N 113°05.5′E. The TS of 25 specimens were measured respectively one by one using Simrad EY60 split-beam scientific echosounder with 120 kHz and self-made tethering-controling device. In this paper, we primarily explored the impact of different parameters on the single target detection for Sthenoteuthis oualaniensis, and the parameters included pluse length determination level (PLDL, dB), min normalized pluse length (min NPL), max normalized pluse length (max NPL) and maximum standard deviation of minor-axis angles (MIA). Then we analyzed comprehensively the stochastic nature of single TS which was highly variable, and concluded the mathematical regression relation between TS of S. oualaniensis and mantle length (ML, cm). The results showed: (1) As the PLDL increased, the number of single target of squid detected increased firstly and then decreased, and the number was the largest when PLDL was 6 dB, while the average TS of all single targets increased monotonously. With increase of min NPL, the number of single squid target decreased; in the range of min NPL<0.7, the average TS of all single targets presented increasing obviously. When the max NPL was less than 1.2, the number of single squid target increased as the max NPL increased, but the average TS decreased; when the max NPL fell in between 1.2 and 1.8, the number of single squid target presented increasing slowly, but the average TS remained relatively stable. As the MIA increased, the number of single squid target increased, but the average TS decreased; (2) In this study, the optimum parameters setting of single target detection were as follows: PLDL=6.00 dB, Min NPL=0.7 dB, Max NPL=1.8, MIA=0.8°; (3) The maximum and minimum value of the average single TS of live Sthenoteuthis oualaniensis specimens were respectively -48.6 dB and -63.63 dB, and the corresponding mantle length of Sthenoteuthis oualaniensis were respectively 25.2 cm and 12.4 cm. The mathematical regression relation between TS of Sthenoteuthis oualaniensis and mantle length (ML) were concluded as follows: TS=34.22 Lg ML–98.23 (N=16, R2=0.603). This TS measurement was the first attempt to investigate the investigate acoustic scattering characteristic and TS of live Sthenoteuthis oualaniensis using tethering controlling method at sea by field work. The results of this paper not only accumulated important information and experience for further investigating the acoustic scattering characteristics and improving the acoustic estimation precision of Sthenoteuthis oualaniensis, also provide the reference for measuring TS of other squid or fish by tethering method at sea.
Ecological characteristics and importance evaluation of fish species in the Alaska Arctic
Qianqian LIN, Guoping ZHU
In Press  doi: 10.11964/jfc.20171111042
[Abstract](872) [FullText HTML](437) [PDF 804KB](15)
Melting sea ice in the Arctic facilitates access of human activities under the context of climate warming and also put forward the opportunities and challenges for sustainable exploitation and conservation of fish species in this region. Currently, loss of synthesis analysis on ecological characteristics of Arctic fish species leads to lack scientific and reasonable measures during exploitation and management processes. So it is urgent to analyze ecological characteristics of Arctic fish species. Based on scientific information available on Alaska Arctic marine fish ecology, the present study analyzes the ecological characteristics and importance of 104 fish species from the following aspects, i.e., depth distribution, trophic level, diel vertical migration, seasonal migration, and ecological, traditional culture and commercial importance. The results indicates, generally, Most of Arctic fish species studied are pelagic or mesopelagic and living in continent shelf or shelf slope that shallower than 1 000 m. Most of the fish in the Arctic Alaska have diel vertical movements with daytime descending and nighttime rising. The trophic level of those species are ranged from 3.0 to 4.5 and very few fish species occupy higher or lower trophic levels. The importance of most of fish species keeps unknown and only a few fish species occupy a key role in the Arctic Alaska marine ecosystem, traditional culture and commercial fishery based on the data available. About half of fish species demonstrates either diel vertical migration or seasonal movement. Based on clustering analysis, 5 ecological groups is classified for 104 Arctic fish species with considering depth of distribution, diel vertical migration, seasonal movement and trophic level as variables; however, 3 importance groups can be identified with ecological, traditional culture and commercial importance as variables. The results from the present study can provide data support for further studying on ecology of Arctic fish species and exploitation potential evaluation of Arctic fish resources and provide reference information for China’s participation in potential exploitation of Arctic fish resources and international negotiation.
Selectivity of different mesh size codends of canvas spreader stow net for Larimichthys polyactis
Guoqiang XU, Hongliang ZHANG, Baocun YU, Feng CHEN, Wenbin ZHU
In Press  doi: 10.11964/jfc.20171011008
[Abstract](700) [FullText HTML](319) [PDF 1388KB](22)
In order to explore the selectivity of different mesh size codends for Larimichthys polyactis, the traditional selective method (covered net method) was adopted, and a selection experiments were carried out with 5 different mesh size (35 mm, 45 mm, 55 mm, 60 mm, 65 mm) codend nets on canvas spreader stow net in the middle of Zhoushan fishery. The Logistic selection model was used to fit the selectivity curve of L. polyactis and the model parameters were estimated by the maximum likelihood method. The results showed that: ① during the experiment, the main catch target was L. polyactis, the number and weight accounted for 66.94% and 84.48% respectively, the body length range was 71 to 190 mm, and the average body length was 124 mm; ② with the increase of mesh size, the dominant body length of L. polyactis in the codend nets was larger than that of the covered nets; ③ when the mesh size for L. polyactis was 35 mm、45 mm、55 mm、60 mm and 65 mm, its L50 was 80.425 mm, 103.496 mm, 119.711 mm, 122.676 mm and 137.026 mm, its SR was 50.202 mm, 52.540 mm, 61.120 mm, 41.730 mm and 63.560 mm, respectively, L50 showed a gradual increase trend; ④ the relationship between the codend nets mesh size of L. polyactis and L50: L50=1.776 m+20.300 (R2=0.980); ⑤ the number escape rate of 5 mesh size were higher than the weight escape rate, when the mesh size of codend nets ≤45 mm, escape rate in number and weight of L. polyactis was less than 5%, when the mesh size of codend nets≥55 mm, escape rate in number and weight of L. polyactis more than 10%, even up to 50%. Comprehensive analysis shows that the mesh size of 55 mm can effectively release juvenile fish of L. polyactis, but the mesh size still need to expand.
Characters of cables tension of submerged circle gravity cage undereffects of waves and currents
Shengcong LIU, Liuyi HUANG, Yi NI, Hui CHENG, Fenfang ZHAO, Xinxin WANG, Zhenlin LIANG, Takeshi KINOSHITA
In Press  doi: 10.11964/jfc.10.11964/jfc.20181211587
[Abstract](511) [FullText HTML](124) [PDF 1381KB](7)
In the paper, the characters of tension in the mooring lines of a single HDPE gravity cylindrical cage under water are studied by experimental tests. The proto type of cage has a perimeter of 40m and a net height of 10m. Based on the Dixon rule of gear tests, two scales are chosen for the experimental model, i.e. λ=10 and λad=2. The horizontal projections of the front mooring lines are in the same direction of current or incident waves. Uniform current is produced in the whole basin at a velocity of 0 cm/s, 5 cm/s, 10 cm/s and 15 cm/s. Regular waves with a period within the range of 1.3 s to 2.2s and a height varying from 10cm to 30cm are generated. Tensions in the mooring lines are measured. The following conclusions might be made from the analysis of measured data.(1) Under the actions of current only, the average tension in the front main cable is greater than that of other cables. Under the actions of waves only, the maximum tension in the rear side main cable is greater than that of other cables. The maximum tension is linearly related to the wave height. The maximum tension of the front main cable decreases with the increase of the period.(2) Under the combining effects of waves and currents, the maximum tension in the front main cable is greater than that of other cables under high flow velocity, low wave height and high wave period; the maximum tension in the rear side main cable is greater than that of other cables under low flow velocity, high wave height and low wave period.(3) Under the combining effects of waves and currents, the maximum tension of each cable increases with the increase of the wave height, the increase of the flow velocity, and the decrease of the wave period.
Effect of sampling transect number on estimation of abundance index for bottom trawl surveys of fisheries resources in the Yellow River estuary
Xingxiang MENG, Jing WANG, Chongliang ZHANG, Ying XUE, Binduo XU, Yiping REN
In Press  doi: 10.11964/jfc.20180511297
[Abstract](890) [FullText HTML](440) [PDF 1136KB](15)
Fishery-independent survey is commonly used for collecting high quality biological and ecological data and provides vital parameters for fish stock assessment and management. For fishery-independent surveys, a small cost in the precision as a result of reduced sample size may lead to a much great reduction of survey mortality for the already depleted fishery populations and the ecosystem of fragile estuarine habitats, so that optimization of sampling design is essential for acquiring cost-effective sampling efforts. Computer simulation is usually applied for evaluating sampling designs in identifying an optimal sampling effort to achieve the goals of a survey program. In this study, the effect of sample size on estimation of abundance index was examined for fishery species including Cynoglossus joyneri, Pholis fangi, Chaeturichthys stigmatias, Loligo sp., Oratosquilla oratoria, Charybdis japonica and small-sized fish species in Clupeidae and Engraulidae in a cluster sampling survey based on the data collected from bottom trawl surveys conducted in August, October 2013 and February, May 2014 in the Yellow River estuary and its adjacent waters. The relative estimation error (REE), relative bias (RB), coefficient of variation (CV), and accuracy change rate (ACR) were used to measure the performances (accuracy and precision) of different sampling efforts. In general, these indices including REE, CV and ACR showed similar trends, decreasing with sample size initially and then becoming stable after certain sample sizes during all the four seasons. Most of RB did not show consistently increasing or increasing trends with sampling size. The absolute value of RB of C. japonica, O. oratoria and P. fangi etc showed an obvious increase when the sample size reduced from 3 to 1. The optimal sampling efforts were different for the selected species because the target species had different spatial variabilities. The effects of reducing sampling effort on the precision of abundance index estimation were low when the species had low spatial variabilities. Therefore, the optimal sampling effort for multiple species fishery resources surveys should consider the spatial and temporal distribution patterns of the target species.
Expression characteristics analysis of major QTL candidate genes in response to high temperature stress in Turbot (Scophthalmus maximus)
Xiaofei LIU, Aijun MA, Zhihui HUANG, Zhifeng LIU, Shuangshuang YANG, Kai YANG
In Press  doi: 10.11964/jfc.20180811413
[Abstract](482) [FullText HTML](148) [PDF 827KB](5)
Turbot (Scophthalmus maximus) is an important economically species extensively cultured in China. In this study, we exerted acute high temperature stress on turbot to identify the expression characteristics of four candidate genes including UBE2H, p53, ZNF469, and MAGI2 genes in the Liver, gill, spleen and skin. These genes are derived from the QTL related to high temperature resistance which located at high-density genetic linkage maps. The relative expression of UBE2H, p53, ZNF469, and MAGI2 mRNA in four experimental groups (temperature 20°C, 23°C, 25°C and 28°C)and one control group (temperature 14°C) were tested using real-time quantitative PCR. Data analysis demonstrated that the four genes were expressed in all tissues, and the expression levels were tissue and temperature specific. The relative expression of UBE2H mRNA firstly increased and then decreased in four tissues. The expression levels in the liver, spleen, and skin tissues increased at rapidly 20°C and reached a peak value (P<0.05), and peaked at 23°C in gill tissue (P<0.05). The expression of P53 mRNA increased first and then decreased in four tissues. However, the expression of p53 increased sharply at 28 °C and peaked significantly (P<0.05). It was speculated that the tissue was severely damaged and the cells are heavily apoptotic. ZNF469 and MAGI2 were abundantly expressed at 20°C in four tissues with over than other temperatures. Therefore, we thought that both can serve as biomarkers for response to high temperature stress in turbot.
