Molecular cloning and mRNA expression analysis of myosin heavy chain (MyHC) from fast skeletal muscle of grass carp, Ctenopharyngodon idella

The myosin heavy chain(MyHC)is one of the major structural and contracting proteins of muscle.We have isolated the cDNA clone encoding MyHC of the grass carp,Ctenopharyngodon idella. The sequence comprises 5 934 bp,including a 5 814 bp open reading frame encoding an amino acid sequence of 1 937 residues.The deduced amino acid sequence showed 69%homology to rabbit fast skeletal MyHC and 73%–76%homology to the MyHCs from the mandarin fish,walleye pollack,white croaker,chum salmon,and carp.The putative sequences of subfragment-1 and the light meromyosin region showed 61.4%–80%homology to the corresponding regions of other fish MyHCs.The tissue-specific and developmental stage-specific expressions of the MyHC gene were analyzed by quantitative real-time PCR.The MyHC gene showed the highest expression in the muscles compared with the kidney,spleen and intestine.Developmentally,there was a gradual increase in MyHC mRNA expression from the neural formation stage to the tail bud stage.The highest expression was detected in hatching larva.Our work on the MyHC gene from the grass carp has provided useful information for fish molecular biology and fish genomics.     

Effect of pollution on DNA damage and essential fatty acid profile in Cirrhinus mrigala from River Chenab

The objective of this study was to evaluate the effect of anthropogenic pollution on DNA damage and the fatty acid profile of the bottom dweller fish( Cirrhinus mrigala), collected from the River Chenab, in order to assess the effect of the toxicants on the quality of the fish meat. The levels of Cd, Hg, Cu, Mn, Zn, Pb, Cr and Sn and of phenols from this river were significantly higher than the permissible limits set by the USEPA. Comet assays showed DNA damage in Cirrhinus mrigala collected from three different sampling sites in the polluted area of the river. Significant differences were observed for DNA damage through comet assay in fish collected from polluted compared to control sites. No significant differences were observed for DNA damage between farmed and fish collected from upstream. The micronucleus assay showed similar trends. Fish from the highly polluted sites showed less number of fatty acids and more saturated fatty acids in their meat compared to fish from less polluted areas. Several fatty acids were missing in fish with higher levels of DNA in comet tail and micronucleus induction. Long-chain polyunsaturated fatty acids, eicosapentaenoic acid(20:5n-3) was found missing in the fish from polluted environment while it was found in considerable amount in farmed fish 7.8±0.4%. Docosahexaenoic acid(22:6n-3) also showed significant differences as 0.1±0.0 and 7.0±0.1% respectively, in wild polluted and farmed fishes.     

ANNUAL CHANGES IN PLASMA LEVELS OF CORTISOL AND SEX STEROID HORMONES IN MALE RAINBOW TROUT, ONCORHYNCHUS MYKISS

The profiles of cortisol, testosterone, ll-ketotestosterone and 17α， 20β- dihydroxy-4-pregnene-3-one in mule rainbow trout reared under constant water temperature and natural photoperiod were determined by radioimmunoassay. Gonads of male rainbow trout reached maturity when the fish were two years old. Changes in the plasma levels of both sex steroid hormones and cortisol were closely related to the GSI. Plasma levels of testosterone, 11-ketotestostemne and 17a, 2013-dihydroxy 4-pregnene-3-one showed a clear peak in the annual breeding season, when the GSI reached their maxima. Plasma cortisol levels also showed clearly seasonal changes in both two- and three-year-old fish. The results suggest that the elevated plasma levels of cortisol may not just be due to stresses during the breeding season but have certain physiological functions in the reproduction of rainbow trout.     

