Abstract:Nicotinamide-Adenine Dinucleotide Phosphate-Dependent Isocitrate Dehydrogenase(IDPc)is an important enzyme essential for survival of all organisms.Many studies have been conducted to isolate IDPc and explore its kinetic parameters in mammals,plants and microorganisms.However,in fish,the related information is very scarce.The aim of the study is to purify and characterize IDPc from yellow catfish hepatic cytoplasm,which will provide some crucial information on the catalytic and regulatory mechanism of the enzyme in liver of yellow catfish.The purification processes include permeation chromatography on Sephadex G-100 gel,chromatographic adsorption by DEAE Sepharose,permeation chromatography on Sephadex G-25 gel and finally chromatographic adsorption by DEAE Sepharose again.The specific IDPc activity is 7.94 U/mg,and its molecular weight after SDS polyacrylamide slab gel electrophoresis is 36.7 ku.The optimum pH,and temperature for the enzyme are 8.0 and 65℃ respectively.Using Arrhenius plots,the enzyme has the activation energy of 81.33 kJ/mol.The Km values of the substrates NADP⁺ and IC are 0.056 mmol/L and 0.175 mmol/L,respectively.The Vmax values of IDPc with NADP⁺ and IC as the substrates are 9.04 U/mg and 10.51 U/mg,respectively.The enzyme of IDPc is inhibited by NADPH in a competitive manner with the K_i value of 0.034 mmol/L.The activity of IDPc is greatly dependent on the binding of divalent metal ions with the active order of Mn²⁺>Mg²⁺>Zn²⁺>Ca²⁺>Cu²⁺.Ca²⁺ and Zn²⁺ are both the activator and inhibitor of IDPc,however,Cu²⁺ showed no effects on the enzyme.The comprehensive information of enzymatic properties may help to better understand the mechanism of catalysis and regulation of IDPc in fish.

Abstract:In order to establish Centropristis striata sperm cryopreservation method, this paper used a computer-assisted sperm motion analysis system(CASA)，and selected six cryoprotectants,such as glycerin(GLY), dimethyl sulfoxide(DMSO), propylene glycol(PG), ethylene glycol(EG), dimethyl sulfoxide(DMA), methanol(METH) at four concentrations (5%, 10%, 15% and 20%, V/V) in HBSS to find the influence of C. striata sperm cryopreservation. Results showed that the best cryopreservation protocol for C. striata was 15% PG as antifreeze protective agent in HBSS with step cooling scheme. After 37°C water bath thawed, motility of the sperm reached (93.1±0.9)%, which was higher than other combination groups and showed no significant difference with fresh sperm (P>0.05).The group of 15% PG performed best in average linear velocity, average velocity, and average path velocity at (88.3±0.3) μm/s, (76.2±0.5) μm/s, and (86.7±0.7) μm/s, which showed no significant difference with fresh sperm (P>0.05). In different cryoprotectants with different concentration, the motility of 15% PG within 1 min had no significant difference with fresh semen. Therefore, 15% PG was the best cryoprotectant for C. striata and could be applied in practice.

Abstract:Body pigment and band are important morphological characteristics of fishes.Pigment variation and band formation during early development are extremely important metamorphic traits,marking the qualitative changes from one stage to another in early development of fish.The formation and changes of fish body color is the result of adaptation to environmental change.It is the important embodiment of environmental change and health,along with the environment,age,health status and physiological changes,fish body color changes accordingly.For understanding the changes of important morphological characteristics during early life stages of Oplegnathus fasciatus,complete artificial propagation and seedling pond rearing were carried out in Raoping,Guangdong Province.Body surface pigment variation and band formation during embryo,larva,juvenile and young of O.fasciatus were observed,and the frequency of bands formation at different life stages was calculated.Results showed that three kinds of pigment cells were found in a chrysanthemum shape,including melanophore,xanthophore and brown pigment.Pigment could be observed in embryonic stages,their appearance and transformation run throughout the early development stages.Pigment cells in embryo and newly hatched larva were small in size,branches short and few,with rare and even distribution,leading light and transparent in body color.With the development of larva,pigment cells increased in size,longer in branches,and some of them connected in net-shape.In late larva stage,especially in band formation period,chrysanthemum shape pigment cells were found to be more,denser and shorter in branches,distributed densely,resulting in deep color on the body.The pigment band development was divided into 8 stages,corresponding to that of non-pigmented band and formation of 7 pigment bands,respectively.Pigment bands appeared first on the head and abdomen at the same time,corresponding to the 3 chrysanthemum shape pigment on anterior 3 pigment bands.The period from 12d to 16d was the formation stage with pigmented cell accumulation.At the water temperature ranging from 22 to 27℃,first band could be found at 17d after hatching,and 7 bands all formed up to 32 d after hatching.Formation of 7 pigment bands was developed in sequence:3^rd band→4^th band→2^nd band →1^st band→5^th band→6^th band→7^th band.Adult fish colored in black from rostrum to eye,black or brown in margin of caudal fin,changeable in body color,white in body in general,becoming yellow-brown when stressed.In conclusion,formation of bands is the important metamorphic trait in larva,juvenile and young of O.fasciatus.Occurrence and sequence of pigment bands vary with the species.Generally,formation of all bands originated from occurrence of one or several pigment cells.

