Effects of base analogues 5-bromouracil and 6-aminopurine on development of zebrafish Danio Rerio

Zebrafish （Danio rerio） genetic screens allow isolation of a wide array of problems in vertebrate biology. The effects of base analogues 5-bromouracil and 6-aminopurine on the development of zebrafish embryos are reported for the first time in this study. The early development of the zebrafish embryos was little affected by 5-bromouracil and 6-aminopurine, while the late development （organogenesis） was significantly impaired. Embryos exposed to 5-bromouracil mainly showed curled tail, wavy body, golden pigmentation and the mouth with protruding lower jaw. 6-aminopurine-treated embryos had defective anterior structures, curled tails and wavy body. RAPD analysis showed that the majority of 5-bromouracil-and 6-aminopurine-treated larvae and fish shared banding patterns in common with the control, suggesting that most mutagenesis induced by these agents are point mutations. However, some fish derived from 5-bromouracil-treated embryos had golden（gol） pigmentation; and RAPD analysis revealed that their band patterns differed from those of the control.Possibly, 5-bromouracil can occasionally cause relatively extensive changes in the fish genome. The results of this study may provide valuable help for detailed studies of mutagenesis.     

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.     

Hybrids between olive flounder Paralichthys olivaceus and stone flounder Kareius bicoloratus： karyotype, allozyme and RAPD analyses

The hybrid between olive flounder Paralichthys olivaceus and stone flounder Kareius bicoloratus was produced by artificial insemination of olive flounder eggs with stone flounder sperm. Sinistral and dextral are two types of hybrid progeny after metamorphosis. Karyotypes of both hybrid flounders are the same as those of the two parental species. Of the 22 loci examined from 12 allozymes, 12 confirmed hybridization of the paternal and maternal loci in hybrids and no difference was found in allozyme patterns of sinistral and dextral hybrid fishes. RAPD patterns of these specimens were also studied with 38 primers selected from 104 tested. Among them, the PCR products of 30 primers showed hybridization of the paternal and maternal bands. Genetic variation between hybrids and their parental stocks was analyzed by RAPD using 10 of the above 38 primers. The average heterozygosity and genetic distance were calculated. The results suggested that the filial generation could inherit a little more genetic materials from paternal fish than that from maternal fish. Supported by National Natural Science Foundation of China (No. 30571445), National High-Technology Research and Development Program (863 Program, No. 2006AA10A404), and Project from the Ministry of Science and Technology of China (No. 2006DKA30470-017)     

Length frequency analysis ofPleuragramma antarcticum,Electrona antarctica,Protomyctophum bolini

British Antarctic Survey (BAS) data collected near the Antarctic Peninsula area were used in this 1988/1989 study on the length frequency of three Antarctic fish species (Pleuragramma antarcticum, Electrona antarctica and Protomyctophum bolini) showing that the standard length (SL) ofP. Antarcticum ranged from 26.0 mm to 161.0 mm, that the 26.0 mm to 54.0 mm SL juveniles could be considered to be the age group 1 fish, and that the SL ofE. antarctica ranged from 39.4 mm to 89.7 mm for the males and 48.0 mm to 118.0 mm for the females, which may contain at least 6 age groups. This is more than previously thought.P. Bolini had 29.2 mm to 60.2 mm SL and complicated age composition.     

Genes of the adaptive immune system are expressed early in zebrafish larval development following lipopolysaccharide stimulation

Information regarding immunocompetence of the adaptive immune system(AIS) in zebrafish Danio rerio remains limited.Here,we stimulated an immune response in fish embryos,larvae and adults using lipopolysaccharide(LPS) and measured the upregulation of a number of AIS-related genes(Rag2,AID,TCRAC,IgLC-1,mIg,sIg,IgZ and DAB) 3 and 18 h later.We found that all of the genes evaluated were strongly induced following LPS stimulation,with most of them responding at 8 d post fertilization.This confirms that a functional adaptive immune response is present in D.rerio larvae,and provides a window for further functional analyses.     

