Quantitative comparison of the expression of myogenic regulatory factors in flounder (Paralichthys olivaceus)embryos and adult tissues

MyoD,Myf5,and myogenin are myogenic regulatory factors that play important roles during myogenesis.It is thought that MyoD and Myf5 are required for myogenic determination,while myogenin is important for terminal differentiation and lineage maintenance.To better understand the function of myogenic regulatory factors in muscle development of flounder,an important economic fish in Asia,real-time quantitative RT-PCR was used to characterize the expression patterns of MyoD,Myf5, and myogenin at early stages of embryo development,and in different tissues of the adult flounder.The results show that,Myf5 is the first gene to be expressed during the early stages of flounder development, followed by MyoD and myogenin.The expressions of Myf5,MyoD,and myogenin at the early stages have a common characteristic:expression gradually increased to a peak level,and then gradually decreased to an extremely low level.In the adult flounder,the expression of the three genes in muscle is much higher than that in other tissues,indicating that they are important for muscle growth and maintenance of grown fish.During embryonic stages,the expression level of MyoD might serve an important role in the balance between muscle cell differentiation and proliferation.When the MyoD expression is over 30%of its highest level,the muscle cells enter the differentiation stage.     

The potential of ocean acidification on suppressing larval development in the Pacific oyster Crassostrea gigas and blood cockle Arca inflata Reeve

We evaluated the effect of pH on larval development in larval Pacific oyster （Crassostrea gigas） and blood cockle （Arca inflata Reeve）. The larvae were reared at pH 8.2 （control）, 7.9, 7.6, or 7.3 beginning 30 min or 24 h post fertilization. Exposure to lower pH during early embryonic development inhibited larval shell formation in both species. Compared with the control, larvae took longer to reach the D-veliger stage when reared under pH 7.6 and 7.3. Exposure to lower pH immediately after fertilization resulted in significantly delayed shell formation in the Pacific oyster larvae at pH 7.3 and blood cockle larvae at pH 7.6 and 7.3. However, when exposure was delayed until 24 h post fertilization, shell formation was only inhibited in blood cockle larvae reared at pH 7.3. Thus, the early embryonic stages were more sensitive to acidified conditions. Our results suggest that ocean acidification will have an adverse effect on embryonic development in bivalves. Although the effects appear subtle, they may accumulate and lead to subsequent issues during later larval development.     

Effect of n-3 HUFA levels in rotifers and Artemia on growth and survival of larval black sea bream （Sparus macrocephalus）

Requirement for dietary n-3 HUFA (n-3 highly unsaturated fatty acid) for growth and survival of black sea bream (Sparus macrocephalus) larvae was studied using rotifers and Artemia at various levels of n-3 HUFA. Five treatments with rotifers and Artemia differing in n-3 HUFA were prepared by enriching them with various oil emulsions. Results indicated that dietary n-3 HUFA significantly influence fish n-3 HUFA levels and are essential for growth and survival of black sea bream larvae. The results also indicated that the incorporation of n-3HUFA TG (triacylglycerols) into tissues of larval black sea bream was more effective from natural fish oil in comparison with n-3 HUFA fatty acid ethyl esters from ethyl-esterified oil.     

EFFECT OF ZINC ON THE GROWTH AND DEVELOPMENT OF LARVAE OF BAY SCALLOP ARGOPECTEN IRRADIANS

The 48-h straight-hinge larvae of Argopecten irradians were exposed for 9 days to various concentrations of zinc in seawater. The growth rate of larvae in the 50 ppb Zn mediums was 77% that of the controls, and nearly zero in the 200 ppb Zn mediums. A progressive decrease in growth and increase in larval deformity and mortality was observed with increasing zinc concentrations from 50 to 200 ppb. 50 and 100 ppb Zn resulted in delayed development of eye spot and metamorphosis and reduced numbers of larvae at both stages. All larvae subjected to higher zinc concentrations died before attaining the eye-spot stage. The zinc concentrations which caused 50% reduction in growth (G C50)and 50% mortality (LC 50)were 109 and 120 ppb respectively. Growing in the medium with 100 ppb Zn, the larvae fed with Isochysis galbana cultured in a medium with 100 ppb Zn showed more suppressed growth and development than those fed with nonnal food. It is suggested that zinc contaminated food has strong influence on the bay sca     