Identification and characterization of LDH gene and its response to seawater acidification in the sea urchin (Strongylocentrotus intermedius)
Dongyao CUI, Liyuan REN, Dongfei XING, Jingxian SUN, Yingying LI, Yaqing CHANG, Yaoyao ZHAN
In Press  doi: 10.11964/jfc.20180811418
[Abstract](585) [FullText HTML](187) [PDF 10584KB](2)
With the attempt to identify and characterize the lactate dehydrogenase (LDH) gene and investigate its expression and activity alterations in response to seawater acidification stress in sea urchins, we used database data mining and rapid amplification of cDNA ends (RACE) to identify a novel LDH gene in the sea urchin Strongylocentrotus intermedius (here designated SiLDH). The full-length cDNA of SiLDH was 1 499 bp, including a 1 017 bp open reading frame encoding 338 amino acid residues. Sequence analysis indicated that the predicted SiLDH protein was similar to the LDH homolog in the sea urchin S. purpuratus. Quantitative real-time PCR analysis showed that SiLDH was ubiquitously expressed in coelomic fluid, gonad, tube foot and intestine of healthy adult S. intermedius, with the highest level of expression identified in the gonad. Total LDH enzyme activity in different tissues of S. intermedius from high to low was as gonad > tubefoot > coelomic fluid > intestine. Both SiLDH mRNA expression profiles and total LDH enzyme activity were altered in the coelomic fluid, gonad, tube foot and intestine after 60-day seawater acidification treatments (Control: natural seawater; OA1: △pH=−0.3; OA2: △pH=−0.4; OA3: △pH=−0.5). Our results provide more information about the characteristics and biological functions of the LDH homolog in sea urchins.
Expression of metabolic enzyme CYP3A65 and its upstream nuclear receptor PXR and AHR2 genes during Zebrafish (Danio rerio) developmental stages
Meijuan QI, Meizhi SHI, Jingxian ZHANG, Junjun CHEN, Xiangqi ZHANG, Jiao YANG, Cheng GUO, Yonglong HAN
In Press  doi: 10.11964/jfc.20180911465
[Abstract](451) [FullText HTML](133) [PDF 712KB](3)
Drug metabolizing enzyme CYP3A65 is the only member of danio rerio CYP3A subfamily. Our study was conducted to selecting appropriate danio rerio developmental stage, by investigate the expression of CYP3A65 and its its upstream nuclear receptor (PXR and AHR2) during developmental stages.The zebrafish in different growth stages were raised, and the whole fish tissues, liver tissues and intestinal tissues were taken. The relative expression levels of CYP3A65, PXR and AHR2 were measured, and the correlation between nuclear receptors and metabolic enzyme mRNA was compared to confirm the regulating effect of nuclear receptors.In whole fish, liver and intestine tissues, the mRNA levels of CYP3A65, PXR and AHR2 were highest in 144 hpf larvae and 75 dpf juvenile danio rerio. The change trends of metabolic enzyme and nuclear receptors gene expression were consistent, and affected by the proportion of liver and intestine relative to whole fish. The 144 hpf and 75 dpf danio rerio may be in the juvenile stage and throughout the growth phase, and are most suitable for CYP3A65 metabolism studies. There were significant correlations between CYP3A65 and PXRCYP3A65 and AHR2 mRNA expression, and correlation between CYP3A65 with AHR2 was better. In our later work, the enzyme activity and protein expression will be further investigated during danio rerio development, which would provide more information for the model organism danio rerio.
Influence of mesh size on sinking performanceand the shape of tuna purse seine
Wei LIU, Liu Xiong XU, Hao TANG, Fu Xiang HU, Cheng ZHOU, Kumazawa Taisei, Hirayama Mamoru
In Press  doi: 10.11964/jfc.20171011004
[Abstract](944) [FullText HTML](439) [PDF 1345KB](15)
The net performance of tuna purse seine is influenced by mesh size, leadline weight, flow velocity and other factors. Most of the purse seine fishing vessels in China are used vessels the age of the vessels is high and the equipment is old. The settling performance of the nets is hardly improved by increasing the weights of the leadline. In order to understand the effect of mesh size on the sinking performance, enclosed effectiveness and net shape, the authors based on our previous research results, net performance measurement at sea and the opinion of the captain, enlarge the mesh size from 260 mm to 390 mm (equivalent to 50%) of the low one third of the main body of the tuna purse seine currently used by the Shanghai Ocean Fishery Co., Ltd. to create a, two model nets with different mesh size in the main body were made by the Tauti rule and the comparative tests were conducted through three shooting patterns at 8 cm/s and one at still water in a professional purse seine test tank of Nichimo Co., Ltd. of Shimonoseki Research Institute. The results show that: both the sinking depth and sinking speed of the experimental net with bigger mesh size (45 mm) are larger than that of the control net with smaller mesh size (30 mm)(Sinking depth increased by an average of 1.6%, and sinking speed increased by an average of 1.1%), indicating that increasing the mesh size can improve the sinking performance of purse seine. The pursing time of the experimental net was 14.3% less than that of the control net, and experimental net also has a good form. The opening areas surrounded by the floatline at the end of shooting are almost identical for both the experimental net and control net; however, at the end of pursing, the opening areas surrounded by floatline of experimental net was 1.33 times than that of the control net and leadlline was 1.79 times than that of the control net, indicating that the bigger mesh netting can provide bigger areas for fish school swimming.
The early-stage fish resources of the four major Chinese carps at Huangshi section in the middle reaches of the Yangtze River
Lei GAO, Xingkun HU, Hao YANG, Daqing CHEN, Xinbin DUAN, Ke WANG, Shaoping LIU
In Press  doi: 10.11964/jfc.20180611333
[Abstract](569) [FullText HTML](149) [PDF 899KB](4)
In order to estimate the early-stage fish resources and distribution of spawning grounds of the four major Chinese carps, we collected samples of fish eggs and larvae at Huangshi section during May to July in 2015 and 2016. A total of 67 fish eggs and 23948 larvae were collected during the survey, Hypophthalmichthys molitrix and Ctenopharyngodon idella accounted for 77.0% and 18.1% of the total numbers, respectively. In 2015 and 2016, we estimated there were 0.20×108 and 4.69×108 ind eggs of the four major Chinese carps drifted through the Huangshi section, respectively; while there were 19.40×108 and 13.65×108 ind larvae, respectively. The spawning grounds of the four major Chinese carps located in Lijiazhou, Ezhou, Daijiazhou and Daoshifu section. Compared with the historical data, the location of spawning grounds was no significant change. It is suggested that enhancement and releasing is needed and the spawning habitat of the four major Chinese carps should been paid attention to the protection in the channel regulation project.
Effects of stocking density of the fishes fed formulated feed and EM product on phytoplankton community in a mussel-fish integrated system
Jinyu TANG, Yan WANG, Gang REN, Youming LI
In Press  doi: 10.11964/jfc.20180511289
[Abstract](524) [FullText HTML](134) [PDF 783KB](3)
Integrated culture of pearl mussel Hyriopsis cumingii and planktivorous fishes, such as Hypophthalmichthys molitrix and Aristichthys nobilis is widely used in commercial freshwater pearl mussel farming. Previous studies reported that stocking Ctenopharyngodon idella and Carassius auratus gibelio in an integrated system comprising of H. cumingii, H. molitrix and A. nobilis and feeding C. idella and C. auratus gibelio with formulated feed could increase pearl production and mussel growth. At present, the integrated system of pearl mussel, C. idella, C. auratus gibelio, H. molitrix and A. nobilis has been widely used as a model for freshwater H. cumingii farming. However, the suitable stocking density of C. idella and C. auratus gibelio remains to be determined. Generally, increasing density of the fishes fed with formulated feed can increase either fish production or waste output, and the increased accumulation of toxic inorganic nitrogen (ammonia and nitrite) and organic matters can negatively affect health and growth of the farmed H. cumingii and fishes. Although microbial products have been recognized as a potential technique to alleviating the environmental impact and enhancing the production performance in aquaculture practices, knowledge about application of microbial products in Hyriopsis cumingii farming is scarce. In the present study, a 93-day experiment was conducted in land-based enclosures to evaluate the effect of stocking density of the fishes (C. idella and C. auratus gibelio) fed with formulated feed and effective microorganisms (EM) on structure and function of phytoplankton community in an integrated system of H. cumingii, C. idella, C. auratus gibelio, H. molitrix and A. nobilis. Four treatments abbreviated as LF0 (20 C. idella + 10 C. auratus gibelio + 40 mussel + 8 H. molitrix + 2 A. nobilis), LFA (20 C. idella + 10 C. auratus gibelio + 40 mussel + 8 H. molitrix + 2 A. nobilis + EM supplementation), HF0 (40 C. idella + 20 C. auratus gibelio + 40 mussel + 8 H. molitrix + 2 A. nobilis), and HFA (40 C. idella + 20 C. auratus gibelio + 40 mussel + 8 H. molitrix + 2 A. nobilis + EM supplementation) were established. The pearl mussel included 20 grafted mussel and 20 non-grafted mussel. Each treatment was three replicated, and total 12 enclosures were used. During the experiment, species and biomass of phytoplankton were monitored at the intervals of four weeks, and chlorophyll a (Chl.a) and primary productivity were measured at the intervals of two weeks. Results showed that more than 83 phytoplankton species that belong to 7 phylum, 32 class and 73 genus were identified. Microcyslis sp. and Scenedesmus sp. dominated in phytoplankton community at the beginning of the experiment, and Microcyslis sp., Merismopedia sp. and Coelosphaerium sp. dominated at the end. The phytoplankton biomass ranged from 3.2×108 to 38.3×108 cells/L. No significant differences were found in the species composition of phytoplankton among treatments LF0, LFA, HF0 and HFA. The increase of stocking density of C. idella and C. auratus gibelio resulted in the increase of phytoplankton biomass and community respiration and the decline of the primary productivity. EM supplementation did not significantly affect species composition and diversity of phytoplankton, but resulted in the decrease of the ratio of blue-green algae in phytoplankton biomass and the increase of the primary productivity. This study reveals that the fishes (C. idella and C. auratus gibelio) fed with formulated feed should not be stocked at high densities in the mussel-fish integrated system.