Genetic differentiation withinEriochoir sinensis (milne, edwards) revealed by allozyme and RAPD

We analyzed 17 allozymes, and 20 primers in order to detect the genetic differentiation between commercial populations (Changjiang River, Liaohe River) ofEriochoir sinensis. Ten allozymes (LDH, MDH, ME, IDH, EST, ALP, AAT, CTL, POD, SOD) showed 21 loci by vertically discontinuos buffer system polyacrylamide gel electrophoresis. RAPD profiles generated by 12 ten-base primers showed 63 loci. The percentage of polymorphic loci and the expected heterozygosity obtained by using allozyme analysis were lower than those obtained by RAPD. The index of similarity between these two populations were 0.955 and 0.932 as revealed by allozyme analysis and RAPD technology. There was gene flow between the above populations. Contribution No. 4025 from the Institute of Oceanology and Limnology, Chinese Academy of Sciences. Project 39470141 supported by NSFC. Work also supported by Climbing Program B (PDB 6-5-3) and No. 863 High-technology Program 863-819-01-05.     

A light and transmission electron microscopic study ofHexamite capsularis sp. nov. (Diplomonadina: Hexamitidae) in fish (Xenocypris dividi)

A diplomonad flagellateHexamita capsularis sp. nov. from the posterior intestine of the fish (Xenocypris dividi) from Liangzi Lake, Hubei Province, is described. The flagellate was oval, with smooth surface and homogenous cytoplasm, 6.21±0.28 μm in length, 3.43±0.48 μm in width and with an elliptical capsule. Two recurrent flagellae arranged between two nuclei ran longitudinally to the posterior end. Fibrillar bands connected the accessory organelles of two cytostomal tubes, the body depressed at the posterior end of the body. This work is a part of the M. S. thesis of Xiao Wu-han and was supported by the National Natural Science Foundation of China (Grant No. 3870489).     

CYTOTOXICITY AND GENOTOXICITY OF POLYETHYLENIMINE AND NICKEL CHLORIDE IN RED SEA BREAM (Pagrosomus major) FIN CELL LINE RSBF

ＩＮＴＲＯＤＵＣＴＩＯＮＡｃｃｕｍｕｌａｔｅｄｅｖｉｄｅｎｃｅｓｓｕｇｇｅｓｔｅｄａｃｌｏｓｅｃｏｒｒｅｌａｔｉｏｎｂｅｔｗｅｅｎｎｅｏｐｌａｓｔｉｃｄｉｓｅａｓｅｏｆａｑｕａｔｉｃｏｒ ｇａｎｉｓｍｓａｎｄｔｈｅｉｎｃｒｅａｓｉｎｇｒｅｌｅａｓｅｏｆｇｅｎｏｔｏｘｉｎｓｂｙａｎｔｈｒｏｐｏｇｅｎｉｃａｃｔｉｖｉｔｉｅｓｉｎｔｏａｑｕａｔｉｃｅｎｖｉｒｏｎｍｅｎｔ(Ｍｉｘ ,1 986;Ｇａｒｄｎｅｒｅｔａｌ.,1 991 ;ＧＥＳＡＭＰ ,1 991 ) .Ｉｔｈａｓ,ｔｈｅｒｅｆｏｒｅ,ｂｅｃｏｍｅｎｅｃｅｓｓａｒｙｔｏｅ…     

CYTOTOXICITY AND GENOTOXICITY OF POLYETHYLENIMINE AND NICKEL CHLORIDE IN RED SEA BREAM（Pagrosomus major）FIN CELL LINE RSBF

A continuous marine fish cell line RSBF(i.e.Red Sea Bream Fin)was utilized to screen the cytotoxicity and genotoxicity of polyethylenimine(PEI)and nickel cholride(NiCl2)in this study on the deleterious effects of aquatic genotoxins on fish.At the 0.01 to 1μgml concentration tested,PEI had acute toxicity to the treated RSBF cells(IC50=1.12,0.92,0.88 and 0.64μg/ml PEI for time 0 h,24 h,48 h and 72 h after treatment,respectively)and markedly inhibited their proliferation in a dose-dependent manner.At the 0.001 to 5 μmol/L concentration tested,NiCl2 posed no acute toxicity but significantly stimulated their growth(107?214?of control).Random amplified polymorphic DNA(RAPD)technique was used to detect the genotoxic effects of PEI and NiCl2 by comparing the RAPD banding patterns of the control and treated cells.RAPD analysis indicated that at the concentrations tested,PEI was more genotoxic than NiCl2 to RSBF cells;that there was a slight dose-dependent response in the genotoxic effect of PEI bue not NiCl2;and that RAPD technique might provide a sensitive,non-specif-ic gentoxic endpoint.And the potent cytotoxicity and genotoxicity of PEI on fish cells showed that we should be cautious in utilizing it as gene vector in fish gene transfer and human gene therapy.     