Changes in fish diversity and community structure in the central and southern Yellow Sea from 2003 to 2015

The central and southern Yellow Sea is an important overwintering ground for many fish species in the Bohai Sea and Yellow Sea. For better understanding the status of the fish community after years of heavy exploitation, variations in fish community structure and diversity were analyzed using data from bottom trawls during 2003–2015. Five fish assemblage indices all showed fluctuations without clear trends from 2003 to 2015, yet there were strong positive and significant correlations(P 0.05) among them. The top-five dominant species accounted for a high weight percentage(49.7%–82.1%) in the annual fish catch. Multivariate analysis showed that two year groups could be pooled for the fish community: Group Ⅰ consisted of the years 2006, 2007, 2008 and 2015, while Group Ⅱ consisted of the years 2003, 2004, 2005, 2009, 2010 and 2014; the groups aggregated with 63.71% similarity, indicating a high level of similarity among all years. The multivariate dispersion values were 1.455 and 0.818 for Groups Ⅰ and Ⅱ, respectively, indicating greater variances in fish assemblage structure in Group Ⅰ than that in Group Ⅱ. Similarity of percentage analysis demonstrated that the average similarities for Group Ⅰ and Group Ⅱ were 71.58% and 67.51%, respectively. Size-spectra analysis revealed no consistent trend in the intercept and slope( P 0.05); there were also no significant differences between the slope of the size-spectra and fishing ef fort. The catch per unit ef fort and mean individual weight analyses of the whole fish assemblage both showed a significantly decreasing trend over time. Overall, the results showed that the fish community structure in the central and southern Yellow Sea was relatively stable from 2003 to 2015 and the study could be used as a reference for supporting ecosystem-based fishery management.     

Effects of temperature, pH and NaCl on protease activity in digestive tract of young turbot, Scophthalmus maximus

1 INTRODUCTION Digestive enzyme activity is one of important issues for learning digestive physiology of fish, and widely applied in commercial fish culture. So far, the data obtained in fish showed that the diges- tive enzymes were qualitatively similar …     

Existence of Microsatellites in Expressed Sequence Tags of Common Carp （Cyprinus carpio L. ） Available in GenBank dbEST Database

Common carp expressed sequence tags （ESTs） were analyzed for the existence of microsatellites, or simple sequence repeats （SSRs）. In the NCBI dbEST database, a total of 10612 sequences were registered before December 31, 2004. A complete search of 2-6 nucleotide microsatellites resulted in the identification of 513 SSR-containing ESTs, accounting for 4.8% of the total. Cluster analysis indicated that 73 sequences of SSR-containing ESTs fell into 27 groups and the remaining 440 ESTs were independent. A total of 467 unique SSR-containing ESTs were identified. These EST-SSRs contained a vari- ety of simple sequence types, and di- and tri-nucleotide repeats were the most abundant, accounting for 42.1% and 27.9% of the whole, respectively. Of the dinucleotide repeats, CA/TG was the most abundant, followed by GA/TC. BLASTx search showed that 38.1% of the SSR loci could be associated with genes or proteins of known or unknown function. BLASTx searches of SSR-containing ESTs also showed high frequencies （98/179） of hits on zebrafish sequences.     

Structure of mitochondrial DNA control region of Pholis fangi and its phylogenetic implication

In this study, the entire mitochondrial DNA （mtDNA） control region （CR） of Pholis fangi was amplified via polymerase chain reaction followed by direct sequencing. The length of the mtDNA CR consensus sequence of P. fangi was 853 bp in length. In accordance with the recognition sites as were previously reported in fish species, the mtDNA CR sequence of P. fangi can be divided into 3 domains, i.e., the extended terminal associated sequence （ETAS）, the central conserved sequence block （CSB）, and the CSB domain. In addition, the following structures were identified in the mtDNA CR sequence of P. fangi：2 ETASs in the ETAS domain （TAS and cTAS）, 6 CSBs in the central CSB domain （CSB-F to CSB-A）, and 3 CSBs in the CSB domain （CSB-1 to CSB-3）. These demonstrated that the structure of the mtDNA CR of P. fangi was substantially different from those of most other fish species. The mtDNA CR sequence of P. fangi contained one conserved region from 656 bp to 815 bp. Similar to most other fish species, P. fangi has no tandem repeat sequences in its mtDNA CR sequence. Phylogenetic analysis based on the complete mtDNA CR sequences showed that there were no genetic differences within P. fangi populations of the same geographical origin and between P. fangi populations of different geographical origins.     