Abstract:In order to recognize the morphogenesis and its application in the taxonomic identification of fish larvae and juveniles in genus Myctophum Rafinesque,1810,larvae and juveniles samples collected during the oceanographical expedition cruises,which were carried out in central and western tropical Pacific during April 1978 to June 1979 and in Kuroshio Current extension regions of the East China Sea during April 1984 to June 1986,were identified separately based on the references related to the morphological and taxonomic features of this genus.The authors identified the larvae and juveniles of Myctophum spinosum(Steindachner,1867),M.nitidulum Garman,1899,M.asperum Richardson,1845,M.punctatum Rafinesque,1818,M.aurolaternatum Garman,1899,M.orientale(Gilbert,1913),M.obtusirostris Tning,1928 and Myctophum sp.,and described their ontogeny in morphological characteristics in detail.Combined with the morphological characteristics of M.phengodes,M.lychnobium,M.selenops,M.brachygnathum,Myctophum sp1.and Myctophum sp2.from the references,the identification key to these fourteen species of Myctophum distributed in the world,including eleven species with morphological characteristics already known and other Myctophum speices which allows identification at the genus level,has been established.The result showed that all but M.aurolaternatum larvae and juveniles in genus Myctophum were observed with enlarged fan-shaped pectoral fins.The head somewhat flat was in a triangular shape with non-spherical eyes on elongate stalks or with well developed conical choroid mass.Pigments were distributed on the jaws,isthmus,opercle,branchiostegal membrance and pectoral fin base.Pigment patterns on lateral gut were evenly distributed on trailing section,except heavier near terminus.The jaws were full of sharp teeth.A single adipose fin was found on the back behind the dorsal fin.All these were the common characteristics of larvae and juveniles in genus Myctophum.M.aurolaternatum larvae had unique morphological characteristics:very flat head,eyes small on distinct elongate stalks,and with straight gut trailing at midbody and extending well beyond caudal fin.The shape of the choroid tissue or whether they own elongate stalks to the eyes;whether there were melanophores at the anterior tip of the lower or upper jaws;pigment pattern on the pectoral fin base,between the pectoral fin rays and in lateral gut;whether there was melanophores distributed on the tip of tail,the trailing edge of the anal fin and adipose fin were the distinct categories identification and morphological characteristics in larvae and juveniles in genus Myctophum.

Abstract:Based on the data of temperature,salinity and DO observed in the Changjiang Estuary covering 54 stations in late summer,2009,vertical structure types of water temperature were classified by the method of vertical gradient,and the parameters including depth and intensity of thermocline and vertical gradient of DO on the water layer of thermocline were calculated.In late summer,the distribution of temperature vertical structure and intensity of thermocline off the Changjiang Estuary was a situation of confluence and mixing by the dilute water of Changjiang,the coastal current of Yellow Sea,the warm upper water of Taiwan Warm Current and the cold deep water of Taiwan Warm Current.Correlation analysis showed that,there was a negative correlation between the depth of thermocline and the vertical gradient of DO on the layer of thermocline,there was a positive correlation between the thermocline depth and the vertical minimum DO,and also a positive correlation between the thermocline intensity and vertical gradient of DO on the layer of thermocline.The thermocline intensity,especially the intensity of upper thermocline which was near sea surface,the origin of DO,has a strong correlation(r=0.69)with the DO vertical gradient on the layer of thermocline.The shallower and the stronger of the thermocline,the greater effect of the thermocline on the hypoxia of deep water.Thermocline,in particular,the upper thermocline hindered the surface water with saturated DO mixed with the deep water,played a role of physical barrier of oxygen vertical transportation.The thermocline was an important environmental control factor of the hypoxia off the Changjiang Estuary from late spring to autumn,covering a period of occurrence,development,maintenance and decay of the hypoxia.