Preparation and characterization of polyclonal antibody againstCyprinid herpesvirus 2 ORF121
Lin YU, LiQun LU, Hao WANG
In Press  doi: 10.11964/jfc.20180911458
[Abstract](496) [FullText HTML](129) [PDF 1323KB](2)
Cyprinid herpesvirus 2 (CyHV-2) is an emerging pathogen of the crucian carp, Carassius carassius, which has caused huge economic loss in China and appears to be spreading worldwide. The objective of this study was to produce and characterize new polyclonal antibodies against CyHV-2 ORF121 that were suitable for diagnostic use and the investigation of ORF121 function. The open reading frame (ORF) of 121 was amplified from the culture supernatant of the CyHV-2. ORF121 was subcloned into the pGEX-4T vector and recombinant protein was expressed in Escherichia coli. The purified recombinant protein ORF121 was used to immunize mice and polyclonal antibodies were obtained. The recombinant protein ORF121 were purified under urea conditions then used to 6-week-old BALA/c mice to prepare its polyclonal antibody. Western Blot and indirect fluorescence assay (IFA) assay were used to validate the ORF121 polyclonal antibody. We report the recombinant protein ORF121(rORF121) could be expressed in E. coli. aggregated in the form of inclusion body. The specificity of the anti-ORF121 polyclonal antibody was confirmed by Western Blot. IFA with anti-ORF121 polyclonal antibody for the detection of CyHV-2 infected RyuF-2 cells was developed in this study. In conclusion, the anti-ORF121 polyclonal antibody will be valuable tools in further studies to elucidate the mechanisms of viral infection and CyHV-2 diagnosis.
Fish community structure and its relationship with environmental factors inthe Ma’an Archipelago and its eastern waters
Xudong HAN, Shouyu ZHANG, Zhenhua WANG, Kai WANG, Jun LIN, Mingxing DENG, Xingchen WU
In Press  doi: 10.11964/jfc.20180211197
[Abstract](931) [FullText HTML](418) [PDF 1331KB](18)
To study the fish community structure in islands area, a fishery-independent survey was conducted in August and December of 2016, February and May of 2017. The cluster analysis, nonmetric multidimensional scaling (nMDS) and biological environment correlation analysis (BIOENV) were used to study the composition of fish community in our study area. A total of 96 species were collected, which belonged to 77 genera, 47 families and 12 orders. The Perciformes has maximum ratio, which accounted for more than 50% of their biomass and abundance. Harpadon nehereus was the dominant species throughout the year. In addition, the dominant species in spring were Psenopsis anomala, Collichthys niveatus, and Cynoglossus robustus; the dominant species in summer were Larimichthys polyactis, Trichiurus lepturus, and Chaeturichthys hexanema; the dominant species in autumn and winter were Collichthys lucidus and Coilia Mystus. The fish community in the Ma’an Archipelago and its eastern waters can be divided into group I (the sites sorrounding island and reef), group II (the sites adjacent to reef), and group III (the sites off to island and reef). There were significant differences in species composition between groups, and seasons (R=0.651~0.977), except the spring group I and group III, group II and group III. BIOENV analysis showed that the bottom water temperature had the most significant impact on the fish community compared to bottom salinity and water depth.
Morphological structure and quality characteristics of cultured Larimichthys crocea in Ningde Area
Yanyan WU, Wenbin TAO, Laihao LI, Xianqing YANG, Shengjun CHEN, Wanling LIN, Hui RONG
In Press  doi: 10.11964/jfc.20180911457
[Abstract](517) [FullText HTML](146) [PDF 903KB](6)
Ningde area is the main producing area of Larimichthys crocea in China. It is a useful reference for revealing the morphology, digestive structure and quality characteristics of cultured L. crocea in Ningde, and for the deep processing and sustainable breeding of L. crocea. In this paper, the biological parameters and food physicochemical analysis, the shape parameters of the L. crocea, the proportion of each part and the rate of meat collection, the organ parameters of the digestive tract, the diameter of myofibrils, texture, color, cooking loss rate, water loss rate, pH, amino acid composition and taste, fatty acid composition, etc. were measured and analyzed. The results showed that the full-featured L. crocea in Ningde area was 2.25%, the fish fillet accounted for 57.32% of the body weight, and the meat yield was high; the fish head, skeleton and fish tail accounted for 27.35% of fish quality. In the digestive system, the fish stomach is more developed, there are 15 ring pyloric caecum, and the length of the fish intestine is moderate, which reflects the digestive characteristics of the feed of cultured L. crocea. Its internal organs accounted for 10.73% of fish quality, while the liver accounted for 36.65% of the visceral weight. The fish maw was thicker, accounting for 15.74% of the visceral quality. The liver and fish maw were worthy of deep processing. The cultured L. crocea has white muscle color, pH 6.66-6.74, muscle fiber diameter of 104.21μm. The muscle fiber density is 153.13 per square millimeter. The fish meat is tender. Its amino acid type is complete, and essential amino acids account for 41.93% of the total amino acids, and the content of lysine is higher, the umami amino acid accounts for 45.92% of the total amino acid, and the fish meat is fresh and sweet. The fatty acids in muscle are mainly unsaturated fatty acids, accounting for 62.04% of the total fatty acids, of which essential fatty acids account for 28.45% and EPA and DHA account for 20.01% of the total content. It is a high-quality food supplement for essential fatty acids and high unsaturated fatty acids.
Effects of supplemental hydrolysable tannin on feeding preference, nutrition digestion and antioxidant ability of obscure puffer (Takifugu fasciatus)
Jingting YAO, Chun KONG, Xueming HUA, Chun SHUI, Yonghai SHI
In Press  doi: 10.11964/jfc.20181211580
[Abstract](585) [FullText HTML](154) [PDF 919KB](3)
The possible roles of supplemental hydrolysable tannin on the obscure puffer (Takifugu fasciatus; initial weight 39.84 ± 3.09 g) were evaluated. Total 720 fish were fed with four isonitrogenous and isoenergetic semi-purified diets for eight weeks: control diet (T0) with hydrolysable tannin-free protein sources (containing 43% fish meal) and three experimental diets supplemented with 0.25%, 0.75%, and 1.25% hydrolysable tannin (called T1, T2, and T3, respectively). In experimental diets, crude protein content was balanced by casein when fish meal level decreased, and the ratio of casein to tannin was nearly equal to that of protein content to tannin content in rapeseed meal (16.8:1).The results showed that the weight gain rate were not influenced, while feed coefficient ratio decreased significantly (P<0.05) with increasing tannin level; the muscle crude lipid of T2 was significantly higher than T0 and T1, and the amino acid level of T0 was significantly higher than other groups (P<0.05); for liver antioxidant index, the activity of catalase (CAT) in T0 was significantly lower than other groups, while total antioxidative capacity (T-AOC) in T0 was significantly higher than other groups(P<0.05), the activity of superoxide dismutase (SOD) and the content of malonaldehyde (MDA) in T3 was significantly higher than other groups(P<0.05); for serum antioxidant index, the activity of SOD showed no significantly difference (P>0.05), while the content of MDA in T0 was significantly lower than other groups(P<0.05), and the activity of catalase (CAT) decreased with increasing tannin level; for serum biochemical indices, the activity of ALT increased, and then decreased at T2, paralleling the result of ALT in liver, the content of album (ALB) in T1 was higher than T3 (P>0.05) and T1 was significantly higher than T0 and T3 groups, the total protein (TP) content of T1 was higher than T0, and T1 was significantly higher than T2 and T3 (P<0.05); the activity of intestinal amylase in T1 and T2 groups were significantly higher than T0 (P<0.05), the activity of protease increased with increasing tannin level in both stomach and intestine(P<0.05), the activity of lipase decreased in stomach (P<0.05); the expression of T2R1mRNA in T3 was significantly higher than T0 both in tongue and intestine(P<0.01); the expression of HSP70 mRNA in liver in T0 was significantly higher than T3 (P<0.05). Furthermore, the preference decreased with increasing tannin level before feeding test (P<0.05), while after 8-week domestication, the fish preferred to diet containing 0.75% tannin in all groups (P<0.05). In conclusion, feeding obscure puffer with diet containing no more than 1.25% supplemental hydrolysable tannin would not decrease growth and feed coefficient ratio, while would change the digestion of protein, lipid, carbohydrate and anabolism of protein, and when the amount of supplemental hydrolysable tannin was up to 1.25%, antioxidant ability and anti-stress ability were both injured. In addition, domesticating obscure puffer with hydrolysable tannin-containing diet could enhance the feeding preference to the diet with 0.75% hydrolysable tannin supplementation and increase the receptivity to bitter.
Effect of different thawing ways and multiple freeze–thaw cycles on myofibrillar protein intermolecular force of Sepia esculenta
Hongchao ZHANG, Zhangzhi XUE, Yuan DING, Xiaorong XU, Yang JIN, Hesheng LI, Hongfei WANG, Feng XU
Accepted Manuscript  doi: 10.11964/jfc.20180711383
[Abstract](456) [FullText HTML](102) [PDF 1953KB](3)
This study explored the change of multiple freeze-thaw cycles on myofibrillar protein intermolecular force of sepia esculenta and the effect of protein properties. Fresh sepia esculenta meat were defrosted by three different ways which is 0 °C,running water and normal temperature for 9 freeze-thaw cycles. The result showed that the effect of defrost ways were significant on intermolecular forces, and thaw at 0 °C is better than running water than normal temperature. With the increase of freeze-thaw times, the content of ionic bond and hydrogen bond showed a significant reduce, the ionic bond decreased 7.71%, 10.64% and 13.61%, respectively, and the hydrogen bond decreased from 31.91%, 31.97% and 31.87% to 26.76%, 25.53% and 23.94%. On the contrary the hydrophobic interaction force, disulfide bond and non-disulfide covalent bond were tend to increase, the hydrophobic forces increased 8.86%, 12.35%, and 14.72%, respectively, and the disulfide bonds increased 1.43%, 1.96%, and 2.85%, respectively, the non-disulfide covalent bonds increased from 1.16%, 1.28%, and 1.55% to 3.75%, 4.05% and 5.50%. The surface hydrophobicity of myofibrillar protein increased from 30.47 μg, 31.31 μg and 32.26 μg to 46.10 μg, 53.51 μg and 58.91 μg, respectively, consistent with hydrophobic force results. Both sulfhydryl and reactive thiol content were decreased, the sulfhydryl groups were reduced 13.07 nmol/mg, 38.99 nmol/mg and 40.32 nmol/mg, respectively, and the active sulfhydryl groups were decreased 6.55 nmol/mg, 24.26 nmol/mg and 43.16 nmol/mg, consistent with disulfide bond generation trends. The changes of the secondary structure of myofibrillar protein were analyzed by Infrared spectroscopy (FTIR), indicated that during the freeze-thaw cycles the spatial conformation of myofibrin made a change. Three thawing ways were treatment after 9 freeze-thaw cycles, the spectral band shifted to different wavelength regularly, and the amide A band and the amide III band move slightly toward the high wavenumber direction, and the amide I band and the amide II band move to the low wave number direction. The secondary structure of the protein changes, as the sum of the content of both α-helix and β-sheet is reduced, and the sum of the content of β-turn and random curl is increased. Repeated freezing and thawing is actually a process of slow oxidation of proteins.