Pharmacokinetics and tissue behavior of enrofloxacin and its metabolite ciprofloxacin in turbot Scophthalmus maximus at two water temperatures

Turbot Scophthalmus maximus, an important aquaculture species in China, currently suffers from epizootic diseases because of high density aquaculture. Enrofloxacin has been used to treat various systemic bacterial fish infections. However, studies concerning the pharmacokinetics of enrofloxacin in turbot are limited. In this study, the pharmacokinetics of enrofloxacin and its metabolite ciprofloxacin, were investigated in the turbot following intravenous and oral administration at 10 mg enrofloxacin/kg body weight, at 16°C and 10°C water temperatures. The concentrations of enrofloxacin and ciprofloxacin in the main tissues (plasma, muscle, liver and kidney) were detected by HPLC. The results show that the plasma concentration-time data for enrofloxacin were best described as a two-compartment open model after intravenous and oral administration. Three pharmacokinetic equations were established between the concentrations and temperatures. The kinetic profile of enrofloxacin was temperature dependent. The absorption half-life of enrofloxacin was 1.99 h and 2.17 h after oral administration, whereas the elimination half-life of the drug was 98.63 h and 136.59 h at 16°C and 10°C, respectively. The peak concentration of enrofloxacin in plasma and tissues was higher at 16°C than that at 10°C, and the peak plasma concentration time in the liver was the shortest at both temperatures among those of other tissues. The plasma C max /MIC ratio varied between 11.08 and 5 540.00 at 16°C; and between 7.92 and 3 960.00 at 10°C. The AUC/MIC ratio was 467.82-280 690.00 at 16°C, and 359.48-215 690.00 at 10°C. These ratios indicate that it is possible to obtain therapeutic efficacy. Very low levels of ciprofloxacin were detected. The AUC ratios of ciprofloxacin and enrofloxacin in plasma suggest that plasma ciprofloxacin might play a minor role in enrofloxacin treatment for turbot.     

Developmental Dynamics of the Larval Muscle System of Bay Scallop (Argopecten irradians)

Though the larval development of bivalves has been extensively studied for commercial purposes,the dynamic development of larval muscle system remains largely unknown.In this study,we characterized the larval muscle system at different developmental stages(D-shaped veligers,umbo veligers and spats)in the bay scallop(Argopecten irradians)by phalloidin staining and under a confocal microscopy.The functional muscles are initially established at the early stage of veligers,which have four pairs of velar retractors and one anterior adductor.At the veliger stage,the velum and posterior retractor muscles are functionally important for velar contractility but undergo an irreversible shrink until they disappear at the end of the larval stage.During metamorphosis,three crucial modifications take place in the larval muscle system.The metamorphosis process involves the gradual degeneration of velum retractors,mantle margin development from an unfolded to a three-fold state,and remodeling of the adductor muscle system from dimyarian(two adductors)to monomyarian condition(one adductor)as in juveniles/adults.All retractor muscles are composed of striated muscle,but both anterior and posterior adductors have smooth and striated components.These findings highlight that the morphological changes at different stages are typical features of myogenesis in scallops.The present knowledge on the developmental dynamics of myogenesis in the bay scallop will not only improve our understanding of phenotypic diversity of larval myoanatomy in bivalves,but also provide useful information on the larval culture in hatcheries.     