Assessment of nitrogen maintenance requirement in juvenile Black Porgy <Emphasis Type="Italic">Acanthopagrus schlegeli</Emphasis>

Nitrogen balance method and nitrogen-free diet were used in this study to determine nitrogen maintenance requirement (NM) and nitrogen maintenance requirement per unit metabolism body weight (NM′) of black porgy Acanthopagrus schlegeli. Fish with body weight (BW) of 50, 80, 120, 160 and 200 g were fed by the diets containing three graded levels of crude protein (380, 420 and 460 g/kg). The results from nitrogen balance experiment showed that the amount of nitrogen deposition varied from 0.15 to 0.31 mg/g BW per day, accounting for 12.2% to 21.1% of nitrogen intake. The amount of fecal nitrogen excretion varied from 0.21 to 0.32 mg/g BW per day, accounting for 16.3% to 21.6% of nitrogen intake. The endogenous nitrogen excretion, a main part of nitrogen consumption varied from 0.79 to 0.97 mg/g BW per day, accounting for 63.3 % to 68.0% of nitrogen intake. Positive correlation was found between NM and body weight, while a negative correlation was found between NM of unit body weight and the growth duration. No significant differences (p>0.05) were found in NM′ among different growth stages. The average of NM′ was 0.485 7mg/g per day. The results from nitrogen-free diet experiment showed that a negative correlation between NM and feed intake of nitrogen-free diet. NM increased with the decrease of feed intake of fish. The average of NM was 0.482 9 mg/g BW per day that was close to 0.483 8 mg/g BW obtained from fish with 120 g BW in nitrogen balance experiment. The nitrogen balance method is recommended to be a better method for determining NM in consideration of fish stress and result stability. This study also provides a calculated result of the protein content in diets, which is necessary for maintaining fish body protein at different growth stages. The calculation was based on the amount of nitrogen required for maintaining body protein per kg BW. Supported by Scientific Research Project Grant (No.2004C100059) from the city government of Ningbo, China.     

PHYLOGENETIC RELATIONSHIPS AMONG PUFFERFISH OF GENUS TAKIFUGU BY RAPD ANALYSIS

AmpLifications with 300 10-base primers under predetermined optimal reaction yielded 2762 reproducible amplified fragments ranging from 200 to 3000 bp. Genetic distances among five species of Takifugu and Lagocephalus spadiceus as outgreup were calculated based on the presence/absence of the amplified fragments. Topological phylogenie trees of the 5 species of Takgfugu and the outgroup were gen-erated by Parsimony and Neighbor-Joining analysis based on RAPD data set. The genetic distance between T. rubripes and T. pseudommus was almost the same as that between individuals within each species, but was much smaller than that among T. rubripes, T. pseudommus and the other species. The molecu-lar data from RAPD analysis convincingly showed that T. rubripes and T. pseudommus should be the same species.     

Effects of Dietary Protein Level on Growth and Utilization of Protein and Energy by Juvenile Mangrove Red Snapper （ Lutjanus argentimaculatus ）