Changes of flavor components of Oreochromis niloticus during the processing of traditional Shanghai smoked fish
Yongxia XUE, Saichao WEI, Ju ZHANG, Shunsheng CHEN
In Press  doi: 10.11964/jfc.20181011481
[Abstract](508) [FullText HTML](138) [PDF 730KB](1)
To investigate the effects of Oreochromis niloticus on flavor components during the processing of Shanghai smoked fish, high performance liquid chromatography (HPLC), amino acid auto-analyzer and gas chromatography-mass spectrometry (GC-MS) were employed to analyze and identify the flavor components of Shanghai smoked fish in four processing stages (fresh O. niloticus, the first pickling, the first pickling followed by frying and Shanghai smoked fish products). The results showed that IMP, as main umami nucleotide, its contents of latter stages were significantly higher than that of the former. Moreover, the contents of free amino acids and taste amino acids had the same trend to the IMP, among them, aspartic acid and glutamic acid had the greatest influence on the flavor of Shanghai smoked fish. In addition, a total of 50, 84, 78 and 82 volatile components were respectively detected in fresh O. niloticus, first pickling, first pickling followed by frying and Shanghai smoked fish products, which were mainly composed of aldehydes, ketones, alcohols and hydrocarbons. Therefore, its fishy smell of O. niloticus was greatly improved by pickling and frying because they were the main reason of its flavor formation.
Effects of temperature on gonadal differentiation of black rockfish(Sebastes schlegelii) and its mechanism
Simin ZHANG, Xiaojie WANG, Jifang LI, Haishen WEN, Likang LÜ
In Press  doi: 10.11964/jfc.20180611335
[Abstract](482) [FullText HTML](133) [PDF 1179KB](3)
In this study, the black rockfish (Sebastes schlegelii) was selected as the research object. Three groups with different temperature treatments were set up, including high temperature group (24 °C), control group (20 °C) and low temperature group (16 °C). Histology, ELISA and qRT-PCR methods were used to investigate that the effect of different temperatures on gonadal differentiation and the potential mechanism. The results showed that at 40 dpb, the gonads developed fastest at 24 °C, and slowest at 16 °C. The female ratio were 70.0%, 42.9% and 33.3%, at 24 °C, 20 °C and 16 °C respectively. 24 °C and 20 °C, high level of E2 was last for long time and T level decreased sharply at 30-35 dpb. At 16 °C, E2 level drops rapidly, and the T was still at a highlevel at 35 dpb. It is indicated that at high temperature, , the E2 level was high whereas T level was fairly low during gonad differentiation, and the gonad tended to be female; on the contrary, the gonad preferred to be male. At 24 °C, the cyp19a1a mRNA expression was significantly up-regulated at 35-40 dpb, which may be associated with the partial female gonad development at high temperature . Under 16 °C, The ERβ2 mRNA expression was significantly decreased at 30-50 dpb. The inhibition of ERβ2 expression may be related to the partial male gonad development at low temperature. At 24 °C and 20 °C, foxl2 mRNA expression was at relative highlevel at 25-35 dpb, and the expression level of low temperature treatment group began to increase at 30 dpb. It is indicated that the high expression of foxl2 may be related to the rate of ovarian differentiation during the early stage of gonadal differentiation. At 30-50 dpb, the general trend of expression levels of sox3, sox9 and dmrt1 was consistent, indicating there is a connection between the expression of the three genes, which might be related to the rate of testis differentiation.
Effects of ecological operation of Three Gorges Reservoir on larval resources of the Four Major Chinese carps in Jianli section of the Yangtze River, China
Xue ZHOU, Ke WANG, Daqing CHEN, Shaoping LIU, Xinbin DUAN, WANG WANG, Lei Gao
Accepted Manuscript  doi: 10.11964/jfc.20180711365
[Abstract](384) [FullText HTML](95) [PDF 907KB](1)
In order to explore the effect of ecological operation of Three Gorges Reservoir on breeding of the Four Major Chinese Carps in Jianli section of the Yangtze River, and evaluate the quantitative range of ecological hydrology index that stimulate spawning behavior of four major Chinese carps. In this study, we investigated the early life resource of the Four Major Chinese Carps in Jianli section of the middle Yangtze River, from May to July,2013-2017. This time period includes analysis on the relationship of the Four Major Chinese carps’ spawning and larvae collected with the ecological hydrology index. The results show that there is a significantly positive correlation between the daily discharge increasing rate and the quantity of fish eggs. When the duration is 4d and more than, and the daily discharge increasing rate is from 1 600 to 2 833 [m3/(s·d)]. Ecological operation is more effective to stimulate the reproduction of the Four Major Chinese Carps in late June. Preliminary results indicated, water rising of ecological operation of Three Gorges Reservoir have improved the hydrological conditions for the breeding of four major Chinese carps in Jianli section. The implement of ecological operation not only reduced the negative impact on fish breeding in the midstream of the Yangtze River, but also maintained fish population resource after the operation of the Three Gorges Reservoir.
Effects of water salinity on the growth, ovarian development, osmoregulation, metabolism and antioxidant capacity of adult female swimming crab(Portunus trituberculatus)
Xiaowen LONG, Renfu WU, Wenjie HOU, Guiping PAN, Yongxu CHENG, Xugan WU
Accepted Manuscript  doi: 10.11964/jfc.20180611338
[Abstract](402) [FullText HTML](97) [PDF 928KB](5)
In order to investigate the effects of long-term salinity adaptation on growth, ovarian development, osmoregulation, metabolism and antioxidant capacity of adult female swimming crab (Portunus trituberculatus). Adult female P. trituberculatus post puberty molt was subjected to four water salinities (10, 15, 20 and 25) for 60 days. The results showed as follows: The survival rate, weight gain rate, specific growth rate and gonadosomatic index of female crabs increased significantly with increasing water salinity. The serum osmolality and the contents of Na+, Cl-, Ca2+, Mg2+ and major free amino acids, and total free amino acids in the serum, as well as the posterior gills Na+/K+-ATPase activity showed an overall increasing trend with increasing water salinity. The contents of serum glucose and hepatopancreas lactic acid decreased significantly with increasing water salinity, while the levels of serum urea nitrogen, uric acid, and hepatopancreas uric acid showed an increasing trend. The higher contents of triglyceride, total cholesterol and urea nitrogen in the hepatopancreas were detected in 20 and 25 salinity treatments. The activity of total superoxide dismutase in the serum and hepatopancreas of females from 10 salinity treatment was significantly lower than other salinity treatments, while the highest levels of total antioxidant capacity, glutathione peroxidase, γ-glutamyl transpeptidase and hemocyanin in the serum, as well as and hepatopancreas glutathione peroxidase and malondialdehyde were detected at 10 salinity treatment. In conclusion, elevating water salinity could promote the growth and ovarian development of adult female P. trituberculatus, and the females had lower levels of metabolism and oxidative stress under the conditions of 20 and 25 salinities.
Optimisation of the Manila clam(Ruditapes philippinarum) breeding technique in diked tidal flat and the impact on physicochemical indices, phytoplankton community and juvenile growth performance
Jianfei QI, Yayou ZHENG, Qisheng WU, Yue Ning, Xiang Guo, Zhinan ZENG, Yuanyuan JIA
Accepted Manuscript  doi: 10.11964/jfc.20180611340
[Abstract](594) [FullText HTML](148) [PDF 895KB](9)
The Manila clam (Ruditapes philippinarum), production of which was 4 million tons in 2015, is a kind of important economic shellfish in China. The breeding technique in diked tidal flat which is the most productive technology, is not particularly reliable. In order to optimize it, a pond (A) was set up to induce phytoplanktonic blooms, which was regularly distributed to the nursery pond (B), and nursery pond (C) without algae superinduced as the control. The water, sediment quality and phytoplankton of A, B, C were compared with those of adjacent natural sea area (D). The growth of juvenile in B and C were also compared. The results showed that, there was no significant difference between A, B and C in water and sediment quality, but the water quality parameters(S, pH, DO, NH3-N, NO3-N, PO4-P) and the sediment quality (organic carbon, sulfide) of this three ponds were significantly different from D; The phytoplankton density followed the order of A > B > C > D, but only A had significant difference with D; the shell length and density of juveniles in B were significantly higher than those in C. This indicated that there was a great difference in the environment between the diked tidal flat ponds and the natural sea area, and phytoplankton supplement can increase the growth and survival of the juveniles, thus ensuring the success rate of the breeding of the manila clam in diked tidal flat ponds.
Comparison of the relation between the length frequency distribution and sample size for several commercial fish species in the Wentai fishing ground
Libin DAI, Siquan TIAN, Weicheng LIU, Chunxia GAO, Jiaqi WANG, Xiaoxue DU
In Press  doi: 10.11964/jfc.20170610861
[Abstract](497) [FullText HTML](120) [PDF 1300KB](7)
Length distribution data provide insight into the dynamics of fish populations. However, it is hard to determine the sample size required to describe size structure. In order to compare the relation between the precision of frequency distributions and sample size for several commercial fish species in the Wentai fishing ground, a resampling approach was used to calculate the mean square difference of length distributions based on actual sampling size and various simulated sampling sizes. The results showed that: ①Factors such as biology difference and seasonal variation would affect the relation of the precision of frequency distributions and sample size. ②The accuracy for estimating the precision of length frequency distributions would be impacted by the numbers of length intervals in iteration calculation. ③The precision of length frequency distributions could be considered as a theoretical basis to optimize sample size. For the reason of lacking domestic researches on sampling design and optimization, the results of this study could provide certain technical reference.