Molecular Cloning,Expression and Characterization of Peroxisome Proliferators-Activated Receptors Gamma in the Sea Urchin(Strongylocentrotus intermedius)

Peroxisome proliferators-activated receptor gamma(PPARγ) plays important regulatory roles in adipocyte differentiation. In this study, we cloned the full-length sequence of the PPARγ gene and analyzed its expression profile in different developmental stages and tissues of Strongylocentrotus intermedius. The full-length cDNA of PPARγ contains 2286 base pairs(bp) with a putative open reading frame of 1755 bp, and the gene encodes encoding a polypeptide of 584 amino acid residues. The predicted molecular mass of the protein is 67.27 kDa, and its theoretical isoelectric point(pI) is 10.07. The protein contains conserved motifs, including an RRM(RNA recognition motif) domain. PPARγ expression with the highest level was observed in the gonad, and the lowest was observed in the tube feet of S. intermedius. Time-course expression measurements at different developmental stages showed that the highest expression level of PPARγ is in the eggs and its weakest expression level is in the 32-cells stage. Knock-down of PPARγ by specific siRNA revealed that UCP2 expression is significantly decreased in the gonads and intestines 48 h post-transfection, indicating that the UCP2 is a downstream target gene of PPARγ.This finding suggests that PPARγ and UCP2 have positive regulatory effects in regulating adipocyte differentiation. Changes in fatty acid levels in the gonads before and after PPARγ interference were assessed, and decreased C18:2(trans, n-6) and C20:3(n-6) levels were observed 48 h after siRNA transfection. The results showed the function of PPARγ in fatty acid anabolism, The data are helpful to improve the current understanding of the fatty acid synthesis pathways and regulatory mechanisms in S. intermedius. They also provide an experimental basis for improving fatty acid synthesis in sea urchins, which is important for cultivating sea urchins with high nutritional value.     

Stable Carbon Isotope and Long-Chain Alkane Compositions of the Major Plants and Sediment Organic Matter in the Yellow River Estuarine Wetlands

Elemental(TOC,TN,C/N)and stable carbon isotopic(δ^13C)compositions and long-chain alkane(n C16-38)concentrations were measured for eight major plants and a sediment core collected from the Yellow River estuarine wetlands.Our results indicate that both C3(-25.4‰to-29.6‰)and C4(-14.2‰to-15.0‰)plants are growing in the wetlands and C3 plants are the predominant species.The biomass of the wetland plants had similar organic carbon(35.5-45.8%)but very different organic nitrogen(0.35-4.15%)contents.Both C3 and C4 plants all contained long-chain alkanes with strong odd-to-even carbon numbered chain predominance.Phragmites australis,a dominant C3 plant contained mainly n C29 and n C31 homologues.Aeluropus littoralis,an abundant C4 plant were concentrated with n C27 and n C29 homologues.Organic matter preserved in the Yellow River estuarine sediments showed strong terrestrial signals(C/N=11-16,δ^13C=-22.0‰to-24.3‰).The distribution of long-chain n-alkanes in sediments also showed strong odd-to-even carbon chain predominance with n C29 and n C31 being the most abundant homologues.These results suggest that organic matter preserved in the Yellow River estuarine sediments were influenced by the wetland-derived organic matter,mainly C3 plants.The Yellow River estuarine wetland plants could play important role affecting both the carbon and nutrient cycling in the estuary and adjacent coastal waters.     

Long interspersed elements in three species of Gracilaria （Gracilariaceae, Rhodophyta）

In order to find out whether long interspersed elements (LINEs) existed in macro-algae ge- nomes or not, we tested the LINE homologues in representative families (species): Gracilaria (G. eucheumoides Harv., G. tenuistipitata Chang et Xia, and G. textorii (Sur) De-Toni), Laminaria (L. longis- sima Miyabe and L. japonica Aresch.), and Ulva (U. lactuca L. and U. pertusa Kjellm.) during 2004 to 2005. Polymerase Chain Reaction (PCR) was carried out with degenerate oligonucleotide primers de- signed from LINEs of rice homologues and Cin4 of maize. Cloning and nucleotide sequencing of the PCR products revealed that 4 clones that derived from 3 species of Gracilaria have LINE homologues. The nucleotide sequences of the 4 LINE homologues diverged greatly, but the amino acid sequences deduced from them were relatively conserved. The endonuclease regions of the LINE homologues greatly di- verged from that of other plants, but they had closer phylogenetic relationship to Zepp elements in Chlor- ella sp., which indicated that sequence divergence by vertical transmission has been a major influence on the evolution of algal LINEs.     