Development of inter-family nuclear transplant embryos by transplanting the nuclei from the loach blastulae into the non-enucleated zebrafish eggs

The developmental fate of the pronuclei in recombined embryos obtained by transplanting the donor nuclei into the non-enucleated eggs remains controversial in the case of fish.In the present study,the nuclei from the loach blastulae were transplanted into non-enucleated zebrafish eggs,the resulting 9 inter-family nuclear transplant embryos developed to larval stages,Although the development timing of the nuclear transplants resembled that of zebrafish,chromosome examination revealed that most of the recombined embryos were diploids with karyotype characteristic of loach,which was also proved by RAPD analysis.Moreover,3 out of the 9 larval fish formed barb rudiments specific to loach.It was therefore concluded that the nuclear transplant larval fish were inter-family nucleo-cytoplasmic hybrids;and that only the donor nuclei were involved in the development of the nuclear transplant embryos,while the pronuclei in the non-enucleated eggs were likely automatically eliminated during the development.     

Embryology of Maldives clownfish, Amphiprion nigripes (Amphiprioninae)

This study investigated the embryonic development of Maldives clownfish Amphiprion nigripes under natural conditions (28 - 30℃) at a lagoon of Agatti Island, Lakshadweep, India. The newly deposited fish egg was capsule-shaped and orange-red, with a (0.73 ± 0.04) mm3 yolk containing 5 - 10 fat globules. The embryonic development of fertilized eggs was divided into 26 stages and the time elapsing for each stage was recorded. Results showed that the cleavage was rapid, with the first division observed 1 h 20 min after fertilization. Blastulation occurred 4 h later, followed by gastrulation 12 h after fertilization, with a yolk volume of (0.61 ± 0.06) mm3. The organogenesis process started 22 h after fertilization when the blastopores closed and notochord formation began. The embryonic stage was recorded 24 h later, with the appearance of forebrain, midbrain, hindbrain, melanophores on yolk-sac and 22 somites, and a decreased yolk volume of (0.54 ± 0.08) mm3. Other organs developed well 31 h after fertilization, whereas the heart started beating and blood circulation began 78 h later. Red pigmentation (erytrophores) appeared 96 h after fertilization, with a small yolk volume of (0.22 ± 0.02) mm3. Mouth developed well and eyes were noticeable 120 h later, with head, pectoral fin and tail frequently moving 144 h after fertilization. The embryo reached the pre-hatching stage 168 h later and started to hatch after 170 - 180 h incubation. This study first detailed the embryonic development and yolk absorption of A. nigripes under natural conditions.     

THE CRITICAL PERIOB OF HIGH MORTALITY OF LAM VAE FESH-A DISCUSSION BASED ON CURRENT RESEARCH

Most fishes suffer high mortality during early development stages. The existence of a "critical period" was hypothesized by Hjort (1914). However, several decades of research have resulted in considerable controversy over the hypothesis. This paper attempts to clarify the critical period concept, demonstrate its usefulness, and resolve some of the controversy surrounding it.Based on our own studies and the literature, we find that within the "aquaculture school" (those concerned with culturing fishes, particulary larval stages), there is a great deal of evidence for the critical period concept. But we redefine the concept of "critical period" of mortality of fish larvae in this way. The deepest, sharpest, and fastest physiological and ecological changes during the development of the larvae are produced with the shift from endogenous nutrition to exogenous. At this stage, the old functions are quickly being replaced by new functions inside the body. Because of this shifting of function and imperfection o     

Effects of temperature, pH and NaC1 on protease activity in digestive tract of young turbot, Scophthalmus maximus

The protease activity in digestive tract of young turbot Scophthalmus maximum was studied, and the optimal pH, temperature and NaCl concentration were determined for different portions of the fish＇s internal organs. The optimal activity in the fish＇s stomach was at pH of 2.2, while that in the intestinal extracts was within the alkaline range from 9.5 to 10.0. In hepatopancreas, the optimal pH was in low alkalinity at 8.5. The optimal reaction temperature was above 40℃ in stomach, intestine and hepatopancreas. With increasing temperature, the pH value increased in stomach, while in the intestine, an opposite tendency was observed due to combined effect of pH and temperature. NaCl concentration showed inhibitory impact on protein digestion in hepatopancreas. The main protease for protein digestion in turbot seemed to be pepsin. Moreover, the maximum protease activity in different segments of intestine existed in the hindgut.     