Polymorphism of seven insert/deletion mutations and their connection with growth traits of juvenile grass carp (Ctenopharyngodon idella)
Meng ZHANG, Yubang SHEN, Xiaoyan XU, Rongquan WANG, Xiangjun LENG, Jiale LI
In Press  doi: 10.11964/jfc.20180911463
[Abstract](466) [FullText HTML](126) [PDF 547KB](4)
When mapping the growth traits of grass carp (Ctenopharyngodon idella), a weight-related QTL was found in the 15 linkage group (LG) by our research group. In this study, we attempt to screen insert/deletion mutations on three scaffolds in the 15 LG, according to the previous consensus linkage map and draft genome of C. idella. At the meantime, we applied the STR genotyping method in genotyping of insert/deletion mutations. Moreover, the association analysis of selected mutations and growth traits of 323 individuals was conducted. Results found that 17 of the 44 selected insert/deletion mutations were SSRs, and 2 loci were failed to design primers; therefore, 25 pairs of primer were successfully designed. PCR amplifications showed that only 22 of the 25 primer pairs were effective. Then STR genotyping method was used for genotyping of those 22 insert/deletion mutations, and 7 polymorphic mutations were found in four parents. Afterwards, PCR products were sequenced to confirm the STR genotyping results. Sequencing results showed STR genotyping method could not only classify insertion/deletion mutant accurately, but also reduce the genotyping costs. Finally, the general linear model was used to analyze the relevance between those 7 insert/deletion mutations and growth traits of 323 juvenile C. idella. Correlation results found that 2 loci (ID-10H and ID-41F) were not related with growth traits; while the other 5 loci were significantly related with one or more growth traits of juvenile C. idella (P<0.05). According to the 5 loci, locus ID-6H was significantly correlated with fatness traits of juvenile C. idella; while locus ID-11F, ID-15F, ID-32F and ID-39F were significantly correlated with body weight or length or width or height. In summary, the inexpensive STR genotyping method could be used in the development of insert/deletion mutations, and the 5 growth related mutations could be used in QTL density-increasing and molecular marker assisted breeding of C. idella.
Genome sequencing and comparative genome analysis of Stenotrophomonas maltophilia isolated from Ictalurus punctatus
Heng XIE, Wenyan WEI, Kaiyu WANG, Tao LIU, Shengyu HE, Qian YANG, jiaxing LIU
In Press  doi: 10.11964/jfc.20180811387
[Abstract](743) [FullText HTML](296) [PDF 9100KB](11)
Stenotrophomonas maltophilia is one of the major pathogens causing high lethality and infectious diseases of Ictalurus punctatus in recent years. In order to provide technical reserves for the prevention and treatment of this disease, S. maltophilia XH.SM.1 strain was subjected to whole genome sequencing and comparative genomics analysis, and its morphological characteristics were observed by scanning electron microscopy. Electron microscopic observation showed that the short rod-shaped bacteria at both ends of XH.SM.1 had a length of about (1.73±0.35)μm and a width of about (0.43±0.04)μm. XH.SM.1 strain contained a chromosome of 4.56 Mb with an average G+C content of 66.60%, 4 087 coding sequences (CDS).The genomic sequence was uploaded to NCBI and obtained accession number: PYBO01000001.1. By comparing with the VFDB database, three highly virulence genes were predicted. The results of collinear analysis showed that XH.SM.1 had good collinearity with 10 S. maltophilia at the level of completion. ARDB database prediction and genomic island analysis found that XH.SM.1 contains many drug resistance genes. The results of pan-genome analysis showed that XH.SM.1 showed an open result, which is consistent with the phenotype of the pathogenic bacteria that are often found in the environment, indicating that it adapts to the environment and the outside world. The ability to communicate with genetic material is better.
Natural infection of Portunus trituberculatus with acute hepatopancreas necrosis disease causing by Vibrio parahaemolyticus (VpAHPND)
Jingwei HAO, Baoquan GAO, Chong WANG, Xianliang MENG, Xiaoyuan WAN, Xiaoping LI, Ping LIU, Qingli ZHANG
In Press  doi: 10.11964/jfc.20181011508
[Abstract](688) [FullText HTML](347) [PDF 1313KB](28)
The prevalence of multiple emerging diseases including acute hepatopancreatic necrosis disease (AHPND) has caused serious economic losses of crustacean aquaculture industry in China in last several years. In order to analyzing the possible pathogenic agents related to the slow growth of Portunus trituberculatus from Weifang City, Shandong Province, the presence and infection of nine suspected pathogens in the disease P. trituberculatus individuals were investigated by using the molecular detection methods in present study. The nine pathogens included white spot syndrome virus (WSSV), Infectious Hypodermal and hematopoietic necrosis virus (IHHNV), shrimp hemocyte iridescent virus (SHIV), AHPND-causing Vibrio parahaemolyticus (VpAHPND), enterocytozoon hepatopenaei (EHP), covert mortality nodavirus (CMNV), yellow head virus (YHV) and hepatopancreatic parvovirus (HPV). Meanwhile, histopathological analysis and in situ hybridization (ISH) were performed on the clinical samples. Results of molecular analysis showed that the P. trituberculatus samples were determined to be positive of VpAHPND and negative of other seven pathogens. Multiple sequence alignment based on the amplicons of VpAHPND nested-PCR of the samples revealed that sequences of the amplicons from P. trituberculatus samples shared 99% similarity to the pirAvp virulence gene from the pathogenic V. parahaemolyticus. Phylogenetic analysis indicated that the sequence from P. trituberculatus samples was clustered into the same branch with the reported PirA of pathogenic V. parahaemolyticus. Histological examination revealed necrosis of epithelial cells in hepatopancreas tubules, myonecrosis of cardiac muscle in heart, necrosis of epithelial columnar cells of gills, severe damage of nerve cell in the thoracic ganglion, as well as nuclear pyknosis in these tissues. Micrographs of ISH showed that blue-violet hybridization signals of the VpAHPND probes were present in the lesions of the hepatopancreas, myocardium, gills, and thoracic ganglia. The results indicated that the P. trituberculatus samples from the shrimp and crab polyculture ponds were naturally infected by VpAHPND, which caused serious pathological damages of the hepatopancreas, myocardium, gills, and thoracic ganglia. The present study revealed, for the first time, that VpAHPND infection in P. trituberculatus and the pathological changes caused by the infection. The results provided basic information for revealing the natural host species of VpAHPND, and for the prevention and control of AHPND of P. trituberculatus.
Construction of G gene nucleic acid vaccine against infectious hematopoietic necrosis, and its effect on serum biochemical indices
Qiyao HE, Wenyan WEI, Kaiyu WANG, Jiaxing LIU
In Press  doi: 10.11964/jfc.20180711378
[Abstract](705) [FullText HTML](276) [PDF 962KB](5)
The aim of this study was to study the effect of infectious hematopoietic necrosis virus (IHNV) acid vaccine on the immune protection of Oncorhynchus mykiss and serum biochemical indices of Oncorhynchus mykiss. Cloning the G gene into pMD19-T vector, transforming the linked product in DH5α, obtaining recombinant plasmid pMD19-T-G, and recovering the G gene fragment. The identified correct G gene fragment was cloned on eukaryotic expression vector pVAX1 using BamH I and Xho I cleavage sites to construct nucleic acid vaccine pVAX1-G. The recombinant plasmid pVAX1-G was injected into Oncorhynchus mykiss at a dose of 8 μg/tail as pVAX1-G group, while 8 μg/tail empty group, PBS control group and blank group were set up. Twenty-one days after immunization, an anti-virus experiment was carried out. After anti-virus, serum was collected and blood indexes were tested. The results showed that there were significant changes in 11 indexes in Oncorhynchus mykiss serum after the attack: Glutamic-pyruvic transaminase (ALT), glutamate transaminase (AST), alkaline phosphatase (ALP), total bilirubin (TBIL), total bile acid (TBA), glucose (GLU), the urea (Urea), creatinine (CREA), total protein (TP), albumin (ALB) and globulin (GLO) compared with normal Oncorhynchus mykiss, and the changes of 11 indexes in no-load group were more significant than those in pVAX1-G group. The cumulative death rate of pVAX1-G group was 19%(19/100)14 days after the attack, while that of no-load group and PBS control group were 82%(82/100) and 85%(85/100) respectively. The protective rate of pVAX1-G nucleic acid vaccine against Oncorhynchus mykiss was 78%. The results showed that pVAX1-G as a nucleic acid vaccine was helpful to reduce IHNV's injury to Oncorhynchus mykiss and had better immune protection effect on IHNV.
Isolation, identification and phylogenetic analysis of an infectious hematopoietic necrosis virus isolated phylogenetic analyses from Oncorhynchus mykiss at PenZhou city, SiChuan province
Tao LIU, Wenyan WEI, Jiaxing LIU, Ma YANG, Qian YANG, Shengyu HE, Qiyao HE, Heng XIE, Kaiyu WANG
In Press  doi: 10.11964/jfc.20180511284
[Abstract](611) [FullText HTML](267) [PDF 979KB](7)
An outbreak of an infectious disease occurred amongst farmed Oncorhynchus mykiss at PenZhou city, Sichuan Province, in October 2017 with heavy mortality of 85%. Major symptoms of the diseased fish included darkening of the body, yellow mucoid fluid fecal casts on anal. Internally, the symptoms were hemorrhage in the swim bladder and peritoneum, gastric distention, enteritis and hydropericardium. Bacteriologic test was negative. Histopathologically, the spleen showed typical coagulative necrosis with marked degeneration and necrosis in the haematopoietic tissue. Hepatocytes showed degeneration and necrosis and edema in some areas. After filtration treatment, the tissue suspension was injected intraperitoneally into 60 O. mykiss. The O. mykiss displayed similar clinical symptoms as fish that were naturally infected. The O. mykiss died acutely in the trial group (cumulative mortality rate of 85%), while there was no abnormal fish in the control group. After filtration treatment, the spleen tissue suspension was inoculated to the Fathead minnow cells (FHM). The typical cytopathic effects were observed after three blind passages in FHM. RT-PCR assay of tissue filtrates from the fish natural infected, the fish artificial infected and diseased cells were IHNV-positive. The amplification products shared 98.2% identity to that of IHNV nucleoprotein gene at nucleotide level. Phylogenetic analysis based on the G gene showed that the viral isolate was classified into Asian isolates, belonged to the JRt genotype.
Management and assessment of spotted mackerel (Scomber australasicus) biomass in the Pacific Ocean
Chang ZHANG, Xinjun CHEN
In Press  doi: 10.11964/jfc.20171211117
[Abstract](776) [FullText HTML](396) [PDF 995KB](11)
Spotted mackerel (Scomber australasicus) is an important economic species in northwest Pacific. The paper evaluates the Pacific stock biomass by aged-structure Virtual Population Analysis (VPA) and Yield per recruit model, and analyses the utilization of stock biomass by using the mackerel catch and CPUE data of the Pacific group which is supplied by Japan's central fisheries research institute from 1995 to 2015. The results show that the biomass of spotted mackerel remains at a high level. The stock biomass is about 650000 tons in 2015, the average fishing mortality shows a trend of falling volatility and fishing mortality of 2015 is 0.15, the current fishing mortality (Fcur) is 0.33, the spawning potential ratio (SPR) remains 36.5%. There is no growth overfishing and recruitment overfishing. It is also discussed that the fluctuations of natural mortality caused by water temperature change and different fishing age affect the resources of spotted mackerel. The study suggests that the fishery is currently utilized in the sustainable way and has great potential for development. It also suggests that the fishery resource could be developed and utilized by using the management reference point F0.1.