SIUDIES ON THE BIOCHEMICAL COMPOSITION,ENERGETICS AND ESSENTIAL AMINO ACIDS OF THREE MUDSKIPPERS IN XIAMEN HARBOUR

Measurements were made on the contents of protein,lipid,glycogen(PLG)and water,and on caloric values and amino acids,in muscle of three mudskippers periophthalmus cantonensis,Scarteiaosviridis and Boleophthalmus pectinirostris collected from Haicang,Xiamen.The essential amino acids(EAA)for these fishes were also studied with radioisotopic trace method.The results showed:(1)Thecontent of each component in tested fish muscles differed slightly,and protein was the most importantcomponent making up from 6.685% to 9.891% of the wet weight (about 44.21%-50.45% of dryweight);(2)Energy calculated from the sum of protein, lipid and glycogen in wet muscle was low (<4.3kJ/g) in these Fishes, especially in B. pectinirostris(<3.1 kJ/g), the ratios of energy to protein content (E/P) also were low(<39.873-45.535kJ/g), (3)Seventeen amino acids were determined in thesethree fishes.The content of the same amino acid(among the seventeen) tested in different species andsexes varied slightly.The amounts of methionine, phenyl     

Effects of Calanoid Copepod Schmackeriapoplesia as a Live Food on the Growth,Survival and Fatty Acid Composition of Larvae and Juveniles of Japanese Flounder,Paralichthys olivaceus

Zooplankton constitutes a major part of the diet for fish larvae in the marine food web, and it is generally believed that copepods can meet the nutritional requirements of fish larvae. In this study, calanoid copepod Schmackeria poplesia, rotifer Brachionus plicatilis and anostraca crustacean Artemia sp. were analyzed for fatty acid contents, and were used as live food for cul- turing larval Japanese flounder, Paralichthys olivaceus. The total content of three types of HUFAs （DHA, EPA and ARA） in S. po- plesia was significantly higher than that in the other two live foods （P〈O.O1）. Three live organisms were used for raising larvae and juveniles of Paralichthys olivaceus respectively for 15 and 10 d. Then the growth, survival and fatty acid composition of the larvae and juveniles were investigated. The results showed that the larvae and juveniles fed with copepods （S. poplesia） had significantly higher growth rate than those fed with the other two organisms （P〈0.01）. The survival of the flounder larvae fed with copepods was significantly higher than that of the others （P〈0.01）, and the survival of the juvenile fish fed with copepods was higher than that fed with Artemia （P〈0.05）. The contents of three types of HUFAs （DHA, EPA and ARA） and the ratio of DHA/EPA in larval and juve- nile flounder P. olivaceus were analyzed. The results showed that the contents of DHA, EPA and ARA in the larvae and juveniles fed with S. poplesia were higher than those fed with a mixed diet orArtemia only, and the ratio of EPA/ARA in larvae and juveniles of P. olivaceus fed with S. poplesia was lower than that in the case of feeding with a mixed diet or Artemia only. The present data showed that copepod is the best choice for feeding the larvae and juveniles of fish considering its effects on the survival, growth and nutrition composition of the fish.     