Abstract:In this paper,the characteristics stability and the cultivation applicability of the improved strain(T-17)of Pyropia yezonensis were evaluated according to the results of laboratory cultivation and pilot cultivation in mariculture farm.The results showed that:in comparison with the wild-type strain(WT)of Pyropia yezonensis,the blades of the T-17 strain have obvious advantages in growth rates,maximum photochemical efficiency,contents of main photosynthetic pigments,blade thicknesses and yields of pilot cultivation.When the blades after being cultured at 18℃ for 50 days were cultured at 18,22 and 24℃ for another 35 days,the absolute growth rates of the blades of T-17 strain were 17.71,15.81 and 33 times higher than those of the WT strain,respectively;the specific growth rates of the T-17 strain were 4.59,4.38 and 9.15 times higher than those of the WT strain,respectively;the maximum photochemical efficiency(Fv/Fm)of the T-17 strain were 1.17,1.29 and 1.58 times higher than those of the WT strain,respectively.The gametophytic blades of the WT strain turned deeply dark,curly hardened and began to decay when the blades of the WT were cultured at 22℃ for 25 days,24℃ for 15 days and 25℃ for 10 days,respectively,while the blades of the T-17 did not harden,did not decay and grew well in the same culture conditions and were cultured for the same period.When the blades were cultured at 18℃ for 65 days,the contents of Chl.a,and phycobiliprotein(PE+PC)of the T-17 strain were 1.45 and 1.54 times higher than those of the WT strain.The mean blade thickness of the T-17 strain was less than the WT strain by 26.4%.The releasing number of conchospores from T-17 has no significant difference with the WT strain.The blades were collected and studied from the first harvest to the fourth harvest in the pilot cultivation.It was found that the fresh yield of the T-17 strain of the total amount of harvests in the first four times increased by 16.3% compared with traditional cultivar(the wild-type,Wt),the maximum photochemical efficiency(Fv/Fm)of the blades of the T-17 strain were 1.06,1.12,1.17 and 1.27 times higher than that of the Wt strain,respectively;the Chl.a contents in the blades of T-17 strain were 1.41,1.49,1.52 and 1.91 times higher than that of the Wt strain,respectively;the phycobiliprotein contents in the blades of the T-17 strain were 1.94,2.04,2.03 and 2.34 times higher than that of the Wt strain,respectively.Compared with the wild-type strain,the T-17 strain had higher yield,stronger resistance to high-temperature and higher quality,and good stability of the characteristics.This strain has probably great potential to be applied in commercial cultivation.

Abstract:Phosphorus is one of the most important minerals required by fish.Due to low concentration of phosphorus in natural waters,and low absorption rate of phosphorus from the water,fish must obtain most of phosphorus from their diets.The optimal amount of phosphorus supplementation in commercial feeds is important not only economically,but also for environmental reasons.Phosphorus metabolism in cultured aquatic species has become a popular research subject,due to rising concerns about phosphorus discharged into aquaculture environment.The present experiment was conducted to evaluate the effects of dietary phosphorus on growth performance,body composition,bone mineralization and blood chemistry index of juvenile rice field eel(Monopterus albus).Five practical diets were formulated to contain graded levels(0.67%,0.91%,1.05%,1.29% and 1.53%)of phosphorus from dietary ingredients and monocalcium phosphate.Each diet was randomly fed to triplicate groups of fish with initial mean weight of (34.4±0.3) g in floating cages(1.5 m×2.0 m×1.5 m)to apparent satiation for 70 days,and each cage was stocked initially with 100 fish.The results showed that:(1)fish weight gain rate(WGR),protein efficiency ratio(PER)and feed efficiency(FE)increased with increasing phosphorus from 0.67% to 1.05%(P<0.05)and then plateau over the level of 1.05%.Broken-line analyses[Y=104.67-46.07(1.10-X),R²=0.944]suggested that optimum dietary phosphorus content is 1.10% based on WGR.(2)whole-body protein contents and condition factor(CF)were significantly elevated,however,whole-body lipid contents as well as hepatosomatic index(HSI)were significantly reduced by increasing dietary phosphorus.(3)Diet phosphorus level can sighnificantly enhance the content of whole-body ash and phosphorus(P<0.05).Broken-line analyses[Y=0.988-0.635 4(1.05-X),R²=0.928]suggested that fish fed diets with phosphorus of 1.05% presented highest whole-body phosphorus content.both vertebrae phosphorus and calcium contents increased linearly with increasing dietary phosphorus levels while vertebrae Ca/P ratio was not influenced.Broken-line analyses[Y=7.696-0.985 5(1.10-X),R²=0.956]indicated vertebrae phosphorus content showed highest values at the diet phosphorus of 1.10%.(4)Dietary TP had distinct effects on P,Ca and alkaline phosphatase contents in serum.This research revealed that 1.10% dietary total phosphorus was required for maximal tissue storage and mineralization as well as optimal growth.