The study on the growth, digestive enzymes and immune enzymes activities in the two growth status of seahorse Hippocampus erectus
Yongchun HUANG, Chenling TU, Hongyi Weng, Xiangri LING
In Press  doi: 10.11964/jfc.20171010988
[Abstract](878) [FullText HTML](402) [PDF 665KB](5)
The paper studied the occurrence time and physiological characteristics of slow growth seahorse. Comparisons of the growth, the ratio of RNA/DNA, digestive enzymes and related immune enzymes activities in normal growth seahorses[(8.57±0.61) cm, (1.51±0.38) g] and slow growth seahorse [(6.68±0.35) cm, (0.81±0.14) g]Hippocampus erectuswere studied in a cultural experiment for 110 d. The results showed that the average daily gain, specific growth rate, condition factor and viscera index in the normal growth seahorses were significantly higher than those of the slow growth seahorse, respectively. The normal growth seahorses grew faster than that of the slow growth seahorses after the body length was5.026 cm. The amylase, protease and lipase activities in normal growth seahorses were as the same tendency as those of the slow growth seahorses under different reaction temperatures (5 °C, 15 °C, 25 °C, 30 °C, 35 °C, 45 °C and 55 °C) and pH (2.0, 3.0, 4.0, 5.0, 6.0, 7.0, 8.0 and 9.0). However, the digestive enzymes activities of the former were higher than those of the latter, except amylase activity at 35-55 °C and protease activity at 5–25 °C. The activities of ACP、AKP、T-AOC and SOD in normal growth seahorses were 12.83%, 48.21%, 44.71%and 65.75% more than those of the slow growth seahorses, respectively. The MDA of the normal growth seahorse was only 61.09% of that in the slow growth seahorse, and the ratio of RNA/DNA in the normal growth seahorse was higher 23.75% than that of the slow growth seahorse. The growth, digestion and immunity of he normal growth seahorses were obviously better than those of the slow growing hippocampus when their body length were more 5.026 cm.
Molecular cloning of multiple vitellogenins in Scatophagus argus and their gene expressions after 17α-ethynylestradiol exposure in vivo and in vitro
Di WU, Meiqin WU, Maoliang SU, Yong ZHONG, Wu WANG, Junbin ZHANG
In Press  doi: 10.11964/jfc.20170410777
[Abstract](907) [FullText HTML](461) [PDF 3559KB](23)
With the aim to explore the molecular mechanism of vitellogenin (Vtg) in Scatophagus argus, we cloned the full-length cDNA sequence of three types of vtg by using reverse transcription-polymerase chain reaction (RT-PCR) and rapid amplification of cDNA ends (RACE) procedures. The length of the vtgAa cDNA is 5360 bp (GenBank No. KY676847), and it encodes a protein consist of 1696 amino acids. The length of the vtgAb cDNA is 5346 bp (KY654346), and it encodes a protein consist of 1699 amino acids. The length of the vtgC cDNA is 4244 bp (KY676848), and it encodes a protein consist of 1275 amino acids. Sequence analysis showed that three vitellogenin homologs of Scatophagus argus have highest homology with Morone americana (83%, 85% and 85%). VtgAa and VtgAb contain all the three major portions, but VtgC lacks phosvitin (PV). Quantitative real-time PCR (qPCR) assay were employed to determine the mRNA expression of vtgAb in the liver after 17 α-ethynylestradiol (EE2) injection in vivo and hepatocytes after exposure EE2 in vitro. Results of qPCR showed that the hepatic expression of vtgAb was higher than vtgAa and vtgC. 48 h after injection, mRNA expression of vtgAb was extremely increased and obviously decreased after 72 h. The expression of vtgAb mRNA was elevated after EE2 exposure to hepatic cells. Based on our results, expressions of vtgAb could be induced by EE2.
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2019, 43(5).  
[Abstract](189) [PDF 3310KB](6)
2019, 43(5).  
[Abstract](200) [FullText HTML](96) [PDF 270KB](3)
Characterization of Carassius auratus herpesvirus ORF31R (CaHV-31R) and the encoded protein colocalization with cellular organs
Zihao WANG, Qiya ZHANG
2019, 43(5): 1263-1270.   doi: 10.11964/jfc.20180311207
[Abstract](771) [FullText HTML](426) [PDF 1381KB](15)
Carassius auratus herpesvirus (CaHV) is a pathogen that can cause crucian carp disease with acute gill hemorrhages and high mortality. Interactions of functional genes with cellular components, such as cellular organs, are needed by viruses to complete infection and replication. In the present study, bioinformatic analysis, PCR amplification, gene cloning, constructing recombinant plasmids, and fluorescence observation of the fish cells (epithelioma papulosum Cyprinid, EPC) cotransfected with cellular organs tagged plasmids were used to analyze the characteristics, subcellular localization and colocalization with cellular organs of the protein encoded by the gene CaHV ORF31R (CaHV-31R). As the results show, the encoding protein CaHV-31R was composed of 313 amino acids, which consists of a transmembrane domain (248−270 aa) and a typical domain of RNase E/G protein family (40−182 aa). The multiple alignment with homology proteins from 5 different fish hepesviruses exhibited higher identity (100% and 80.7%) with the Janpanese strain ST-J1 and Chinese strain SY-C1, which were both the members of type II Cyprinid hepesviruses, centered identity (26.5%) with CyHV-1 which was the representative member of type I Cyprinid hepesviruses, and the lowest identity (20.7% and 18.2%) with the Germanic strain CyHV-3(also named Koi herpesvirus, KHV) and Chinese strain (CyHV-3-GZ11), which were both the members of type III Cyprinid hepesviruses. The plasmids pEGFP-31R was constructed by PCR amplification, gene cloning, and then used to transfect EPC cells. pEGFP-31R dispersed distribution in cytoplasm and a small part of spotty distribution were found circling around outside the nucleus. CaHV-31R was used to colocalize with two celluar organs with monolayer membrane in cytoplasm, which was endoplasmic reticulum and Golgi apparatus. The results indicated that CaHV-31R was a gene of Carassius auratus herpesvirus, and was able to encode a protein that colocalizes with cellular organs.
Antimicrobial activity and proteomic analysis ofBoleophthalmus pectinirostris skin mucus
Honghan LIU, Yuting JIANG, Meihua FAN, Rixin WANG, Zhi LIAO
2019, 43(5): 1271-1287.   doi: 10.11964/jfc.20180511293
[Abstract](823) [FullText HTML](471) [PDF 883KB](38)
Fish skin mucus serves as the first line of defense against pathogens and external stressors. The mudskipper Boleophthalmus pectinirostris inhabit intertidal mudflats containing abundant and diverse microbial population, thus, the skin together with the mucus of B. pectinirostris are very important for the immune defense, osmotic pressure maintenance and adaptation to amphibian life. For exploring the proteomic profile of the skin mucus and understanding the molecular mechanism of B. pectinirostris adaptation to amphibious environments, the antibacterial activity was determined by agar diffusion plate method for the mucus and the serum samples. In addition, a growth curve inhibition method was used to compare the antibacterial activities of B. pectinirostris mucus before and after vibrio induction. Furthermore, the proteomic profile of natural B. pectinirostris mucus was identified by Shotgun mass spectrometry technology combining with skin transcriptome searching. The interaction network analysis of the identified proteins from mucus was performed by String software. Skin mucus was collected from B. pectinirostris after electrical stimulation. The mucus of B. pectinirostris showed a broad spectrum of antimicrobial activity more than serum of the same species, indicating that the mucus has a stronger antimicrobial activity than the serum. After vibrio induction, the antibacterial activity of mucus was slightly stronger for some gram-negative bacteria than that of un-induced mucus. A total of 97 proteins were identified from natural mucus of B. pectinirostris with a similar results from studies of other fish species, including actins, keratins, apolipoproteins, transferrins, calmodulins, ubiquitins, pentraxins, and various enzymes. However, some proteins, such as ubiquitin-like proteins and thymosin, were identified first from fish mucus. The identified proteins can be clustered into structural proteins, enzymes, material transport related proteins, immune proteins, and other proteins. Most of these proteins are known to be involved in immune and/or stress responses. Protein interaction analysis showed strong interactions among the identified proteins, such as actin, myosin, cofilin, filamin, apolipoprotein, transferrin, calmodulin, and superoxide dismutase. The proteomic profile established in this study could not only provide knowledge on the routes involved in mucosal innate immunity, but also put forward a non-invasive technique based on locating immune markers with a potential use for prevention and/or diagnosis of fish diseases.
Effects of miRNA on snakehead vesiculovirus (SHVV) infection in stripped snakehead (SSN-1) cells
Lizhu YI, Zhen XU, Li LIN, Jiagang TU
2019, 43(5): 1288-1297.   doi: 10.11964/jfc.20171211098
[Abstract](734) [FullText HTML](401) [PDF 765KB](19)
In order to study the effects of miRNAs in stripped snakehead (SSN-1) cell line upon snakehead vesiculovirus (SHVV) infection, SHVV-infected and mock-infected SSN-1 cells were analyzed by miRNA high-throughput sequencing. The expression levels of twenty-two miRNAs with levels equal to or higher than 1 000 reads per million (TPM≥1 000) were determined by qRT-PCR with U6, β-actin, and 5S rRNA as internal control. When U6 gene was used as internal control, no significant changes were observed for β-actin upon SHVV infection, while 5S rRNA was upregulated in SSN-1 cells upon SHVV infection. The results showed that U6 gene or β-actin could be used as internal control genes for miRNAs in SSN-1 cells. Furthermore, fourteen miRNAs were selected and their effects on SHVV replication were analyzed. The results showed that eleven miRNAs (miR-27a-3p, miR-26a-5p, miR-30e-3p, etc.) promoted, whereas miR-150 and miR-216b suppressed, SHVV replication. Next, we found that high-expression miR-100-5p was upregulated at early stage of SHVV infection, while downregulated at later stage. Overexpression of miR-100-5p exhibited significant suppression of SHVV replication, while inhibition of expression of miR-100-5p promoted SHVV replication. Our findings presented valuable information for the study of molecular drugs against SHVV.