Administration of Yeast Glucan on Immunity of Offspring in Turbot Scophthalmus maximus: A Trans-Generational Immune-Enhancing Effect

β-glucan has been shown to increase immunity and survival in various juvenile and adult fish both in freshwater and marine aquaculture species as well as in marine fish larvae. However, information about the trans-generational immune-enhancing effects of β-glucan remains rather limited. Here we clearly show that dietary intake of β-glucan enhanced the levels of C3, Bf and lysozyme in the serum of turbot as well as in their eggs released. We also show that yeast glucan induced a significant increase in lysozyme activity in both the serum and eggs. Moreover, the embryos derived from yeast glucan-treated turbot were more resistant to bacterial challenge than control embryos. By contrast, the administration of yeast glucan on female turbots had little influence on the egg development and embryonic development. Collectively, these data indicate that yeast glucan can be safely used to promote the non-specific trans-generational immunity in offspring of turbot.     

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.     

THE CYTOCHEMISTRY OF OOCYTES OF CHINESE SHRIMP PENAEUS ORIENT A US

In the growth of oocytes of Penaeus orientalis Kishinouye, five stages were distinguished . Histochemical tests showed the presence of DNA in the chromatin and nucleolus of the cell. The cytoplasm at the previtellogenetic stage and the nucleolus are rich in RNA and the proteins abounding with cysteine, tyrosine and tryptophan. The yolk consists mai nly of proteins and phospholipids. The 1, 2-glycol groups of carbohydrate occur in the cytoplasm at stage II , and aggregate mostly into cortical rods at stages IV and V. Neutral lipid droplets, and protein containing disulfides, appear in the cytoplasm at stages III and IV respectively. The proteins in the cortical rod differ from those in other components of the cell in the presence of cystine and absence of arginine.     

Proline with or without Hydroxyproline Influences Collagen Concentration and Regulates Prolyl 4-Hydroxylase α(I) Gene Expression in Juvenile Turbot(Scophthalmus maximus L.)

This study was conducted to investigate the effect of dietary proline(Pro), and Pro and hydroxyproline(Hyp) in combination on the growth performance, total Hyp and collagen concentrations of tissues, and prolyl 4-hydroxylase α(I)(P4H α(I)) gene expression in juvenile turbot feeding high plant protein diets. A diet containing 50% crude protein and 12% crude lipid was formulated as the basal and control, on which other two protein and lipid contents identical experimental diets were formulated by supplementing the basal with either 0.75% Pro(Pro-0.75) or 0.75% Pro and 0.75% Hyp(Pro+Hyp). Four groups of fish in indoor seawater recirculating systems, 35 individuals each, were fed twice a day to apparent satiation for 10 weeks. The results showed that dietary Pro and Hyp supplementation had no significant effect on growth performance and feed utilization of juvenile turbot(P 0.05). Total Hyp and collagen concentrations in muscle were significantly increased when dietary Pro and Hyp increased(P 0.05), and fish fed diet Pro+Hyp showed significantly higher free Hyp content in plasma than those fed other diets(P 0.05). The expression of P4 H α(I) gene in liver and muscle was significantly up regulated in fish fed diet Pro-0.75 in comparison with control(P 0.05); however the gene was significantly down regulated in fish fed diet Pro+Hyp in muscle in comparison with fish fed diet Pro-0.75(P 0.05). It can be concluded that supplement of crystal L-Pro and L-Hyp to high plant protein diets did not show positive effects on growth performance of juvenile turbot, but enhanced total collagen concentrations in muscle.     