Abstract:Full length cDNA sequences of gonadotropin hormone β subunits were cloned from the pituitary of Oplegnathus fasciatus by homology cloning and RACE techniques.The expression of GtHβ mRNA was analysed in different tissues and different ovarian development stages of pituitary,ovary,and brain.The FSH cDNA sequence is 554 bp and its open reading frame is 363 bp,which encoded a precursor protein of 120 amino acids preceded by a signal peptide of 18 amino acids residues.The LH cDNA sequence is 857 bp and its open reading frame is 450 bp,which encoded a precursor protein of 149 amino acids preceded by a signal peptide of 24 amino acids residues.Both of the putative peptide of FSH and LH contained 12 cysteine residues and 1 N-glycosylation sites residues.Moreover,there was a special area of Cys-Ser-Gly-His(CSGH)in the middle of 4 and 5 cysteine of LH.Sequences comparison and phylogenetic analysis showed that the O.fasciatus FSH and LH had the close relationship with Perciformes.The Real-time Quantitative PCR results suggested that the expression levels of FSH and LH mRNA were the highest in pituitary,followed by ovary.The FSH mRNA was not found in kidney and head kidney and the LH mRNA was not found in Head kidney.The GtHβ expression level in the reproductive cycle showed that the level of pituitary FSH mRNA was maximum in the Ⅳ ovarian development stage and the LH mRNA was maximum in the Ⅴ ovarian development stage.In ovary,the level of FSH mRNA was the lowest in the Ⅴ ovarian development stage,however,the LH mRNA was maximum in the Ⅴovarian development stage.Both of the brain FSH and LH mRNA were the lowest in theⅤovarian development stage.The hormone analysis indicated that the concentration of FSH was maximum in the Ⅳovarian development stage in serum,but the concentration of LH was maximum in the Ⅴ ovarian development stage in serum.The content of FSH and LH in serum showed a consistent trend of regular changes with the level of FSH mRNA and LH mRNA in pituitary.The research is beneficial to the technique of artificial breeding in O. fasciatus.and provides a certain reference value to the reproductive regulation of O. fasciatus.

Abstract:To determine dietary leucine(Leu)requirement of tilapia(GIFT Oreochromis niloticus),tilapias with an initial body weight(53.65±0.05)g were randomly divided into seven treatments in triplicates with 14 fishes per repetition.Seven isonitrogenous(crude protein 32%) and isoenergetic semi-purified diets containing different levels(1.11%,1.51%,1.90%,2.29%,2.69%,3.08% and 3.48%) of dietary Leu were fed to tilpias at the feeding rate of 4%-6% for 56 d. Quadratic regression analysis showed that the optimal dietary Leu levels of tilapia were 2.28%-2.33% base on weight gain rate(WGR),specific growth rate(SGR),feed conversion ratio(FCR). With an increase of dietary Leu level,the crude protein and crude lipid content of whole body and muscle initially increased and then decreased, but there were no significant differences in moisture content of muscle.With an increase of dietary Leu level,activities of intestinal amylase activity,intestinal protease ,Na⁺-K⁺-ATPase initially increasing and then decreased,and the lipase activity get maximum at 2.69% level and the others got maximum at 2.29% level.With the increase of dietary Leu level,the concentrations of EGF and EGFR,and the activities of SOD,LZM and AKP also initially increased and then decreased,all got maximum at 2.29% level.In conclusion,the optimal dietary Leu level for 50-200 g GIFT tilapia was 2.28%-2.33%(or 7.11%-7.26% of dietary protein).This Leu level can significantly promote the development of intestinal mucosa,enhance gastro-intestinal digestion and absorption capacity,thus improve the body's nonspecific immunity,and then promote the growth and utilization of tilapia.