Research on epidermis ulcer syndrome caused by Vibrio harveyi in Hippocampus erectus
Ying LI, Bo WANG, Peiyu ZHANG, Hongzhan LIU, Fengrong ZHENG, Zongxing WANG, Zongjun XU, Chao ZHAO
2019, 43(5): 1298-1307.   doi: 10.11964/jfc.20180611339
[Abstract](581) [FullText HTML](272) [PDF 935KB](12)
Hippocampus spp. is unique marine fish with special body morphology and life history traits. Compared with other marine fishes, the most special part of Hippocampus spp. is male breeding and strict monogamy. In addition, its unique body morphology, including curvaceous torso and horse-shaped head, has given them higher ornamental value. The trade volume of Hippocampus spp. is huge in the world, and the majority of the trade in Hippocampus spp. is for traditional Chinese medicine. In recent years, the destruction of wild habitats and overfishing lead to a sharp decline of the wild populations. Aquaculture of Hippocampus spp. has been considered as an effective way to solve the problem of decline of the wild populations and the unsustainable trade for traditional Chinese medicine. Therefore, the development of the Hippocampus spp. aquaculture is essential. However, the threat of various diseases has seriously affected the healthy development of the aquaculture industry. Since 2017, in the breeding process of the H. erectus in northern China, epidermis ulcer syndrome has frequently occurred, the morbidity is rapid and the mortalily is high. Epidermis ulcer syndrome has become a high incidence disease in the aquaculture process. Diseased H. erectus showed obvious symptoms, such as large rot area in body surface, darker and lackluster of the gills and liver, diseased H. erectus would die within 3 days. If the diseased H. erectus were not removed in time, a large number of seahorses would be infected. Although most fish diseases can be treated with antibiotics, the overuse of antibiotics may produce many potential threats, which are not conducive to the sustainable and healthy development of the aquaculture industry. Therefore, it is essential to further study the pathogens of various fish diseases and then we can develop biological control on this basis. Accordingly, the priority of research should be disease treatment and health management, especially disease treatment. Specific vaccines can be developed from isolated pathogenic bacteria, which provide effective way for disease treatment. In this study, dominant strains were isolated from the lesion tissue and intestine of H. erectus with epidermis ulcer syndrome. In order to determine the pathogenic bacteria, we carried out artificial infection, the infection results suggested that strain HDM-2 was the pathogen causing epidermis ulcer syndrome, and the LD50 of strain HDM-2 was about 2.89×108 CFU/mL. We identified strain HDM-2 with physiological and biochemical identification and molecular biological identification methods, and we observed strain HDM-2 under transmission electron microscope. 16S rDNA gene sequence analysis by BLAST in GenBank suggested that the similarity between strain HDM-2 and V. harveyi reached 100%. The observation results showed that HDM-2 was about 2.2 μm×4.75 μm, with peritrichous flagella and capsule. In order to study antibiotic susceptibility of strain HDM-2, we implemented the antibiotic sensitivity test by using Kirby-Bauerdiffusion method, and results showed that strain HDM-2 was highly sensitive to florfenicol. In order to study the pathogenicity of the pathogen to the host, we carried out histopathological observation of the diseased H. erectus. Observation results suggested that skin, liver and gills were severely damaged by HDM-2. This study would lay a foundation for the prevention and further study of the epidermis ulcer syndrome of H. erectus.
Virulence evaluation of Streptococcus agalactiae isolated from tilapia(Oreochromis niloticus) using BALB/c mice
Ling LIU, Jun XU, Pan MA, Anxing LI
2019, 43(5): 1308-1316.   doi: 10.11964/jfc.20180311213
[Abstract](475) [FullText HTML](240) [PDF 941KB](11)
Streptococcus agalactiae is one of the major pathogens of tilapia (Oreochromis niloticus). At present, the virulence of strains was evaluated mainly by comparing the median lethal dose (LD50) to tilapia. However, tilapia as experimental animals for evaluation of the virulence of S. agalactiae was often unstable. In this experiment, BALB/c mice were used as experimental animals, in order to establish a method for the determination of virulence of S. agalactiae. BALB/c mice were injected intraperitoneally with tilapia Streptococcus agalactiae to establish an infection model. The LD50 of Streptococcus agalactiae to tilapia and mice were tested 3 times and the virulence of different Streptococcus agalactiae to tilapia and mice was determined. Results show that: by intraperitoneal injection, S. agalactiae can cause the death of mice within 24 hours. S. agalactiae could damage brain, liver, spleen, kidney and other tissues in mice. The LD50 of S. agalactiae to tilapia and mice 3 times was 7.7×107, 2.2×108, 3.5×109 CFU and 405, 361, 419 CFU, respectively. When S. agalactiae TFJ0901 and THN0901 infected tilapia (1.0×107 CFU) and mice (100 CFU), the survival rates of tilapia and mouse were 100.0%, 6.7%±5.8%, and 100.0%, 0%, respectively, both of which were significantly (P<0.05) different. When S. agalactiae TFJ0901 and TFJ-F infected tilapia (3.0×108 CFU) and mice (2 500 CFU), the survival rates of tilapia were 73.3%±11.5% and 80.0%±10.0%, respectively, which were not significantly (P>0.05) different from each other, while the survival rates of mice were 13.3%±11.5% and 100.0%, respectively, which were of significant (P<0.05) difference. Taken together, BALB/c mice were successfully established as a stable model for virulence determination of S. agalactiae in tilapia. The determination of the virulence of different S. agalactiae in mice is consistent with tilapia, and this model was able to distinguish S. agalactiae with similar virulence that was difficult to be distinguished by using tilapia model.
Variance analysis of intestinal bacterial community between healthy and diseased Trachinotus ovatus
Xiangying XIONG, Xianfeng WANG, Yinhui PENG, Zhicheng WANG
2019, 43(5): 1317-1325.   doi: 10.11964/jfc.20180411227
[Abstract](760) [FullText HTML](442) [PDF 818KB](52)
In order to investigate the difference of the intestinal community structure and its relationship with environmental factors in response to disease, the intestines of healthy and diseased Trachinotus ovatus were used to analyze the bacterial community composition and diversity as well as the culture waters and pellet feed. The five samples were studied by using Illumina HiSeq high-throughput sequencing and biological information analysis method. Compared with the intestinal microflora of the healthy pompano, the relative abundances of Spirochaetes significantly increased in the diseased pompano, whereas, the Firmicutes and Bacteroidetes exhibited an opposite pattern. The bacteria species in diseased pompano intestine only accounted for 54.94% in healthy pompano. There was 73.46% of the OTUs in the healthy ovate intestine, the same as that in the culture waters, and 70.58% in the feed, while the percentage dropped to 17.98% and 38.95% in the diseased pompano. Notably, the disease pompano had a higher relative abundance of Vibrio ponticus than healthy pompano being 78.90% and 17.19%, respectively. In addition, the relative abundance of Photobacterium leiognathi in healthy pompano was 54.53%, but absent in diseased pompano. The intestinal bacterial composition was relatively stable but there were still some difference between healthy and diseased pompano. After the occurrence of disease, the bacterial diversity declined markedly. The culture water and feed had a close correlation with the healthy pompano intestine but low influence on diseased pompano in bacterial species composition.
Pathogenicity of Cryptocaryon irritans to Larimichthys crocea and Chinese herbal inhibition on the parasitic generation reproduction
Kaiqiang JIAO, Yingeng WANG, Xiaojun RONG, Jia CHEN, Zheng ZHANG, Meijie LIAO, Bin LI, Luyu SUN, Yuezhong SU
2019, 43(5): 1326-1337.   doi: 10.11964/jfc.20180311206
[Abstract](666) [FullText HTML](373) [PDF 1099KB](12)
The "white spot disease" caused by the parasitic infection of Cryptocaryon irritans has become one of the most serious diseases in the production of Larimichthys crocea. This study aims to investigate the pathogenicity of C. irritans to L. crocea, as well as the effect of Chinese herbal compound HD-2 on inhibiting the generation reproduction of parasite C. irritans. The number, size, the hatching rate and number of hatched theronts of tomonts produced by natural sick L. crocea were investigated. Then the survival time and infectivity of theront was determined in vitro. The effects of Chinese herbal compound HD-2 on theront, trophont, and tomont, and the therapeutic effects of oral HD-2 on diseased L. crocea were studied. The results showed that the total number of tomonts produced by L. crocea was positively correlated to the fish body weight; the average diameter of tomonts were (340.8±64.9) μm and the hatching rate was 76.4%, and each tomont could produce (280±42.5) theronts; the theront could survive for more than 24 h in vitro and lost their infectivity in 20 h after hatching. The 90% lethal dose (LD90) of L. crocea infected with C. irritans in 7 days was 9 712 theronts/fish and the median lethal dose for 50% (LD50) was 4 366 theronts/fish. HD-2 could kill theront effectively. After oral administration with different doses of HD-2 (control, 0; G1, 5 g/kg; G2, 10 g/kg; G3, 15 g/kg) for 30 days, the relative infection intensity of the L. crocea groups challenged with LD50 decreased by 32.0%, 44.5% and 51.8%, respectively. Meanwhile, the volume of tomonts reduced by 35.4%, 36.1% and 37.3% and the hatching rate reduced by 16.3%, 23.3% and 27.9%, respectively. The fish survival rate of G1, G2 and G3 challenged with LD90 was 40.0%, 55.0% and 58.3%, which was significantly higher than the control group (only 8.3%). Using HD-2 oral treatment for 15 d, the tomonts produced from diseased fish reduced 73.1%, 87.7% and 93.8%, and the fish mortality rate of the three groups were 58.3%, 36.7% and 21.7%, which were significantly lower than the control group (83.3%). The physiological status of L. crocea in the treated group was significantly better than that of the control group. Therefore, HD-2 oral treatment could effectively interrupt the secondary infection of C. irritans, suppress the formation and development of tomonts, reduce the tomont hatching rate and the number of hatching theronts, and then inhibit the generation reproduction of parasite C. irritans.
Effects of traditional Chinese compound prescription and antibiotics treating in Vibrio alginolyticus diseases of Epinephelus coioides
Xiaojin XU, Xiuhua LI, Yifan MA, Huiyao LI, Xin QI, Jiang ZHENG, Hailong LÜ, Qingpi YAN
2019, 43(5): 1338-1346.   doi: 10.11964/jfc.20181211565
[Abstract](589) [FullText HTML](242) [PDF 714KB](7)
The antibacterial effect of the drugs against Vibrio alginolyticus in vitro and the drugs on the resistance to diseases of Epinephelus coioides were studied. This study aimed to screen traditional Chinese medicines formulas and Chinese and western medicines formulas which can prevent V. alginolyticus diseases of E. coioides. We had the bacteriostatic test on traditional Chinese medicines formulas against V. alginolyticus in vitro. The ratios of Terminalia chebula, Radix paeoniae and Glycyrrhiza uralensis of Chinese medicines formulas Ⅰ, Ⅱ, Ⅲ were 0.9∶1.3∶0.9, 1.2∶0.9∶1.0, and 1∶1∶1, respectively. The groups with 3 replicates per treatment of feed containing the drugs feed additives at 2.2% respectively, were fed to E. coioides. The ratio of T. chebula, R. paeoniae and G. uralensis were different in Chinese medicines formulas trial groupⅠ, Ⅱ, Ⅲ. The ratios of T. chebula, R. paeoniae, G. uralensis and enrofloxaci were different in Chinese and western medicines formulas trial groupⅣ, Ⅴ. Results revealed that the concentration of Chinese medicines formulas Ⅲ (200 mg/mL) was the best formula. The mortality for 40 d in test groups were as follows: control groupⅡ>control group Ⅳ>control group Ⅲ>trial group Ⅲ>trial groupⅡ>trial groupⅠ>trial group Ⅴ>trial group Ⅵ>trial group Ⅳ>control groupⅠ. The ratio of T. chebula, R. paeoniae and G. uralensis of Chinese medicines formulaⅠwas 0.9∶1.3∶0.9. The ratios of T. chebula, R. paeoniae, G. uralensis and enrofloxaci in Chinese and western medicines formula Ⅳ were different. These are the best formulas. The Chinese and western medicines formula is better than the Chinese medicines formula. Results showed that by adding the formulas, the capacity of disease resistance can be improved.