A feeding trial was conducted in a recirculating water system to investigate the effects of dietary protein levels on growth, feed utilization, hepatosomatic index and liver lipid deposition of juvenile red snapper, Lutjanus argentimaculatus (average initial wet weight 8.0 ± 0.39 g and total length 3.14 ± 0.3 cm). In the experiment, six fishmeal-based diets were formulated to contain various protein levels (20% to 45% in 5% increments), with dietary energy ranging from 2210.7kJ lOOg to 2250.2kJlOOg dry matter. The protein to energy ratios of diets ranged from 8.58 mg protein kJ⁻¹ to 20.03 mg protein kJ⁻¹. Diets were fed for 90d to triplicate groups of fish stocked in 0.128m³ seawater tanks, 25 individuals each. The daily ration of 2% wet body weight was offered to the fish thrice a day. The fish at the end of the study had more than ten-fold (77.0g) increase in weight compared to the initial (8.0g). Fish fed diets of 40% and 45% protein produced significantly (P<0.05) higher weight gain of 77.2g and 76.5g, and specific growth rate (SGR) of 2.65% and 2.62% than those of 67.0 g and 68.3g, and 2.49% and 2.51% of the other diets. The broken-line regression of SGR against dietary protein level yielded an optimum dietary protein requirement of 42.6% (Y=−1.6295 + 0.1114 X ²,P<0.05). Survival remained 100% among groups. Feed conversion ratio decreased from 0.45 for fish fed 20% dietary protein to 0.35 for fish fed 45% dietary protein. Nitrogen intake increased with an increase in dietary protein, which in turn resulted in an increase in nitrogen gain of fish whole body. Fish fed 40% and 45% protein diets showed higher (P<0.05) nitrogen gain (0.27g and 0.26g) than those (0.23g and 025g) fed all other diets. Gross energy intake (GEI) in fish fed 45% protein was lower (600.67kJ) than that (607.97 kJ) of 40% protein diet, though the differences were not statistically significant (P>0.05); GEI ranging from 677.31 kJ to 663.20 kJ at remaining four diets (20% to 35% protein) did not appear to differ significantly (P>0.05). The highest energy gain of 518.33 kJ was obtained with fish fed 40% protein, resulting in the highest energy retention efficiency of 85.26%. The hepatosomatic index of fish fed diets of 20%, 25%, 30% and 35% protein were significantly (P<0.05) higher (2.09% to 2.57%) than those (1.44% and 1.41%) of fish fed diets containing 40% and 45% protein. Liver lipid contents decreased from 8.72% to 7.0% in fish fed dietary protein of 20% to 45% in 5% increments. Results suggest that the diet containing 40% to 42.6% protein with a P/E ratio of 17.6 mg protein kJ⁻¹ is required for good growth of L. argentimaculatus weighing between 8.0 g and 85.2 g under the culture conditions of the present study.     

Genetic study of Kelp"901"strain

Based on DNA extraction and optimization of random amplified reaction (RAPD) to the gametophytes and sporophytes of Kelp “901” strain, genetic study on variation was conducted to its parents and offsprings of F₆, F₇, F₈, and F₉ generation. RAPD results have shown that among 30 selected primers for gametophytes, 297 loci ranging from 200 to 3 000 bp were obtained in the average of 9.9 loci for each primer. This indicated a high polymorphic rate with RAPD detection. UPGMA (unweighted pair-group method arithmetic average) analysis showed that each male and female gametophyte of a generation could be clustered into one pair separately. The genetic distances of the Kelp 901 generation were 0.3212–0.4767, and the maximum was between F₇ and F₈ (0.4767). Identity analysis showed that F₆ generation was more close to the female parent (0.6593), and F₇ generation was more close to the male parent (0.578 8). To the sporophytes study in 24 selected primers for RAPD amplification, 191 loci ranging from 230–2800 bp were obtained, in the average to each primer of 8.0 loci. The heterozygosity to six populations were male parent (0.2239), female parent (0.1072), F₆ (0.2164), F₇(0.2286), F₈(0.2296) and F₉(0.3172). The nearest genetic distance was 0.083 5(F₈, F₉). Total heterozygosity (H_T) of F₆, F₇, F₈ and F₉ generations was 0.3186, the average heterozygosity (H_S) for F₆, F₇, F₈ and F₉ generations was 0.2480, and deduced coefficient of population differentiation (G_st) was 22.2%. Six sequence characterized amplified regions (SCAR) were preliminary screened through RAPD analysis. It needed to be verified in detail as they are significant for molecular marker assistance in breeding and selectingLaminaria.     

ENCLOSURE EXPERIMENTS ON AND LACUSTRINE PRACTICE FOR ELIMINATING MICROCYSTIS BLOOM

Microcystis bloom, one of the most objectionable characteristics of eutrophication in tropical and subtropical waters, occurred in Donghu Lake (East lake) of Wuhan every summer from the 1970s up to 1984, but from 1985 up to now failed to occur there. The cause of its disappearance rema-ined in obscurity until recently. In situ enclosure experiments in the lake for three years showed that the stocking of the filter-feeding silver carp ( Hypophthalmichthys molitrix ) and big-head carp (Aristichthys nobilis ) played a decisive role in eliminating Microcystis bloom from the lake; but that recurrence of the bloom is possible under certain conditions. This paper presents the details and the results of enclosure ex-periments. The authors‘ analysis of fish biomass data obtained by echo-sounding and the fishery produc-tion of the lake over the years, revealed that the recurrence of Microcystis bloom can be prevented so long as the combined biomnss of silver carp and big-head carp remains at or exceeds 50 g per cubic meter of lakewater, as was the case in the lake‘s 1985 fish yield of 1015 t.     