Inbreeding Depression on Growth and Survival of Full-Sib Family of Manila Clam(Ruditapes philippinarum)

In present study, the inbreeding depression(ID) of growth and survival of Manila clam(Ruditapes philippinarum) was investigated at larval and juvenile stages. Nine inbred families(A_2, B_2, C_2, D_2, E_2, F_2, G_2, H_2 and I_2) were established by mating within nine full-sib families with expected inbreeding coefficient of 0.25. Inbred families showed significant differences in shell length and hatching rate of D-larvae(straight-hinged larvae). The larvae of the nine inbred families grew slower than those of control group(CG), and their ID value ranged from 0.81% ± 6.09% to 16.10% ± 1.49%. The ID value of larval survival rate varied between 27.47% ± 9.36% and 70.50% ± 13.66%. The ID was also detected for juvenile growth in A_2, B_2, C_2, and D_2, which ranged from 4.60 ± 2.21 to 17.71 ± 7.73. The A_2 family maintained the highest juvenile survival rate, whereas the other inbred families exhibited ID values varying between 62.79% ± 4.54% and 96.14% ± 0.87%. The linear relationship of estimated ID between growth and survival was negatively correlated(R =-0.434, P 0.05). The results of this study suggested that the ID of growth was common at the larval stage but was less prevalent at juvenile stage. In contrast, the ID of survival increased from larval to juvenile stage. A better understanding of the effect of inbreeding may aid to selective breeding of Manila clam.     

COMPARATIVE STUDY ON RESIDENTS＇ PERCEPTION OF TOURISM IMPACT AT TOURIST PLACES—A Case Study of Xidi, Zhouzhuang and Jiuhua Mountain

Based on the questionnaire investigation, the authors make a comparative study on local residents‘ perception of tourism impacts in Xidi, Zhouzhuang and Jiuhua Mountain. The result shows that residents of the three places tend to have strong and consistent perception of its positive economic, socio-cultural and environmental impacts, but distinctly diverse ones of its negative impacts. Residents in Xidi and Zhouzhuang also have noticeable perception of its negative environmental impacts while those in Jiuhua Mountain have weaker perception of them.The research confirms the fact that residents are more liable to have positive perceptions at the early stage of the life cycle of the tourist places. The authors conclude that there exists a nonlinear relationship between residents‘ perception and tourism development stages, and that tourism socio-cultural impact may appear at an early stage of tourism development instead of being no universal.     

Low temperature stress on the hematological parameters and HSP gene expression in the turbot Scophthalmus maximus

To study the effect of low temperature stress on hematological parameters and HSP gene expression in the turbot( Scophthalmus maximus),water temperature was lowered rapidly from 18 to 1°C.During the cooling process,three individuals were removed from culture tanks at 18,13,8,5,3,and 1°C.Blood samples and tissues were taken from each individual,hematological indices and HSP gene expression in tissues were measured.The red blood cell count,white blood cell count,and hemoglobin concentration decreased significantly( P 0.05) as temperature decreased.Enzyme activities of plasma alanine transaminase and creatine kinase increased as temperature decreased,whereas aspartic transaminase and γ-glutamyl transpeptidase activities displayed no obvious changes above 1°C and lactate dehydrogenase activity increased first and then decreased.Blood urea nitrogen and uric acid levels were highest at 8°C,and creatinine concentration was highest at 3°C.The concentrations of plasma cortisol,cholesterol,and triglyceride all increased significantly( P 0.05) as temperature decreased.The serum glucose concentration increased first and then decreased to the initial level.The HSP70 m RNA expression showed various patterns in different tissues,whereas HSP90 m RNA expression showed the same tendency in all tissues.Overall,these results indicate that temperature decreases in the range of 8 to 5°C may induce a stress response in S.maximus and that temperature should be kept above 8°C in the aquaculture setting to avoid damage to the fish.