Diversity of gut microbiota and antibiotic resistance genes inLitopenaeus vannamei and Macrobrachium rosenbergii
Bin HONG, Ben NIU, Ping CHEN, Wei LI, Haiquan LIU, Yingjie PAN, Yong ZHAO
2019, 43(5): 1347-1358.   doi: 10.11964/jfc.20180811404
[Abstract](730) [FullText HTML](419) [PDF 906KB](29)
To compare the differences in gut microbiota and antibiotic resistance genes (ARGs) between Litopenaeus vannamei and Macrobrachium rosenbergii, microbiota community structure and microbiota diversity of two shrimps were analyzed by high-throughput sequencing and denaturing gradient gel electrophoresis (DGGE), and 38 kinds of ARGs in the gut bacteria of two shrimps were detected. The effective bacterial sequences of L. vannamei and M. rosenbergii were 42 795 and 40 713. The results showed that the numbers of operational taxonomic units (OTUs) were 124 and 82, and the bacterial types with clear classification status were 5 phyla, 17 genera and 5 phyla, 16 genera. The dominant group in the gut bacteria of L. vannamei is Proteobacteria, which accounts for 75.45%. The dominant genera are Paracoccus (25.83%) and Acinetobacter (25.24%). The dominant group in gut bacteria of M. rosenbergii is Firmicutes (49.74%), and the dominant genera are Lactococcus (49.01%) and Vibrio (29.98%). The Shannon index of the gut bacteria (2.19) of L. vannamei was higher than that of M. rosenbergii (1.78), indicating that the gut bacteria diversity of the former was greater than that of the latter. The analysis results of DGGE fingerprint were consistent with those of high-throughput sequencing, and the bacterial diversity of the two shrimps was significantly different. The PCR results showed that the gut bacteria of L. vannamei carried 15 ARGs and gut bacteria of M. rosenbergii carried 14 ARGs. Our study revealed that the community diversity, OTU richness, total number of species and ARGs in the gut bacteria of L. vannamei were higher than those of M. rosenbergii, which provided a theoretical basis for the subsequent excavation of gut microbial resources.
Isolation and identification of Vibrio vulnificus from Cynoglossus semilaevis and establishment of fluorescence quantitative PCR technique
Xiucai HU, Yingjie WANG, Aijun LÜ, Jingfeng SUN, Qingkui WANG, Zhengyi LIAN, Xiaoxue LIU, Yajiao SONG
2019, 43(5): 1359-1368.   doi: 10.11964/jfc.20181011505
[Abstract](290) [FullText HTML](164) [PDF 825KB](3)
In order to investigate the pathogen of the diseased tongue sole Cynoglossus semilaevis, three strains of Gram-negative bacteria isolated from diseased C. semilaevis were tentatively named strains ST-1, ST-3 and ST-6, which were identified by physiological and biochemical characteristics, antimicrobial susceptibility test, 16S rDNA gene sequences analysis and histopathology examination. Furthermore, the SYBR Green Ⅰ quantitative real-time PCR (qRT-PCR) targeting the outer membrance protein (OMP) gene was established. The biochemical results of the isolate ST-1, ST-3 and ST-6 were consistent with the properties of V. vulnificus. The BLAST alignments of the 16S rDNA gene sequence showed that the isolates ST-1, ST-3 and ST-6 shared the identities above 99% with type strain Vibrio vulnificus ATCC 29307 (X74727). The phylogenetic tree indicated the isolates clustered with V. vulnificus ATCC 29307. The isolates were eventually identified as V. vulnificus. Antimicrobial susceptibility testing showed that the isolates were highly susceptible to cefixime, cefoperazone, streptomycin, imipenem and florfenicol. The challenge experiments showed that the isolated strains were pathogenic on C. semilaevis and zebrafish(Danio rerio), and similar symptoms were observed with the natural infection. Histopathology examination revealed the hyperplasia of the epithelial cells in the primary lamella, the swelling of the secondary lamella and the increase of the number of granulosa cells and mucus cells, hepatic sinusoid and the congestion of central vein in the liver, hemorrhagic-necrotic lesion in the kidney, the atrophy of the lamina propria and the increase of the number of mucus cells in the intestine. The SYBR Green I quantitative PCR (qPCR) method based on the OMP gene was further established and the results showed that the correlation coefficient of standard curve was 0.995 and the lowest limit of detection was 1.88×102 CFU/mL. The results of this study will lay a foundation for the pathogenesis and prevention of C. semilaevis vibriosis, which can provide scientific reference for the molecular diagnosis of the disease.
Identification and antimicrobial resistance of Vibrio cyclotrophicus isolated from aquatic animals
Shu ZHAO, Lan JIANG, Junfang ZHOU, Yuan WANG, Wenhong FANG
2019, 43(5): 1369-1377.   doi: 10.11964/jfc.20180211180
[Abstract](682) [FullText HTML](385) [PDF 652KB](15)
This paper studied the identification, drug resistance and the molecular resistance mechanism of five Vibrio strains isolated from diseased Cynoglossus semilaevis. The five strains were identified as V. cyclotrophicus by a biochemical test combined with a molecular method (16S rRNA, HSP60 and mreB genes). The broth dilution method was used to determine the antimicrobial susceptibility of the strains to 14 antimicrobials. The PCR amplification and DNA sequencing were used to detect antimicrobial resistance genes, integrons and the gene cassettes. The results indicated that the five strains were resistant to ciprofloxacin, streptomycin, erythromycin, florfenicol, ampicillin, SMZ, TMP/SMZ and rifampicin, and carried qnr VC, blaCARB-17, strA, strB, sul genes. Class I intergron, qacEΔ1 and sul1 genes were present in JS291 and JS295 strains. The integron-gene cassettes of JS291 were encoding dihydrofolatereductase (dfr16), aminoglycoside-(3″)(9)-adenylyltransferase (aadA2), and the cassettes of JS295 were encoding rifampin ADP-ribosylating transferase (arr-3) , dihydrofolate reductase (dfrA27), respectively. These findings suggested that the five strains exhibited multi-drug resistance and the multi-drug resistance phenotype may be closely related to the antimicrobial resistance genes.
Isolation, identification and biological properties of 3 lytic bacterio phages against AHPND-associated Vibrio parahaemolyticus
Huan QIAO, Xuling XU, Wenbin FEI, Jie HUANG, Hai CHEN, Yilin HU, Yu CONG
2019, 43(5): 1378-1396.   doi: 10.11964/jfc.20180711345
[Abstract](677) [FullText HTML](362) [PDF 1522KB](20)
To isolate and identify lytic bacteriophages against AHPND-associated Vibrio parahaemolyticus and analyze their biological properties, lytic bacteriophages were isolated from Fujian and Mexico seawater samples by double-layer agar culture method, and one strain of V. parahaemolyticus strain associated with AHPND was used as the host bacterial strain for phage isolation and amplification. The phages were classified and identified by plaque shapes, restriction endonucleases, whole genome sequence, phylogenetic tree building and transmission electron microscopic technique. The lysis range and biological properties including optimal multiplicity of infection (MOI), thermal stability, pH stability, stability to peracetic acid and survival stability at different temperature were determined. The results showed that 3 lytic bacteriophages against AHPND-related V. parahaemolyticus were isolated, named P46, P48 and VP7. Their plaques were translucent circle. The plaque diameter of P46 was 4-5 mm. The plaque diameter of P48 and VP7 were 5-6 mm. The nucleotides of 3 bacteriophages were all dsDNA. P46, P48 and VP7 all had an icosahedral head with about 60 nm diameter. Their tails were about 18-20 nm in length and 8 nm in width. 3 phages belonged to the familiy Podoviridae. The cocktail of P46, P48 and VP7 lysed 91.5% of AHPND-associated V. parahaemolyticus strains and non-VPAHPND 92.3% respectively. And they all could not identify non-V. parahaemolyticus bacteria. The optimal MOI of P46, P48 and VP7 were 0.001, 0.1 and 0.001 respectively. The optimal pH of P46 and VP7 were 6-8 and P48`s was 6-9. Three bacteriophages were stable at 60 °C and had higher survival rate at 4 °C for 50 weeks. P46, P48 and VP7 also tolerated peracetic acid with universal bactericidal concentration. Alignment with the sequences of the conserved protein sequences of NCBI showed that P46, P48 and VP7 had low homology with other bacteriophages. Bacteriophages P46, P48 and VP7 might be 3 novel Podoviridae bacteriophages. This study is the first report of lytic Podoviridae bacteriophages against AHPND-associated V. parahaemolyticus in China. The results may enrich the resources of bacteriophages against AHPND-associated V. parahaemolyticus and provide potential application of phages as a biocontrol agent.
Anaesthetic effects of zolazepam and tiletamine on Chinemys reevesiis and pharmacokinetics of zolazepam and tiletamine
Yan MA, Liande PAN, Jianping XU, Chuan ZHENG
2019, 43(5): 1397-1406.   doi: 10.11964/jfc.20170910958
[Abstract](610) [FullText HTML](372) [PDF 1019KB](4)
The current study aimed to solve the problems of anaesthesia in clinical veterinary medicine of turtles. According to the results of pre-experiment, we selected the animal medicine recommended clinical anaesthetic drugs Telazol(tiletamine:zolazepam=1:1). We compared the anaesthetic effects of different dosages of Zoletil on Chinemys reevesiis, and the concentrations of zolazepam and tiletamine in plasma were detected by High Performance Liquid Chromatogram (HPLC), and analyzed with software PKsolver 2.0 to research the features of pharmacokinetics. Zoletil at a rate of 25 mg/kg was administered intramuscularly to C. reevesiis.The induction time, anaesthesia time and recovery time were 0.42, 4.33 and 4.14 h respectively. Zoletil on C. reevesiis anaesthesia was better, in line with the requirements of clinical surgery. The concentration-time curves of zolazepam and tiletamine in plasma all were conformed to a two-compartment open model. And the concentration-time equations of zolazepam and tiletamine were C=13.462e–0.223t+2.127e–0.055t, C=10.619e–0.203t+0.279e–0.047t, respectively. The major pharmacokinetic parameters were as follows: Cmax=11.695 μg/mL, Tmax=1.060 h and AUC=92.470 μg/(mL·h) in zolazepam; Cmax=9.654 μg/mL, Tmax=0.482 h and AUC=56.348 μg/(mL·h) in tiletamine. It suggested that blood concentration of zolazepam and tiletamine all reached 8.12 μg/mL and 8.96 μg/mL, respectively, which could cause healthy C. reevesiis into anaesthesia. To maintain anaesthesia, the blood concentrations of zolazepam and tiletamine should not be less than 7.18 μg/mL and 4.73 μg/mL.

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