Abstract:This research of the backcross progeny of female diploid F₁ hybrid(blunt snout bream×topmouth culter)×male blunt snout bream(BTB for short)and its parents topmouth culter(TC),blunt snout bream(BSB)and BT(BSB×TC)investigated the chemical composition of muscle nutrients including moisture,fat,protein and ash,as well as the composition of fatty acid and amino acid.The observations suggested that,compared to the original parents,BTB displayed high protein content and low carbohydrate level.Further analysis of fatty acid revealed that the UFA(unsaturated fatty acids)content in BTB,especially oleic acid,DHA,etc.(P<0.05),was significantly higher than that in other kinds of fish.In terms of amino acids,BTB had higher flavor amino acid ratio than its parents.Taken together,the results provided biochemistry evidences for proving the advantages of BTB in nutritional value and taste.

Abstract:Suppressors of cytokine signaling(SOCS)is an important inhibitor of JAK/STAT signaling pathway,which plays an important role in immunity,growth and development.The results of our previous transcriptome showed that the expression profile of Lv-SOCS fluctuated significantly in response to WSSV infection.It is suggested that Lv-SOCS might be involved in innate immunity in L.vannamei in response to WSSV infection.In this research,the full length cDNA of SOCS gene(Lv-SOCS,GenBank accession number:KJ000426)was cloned from hemocytes of L.vannamei.Lv-SOCS has a 1 191 bp ORF encoding 397 aa with a predicted molecular weight of 42.30 ku and calculated isoelectric point of 8.62.The SMART online software analysis shows that Lv-SOCS has two functional domains(SH2 domain and SOCS-box).According to the multiple sequence alignment,Lv-SOCS shows the highest similarity with Marsupenaeus japonicas SOCS which is in a same branch of phylogenetic tree with Lv-SOCS.Semi-quantitative RT-PCR revealed that Lv-SOCS was mainly expressed in hemocytes,intestine and hepatopancreas.The expression of Lv-SOCS could be up-regulated significantly after WSSV infection.The above results give important clues for further study on the function and regulation mechanism of Lv-SOCS in anti-WSSV immune response.

Abstract:The effect of freeze and frozen storage on Eriocheir sinensis was studied by evaluation of the characteristics of protein,the contents of free amino acids and flavor nucleotides,the activity of Ca²⁺-ATPase and the thawing loss rate.The results showed that E.sinensis had freeze resistance but no frozen storage resistance and the elasticity decreased after 2 weeks' frozen storage.The contents of free amino acids and flavor nucleotides,the activity of Ca²⁺-ATPase and the thawing loss rate changed significantly during frozen storage.After frozen storage of 12 weeks,the contents of free amino acids and flavor nucleotides,the activity of Ca²⁺-ATPase decreased by 25.3%,100%,and 41.6% respectively,compared with those of fresh crabs.While the thawing loss rate increased by 9.2%.The high activities of autolytic enzyme and ATP correlative catabolic enzyme accelerated the decomposition of protein and ATP correlatives at common freezing(-20℃).The protein of E.sinensis was easy to be denatured,which resulted in serious protein denaturation and thaw loss of crab meat.The reason that E.sinensis had no frozen storage resistance might have connection with its autolytic enzyme and ATP correlative catabolic enzyme.

Abstract:To understand the physiological change processes in adaptation to salinity and alkalinity water,Oreochromis niloticus were divided into four groups and directly transferred into four salinity-alkalinity treatments(A:salinity 10,1 g/L NaHCO₃;B:salinity 10,2 g/L NaHCO₃;C:salinity 15,1 g/L NaHCO₃;D:salinity 15,2 g/L NaHCO₃)from freshwater,and the acute changes of serum osmolality,serum Na⁺,K⁺,Cl^- concentration and mRNA expression of Na⁺-K⁺-ATPase(NKA)and carbonic anhydrase(CA)in gill of different treatments were observed at 0,6,12,24,36,48,72 and 96 h post-transfer,respectively.Results showed that the changes of serum osmolality,serum Na⁺,K⁺,Cl^- concentration,and NKA,CA gene mRNA expression were relevant to the salinity-alkalinity stress levels.Meantime,these parameters shared a similar “up-peak-down” change trend.The serum osmolality in B,D treatments reached the peak at 24 h,while A,C treatments at 36 h.Serum Na⁺,K⁺,Cl^- concentration in all treatments reached the peak at 24 h.The highest NKA gene mRNA expression in B,D treatments was detected at 36 h,while 24 h in A and C treatments.The highest CA gene mRNA expression in B,C and D treatments was observed at 24 h.These results showed O.niloticus could adapt to salinity-alkalinity water to a certain degree,and NKA and CA were involved in ion transportations and osmolality balance under salinity-alkalinity stress.