EFFECTS OF FISH OIL,DHA OIL AND LECITIHIN IN MICROPARTICULATE DIETS ON STRESS TOLERANCE OF LARVAL GILTHEAD SEABREAM（SPARUS AURATA）

The effects of natural fish oil,DHA oil and soybean lecithin in microparticulate diets on stress tolerance of larval gilthead seabream(Sparus aurata)were investigated after 15 days feeding trials.The tolerance of larval gilthead seabream to various stress factors such as exposure to air(lack of dissolved oxygen),changes in water temperature(low)and salinity(high) were determined.This study showed that microparticulate diet with natural fish oil and soybean lecithin was the most effective for in-creasing the tolerance of larval gilthead seabream to various stresses,and that microparticulate diet with natural fish oil and palmitic acid(16:0)was more effective than microparticulate diet with DHA oil and soybean lecithin.     

Assessing the germplasm of Laminaria (phaeophyceae) with random amplified polymorphic DNA (RAPD) method

Eighteen gametophytes includingL. japonica, L. ochotensis andL. longissima, were verified with random amplified polymorphic DNA (RAPD) technique. Eighteen ten-base primers were chosen from 100 primers selected for final amplification test. Among the total of 205 bands amplified, 181 (88.3%) were polymorphic. The genetic distance among different strains ranged from 0.072 to 0.391. The dendrogram constructed by unweighted pair-group method with arithmetic (UPGMA) method showed that the female and male gametophytes of the same cell lines could be grouped in pairs respectively. It indicated that RAPD analysis could be used not only to distinguish different strains ofLaminaria, but also to distinguish male and female gametophyte within the same cell lines. There is ambiguous systematic relationship if judged merely by the present data. It seems that the use of RAPD marker is limited to elucidation of the phylogenetic relationship among the species ofLaminaria. Contribution No. 4389 from the Institute of Oceanology, Chinese Academy of Sciences. This research was supported by the State's High-tech Project of China, Marine 863 project (No. 819-03-02) and (2001AA621090).     

An experiment on induction of triploidy inEriocheir japonicus hepuensis Dai by heat treatment

Temperature treatment to inhibit extrusion of the polar body of the egg was first used on a lower crustacean,Artemia salina (Gross, 1932), and then was used for inducing triploids of amphibian (Frankhauser and Griffiths, 1939), fish (Svardson, 1945), and mammal (Beatly and Fishchberg, 1949). So far, induction of triploidy has been extensively used to obtain sterile or quick-growth individuals in fish (Swarup, 1956; Lincoln, and Scott, 1983 and Thorgaard, 1986) and mollusk (Stanley et al., 1984), but similar work has not been reported on crab, a higher crustancean.     

Effects of Body Weight and Water Temperature on Maximum Food Consumption of Juvenile Sebastodes fuscescens （Houttuyn）

Maximum rate of food consumption ( Cmax ) was determined for juvenile Sebastodes fuscescens (Houttuyn) at water temperature of 10, 15, 20 and 25℃. The relationships of Cmax to the body weight (W) at each temperature were described by a power equation: lnCmax = a b lnW. Covariance analysis revealed significant interaction of the temperature and body weight. The relationship of adjusted Cmax to water temperature (T) was described by a quadratic equation: Cmax = -0.369 0.456T - 0.0117T^2. The optimal feeding temperature calculated from this equation was 19.5 ℃. The coefficients of the multiple regression estimation relating Cmax to body weight (W) and water temperature (T) were given in the Table 2.     

波纹龙虾胚胎的离体培养及发育观察

对波纹龙虾不同发育时期的胚胎进行离体培养和显微观察。结果表明,初始培育的离体胚胎愈接近孵化期,其孵化率愈高(最高达80.0%);波纹龙虾胚胎发育经历受精卵、卵裂期、囊胚期、原肠期、中眼色素形成期、复眼色素形成期、心跳期、破膜前期和出膜期;胚胎在发育过程中颜色变化:橙红→深橙红→砖红→灰白→接近透明。