Ontogenetic changes in isotopic signatures of an omnivorous fish Cultrichthys erythropterus in East Lake Taihu,China

The relationship between body size and stable isotopic signatures of the omnivorous Redfin Culter(C ultrichthys erythropterus),commonly found in East Lake Taihu,was investigated. Previous analyses of C. erythropterus stomach contents have shown that this species undergoes a diet switch from being predominantly zooplanktivorous to piscivorous during its life history. This was confirmed by stable carbon isotopic signature(δ 13 C) in this study,in which δ 13 C was positively correlated with both standard length and weight. The importance of littoral-benthic resources in supporting C. erythropterus during its lifespan was also demonstrated using a two-source mixing model,the results of which showed a significant increasing trend in the contribution of littoral-benthic energy. However,the stable nitrogen isotopic signature(δ 15N) exhibited an unusual pattern compared with previous studies. The δ 15 N of C. erythropterus showed no relationship with body size,even though dietary changes were observed. This indicated that δ 15 N alone cannot fully reflect a diet shift in a species and possible variability in isotopic signatures over its life history. This should be considered when using stable isotopic signatures to investigate intra-specific variations and the timing of life-history events,such as estimating the trophic positions of fish species.     

Experimental Study on the Hydrodynamic Characteristics of a Submersible Fish Cage at Various Depths in Waves

Submersible fish cages can be submerged under the water to mitigate the negative effects that arise from severe sea conditions and improve the growing environment for the farmed fish. Thus they are increasingly applied in offshore aquaculture. To ensure both safety and economic efficiency of submersible fish cages, it is important to determine the optimum submergence depth. In this study, a series of physical model experiments were conducted to investigate the hydrodynamic performance of a submersible fish cage at various submergence depths(1/6, 1/4, 1/3, and 1/2 of the water depth as well as the floating condition for reference) with a model scale of 1:20. The results of the physical model experiment for the different depths were compared to analyze the effects of submergence depths on the mooring line tension and the movement of the floating collar. The results showed that the mooring line tension and the floating collar movement significantly attenuated with increasing submergence depth. However, the attenuation tendency became stable when the fish cage reached a certain depth. According to the results, 1/3 of water depth was determined as the optimal submergence depth of the fish cages. Deeper submergence depths showed no significant advantage from a perspective of the hydrodynamic characteristics of the fish cage. The determination of the optimum submergence depth is beneficial for the structural design and operation safety of submersible net cages.     

Lipid and Fatty Acid Compositions of Cod (Gadus morhua),Haddock (Melanogrammus aeglefinus) and Halibut (Hippoglossus hippoglossus)

This study was conducted to compare lipid and fatty acid composition of cod, haddock and halibut.Three groups of cod (276 g±61 g), haddock (538 g±83 g) and halibut (3704 g±221 g) were maintained with commercial feeds mainly based on fish meal and marine fish oil for 12 weeks prior to sampling.The fatty acid compositions of muscle and liver were determined by GC/FID after derivatization of extracted lipids into fatty acid methyl esters (FAME).Lipids were also fractionated into neutral and polar lipids using Waters silica Sep-Pak.The phospholipid fraction was further separated by high-performance thin-layer chromatography (HPTLC) and the FAME profile was obtained.Results of the present study showed that cod and haddock were lean fish and their total muscle lipid contents were 0.8% and 0.7%, respectively, with phospholipid constituting 83.6% and 87.5% of the total muscle lipid, respectively.Halibut was a medium-fat fish and its muscle lipid content was 8%, with 84% of the total muscle lipid being neutral lipid.Total liver lipid contents of cod, haddock and halibut were 36.9%, 67.2% and 30.7%, respectively, of which the neutral lipids accounted for the major fraction (88.1%-97.1%).Polyunsaturated fatty acids were the most abundant in cod and haddock muscle neutral lipid.Monounsaturated fatty acid level was the highest in halibut muscle neutral lipid.Fatty acid compositions of phospholipid were relatively constant.In summary, the liver of cod and haddock as lean fish was the main lipid reserve organ, and structural phospholipid is the major lipid form in flesh.However, as a medium-fat fish, halibut stored lipid in both their liver and muscle.     

Field experiments to improve the efficacy of gargoor (fish trap) fishery in Kuwait’s waters

Fish traps were investigated to understand the effects of season, bait type, trap size, and trap soak time on catch rates, catch composition, and trap loss rates from March 2004 to September 2005, to improve the performance and management of Kuwait’s gargoor (cage style fish trap) fishery, which used to be the nation’s most important one in terms of value and landings volume. Catch rates were the highest in April/May (5-8 kg/trap haul) and again in December (7 kg/trap haul). Bait type and trap size also affected catch rates and species composition. Of the seven baits tested, the best catch rates, >5 kg/trap haul, occurred with cuttlefish (Sepia pharaonis), but wolf-herring (Chirocentrus dorab) and mullet (Liza klunzingeri) also produced good results (4-5 kg/trap haul). Within the five tested sizes, the two largest-sized traps captured more fish and larger size fish. Analysis of variance (ANOVA) showed significant differences of catch rate among traps with different baits as well as among traps of different sizes. Duncan test further revealed these differences between two specific baits and sizes. Cluster Analysis of species composition showed more differences among different baits than among different trap sizes. Longer soak times did not result in larger catch rates, but increased trap loss. About 10-day soak time resulted in trap loss 7%, while 40-day soak time could result in a loss of around 20%. Consequently, it is recommended that the gargoor be checked every 10 or fewer days. The average overall catch rate during the study period was lower than that of 1980s (4.5 vs. 5.8 kg/trap haul), indicating a possible decline of fish abundance in Kuwait’s waters. It is recommended that the number of gargoor fishing boats and gargoors from each boat should be limited to allow stock rehabilitation.     

Effects of dietary genistein on GH/IGF-I axis of Nile tilapia Oreochromis niloticus

There is considerable concern that isoflavones,such as genistein in fish feed composed of soybean protein,affects somatic growth in fish.Our previous works demonstrated that 30 and 300 μg/g dietary genistein had no significant effect on growth performance in Nile tilapia(Oreochromis niloticus),but the higher level of genistein(3 000 μg/g) significantly depressed growth.This study was conducted to further examine the effects of dietary genistein on the endocrine disruption on growth hormone/insulin-like growth factor-I(GH/IGF-I) axis in Nile tilapia(O.niloticus).Juvenile fish were fed by hand twice daily to satiation with one of four isonitrogenous and isoenergetic diets,each containing either 0,30,300 or3 000 μg/g genistein.Following an 8-week feeding period,plasma GH and IGF-I levels were investigated by radioimmunoassay and gene expression levels of gh,ghrelin,gnrhs,ghr,npy,npyrs,pacap,ghrs,igf-I,igf-Ir,and igfbp3 were examined by real-time PCR.The results show that no significant change in plasma GH and IGF-I levels in fish fed with diets containing 30 μg/gand 300 μg/g genistein.mRNA expression of genes along the GH/IGF-I axis remained unaffected,except for igf-Ir,which was stimulated by the 300 μg/g genistein diet.While in fish fed the 3 000 μg/g genistein diet,the plasma GH and IGF-I levels decreased,and mRNA expression of gh,ghr2,npyr1,igf-I,and igf-Ir were also significantly depressed.In contrast,npy and igfbp3 mRNA expression were enhanced.This study provides convincing evidence for growth impediment by genistein by disturbing the GH/IGF-I axis in Nile tilapia O.niloticus.     

Construction and characterization of a normalized cDNA library of Nannochloropsis oculata(Eustigmatophyceae)

We constructed and characterized a normalized cDNA library of Nannochloropsis oculata CS-179,and obtained 905 nonredundant sequences(NRSs) ranging from 431-1 756 bp in length.Among them,496 were very similar to nonredundant ones in the GenBank(E ≤1.0e-05),and 349 ESTs had significant hits with the clusters of eukaryotic orthologous groups(KOG).Bases G and/or C at the third position of codons of 14 amino acid residues suggested a strong bias in the conserved domain of 362 NRSs(60%).We also identified the unigenes encoding phosphorus and nitrogen transporters,suggesting that N.oculata could efficiently transport and metabolize phosphorus and nitrogen,and recognized the unigenes that involved in biosynthesis and storage of both fatty acids and polyunsaturated fatty acids(PUFAs),which will facilitate the demonstration of eicosapentaenoic acid(EPA) biosynthesis pathway of N.oculata.In comparison with the original cDNA library,the normalized library significantly increased the efficiencies of random sequencing and rarely expressed genes discovering,and decreased the frequency of abundant gene sequences.