Molecular Cloning and Expression ofPoIR2, a Novel Gene Involved in Immune Response in Japanese Flounder （Paralichthys olivaceus）

A novel immune-related gene was expressed in Japanese flounder (Paralichthys olivaceus) injected with Vibrio anguillarum. The complete cDNA contained a 169 bp 5’UTR, a 336 bp open reading frame (ORF) encoding 111 amino acids and a 556bp 3’UTR. Six exons and five introns were identified in the PoIR2 gene. Blastp similarity comparison showed its encoding protein had 50% similarity to Danio rerio neuromedin S (NMS), but further alignment indicated they did not have NMS C-terminal conservational signature domain. So it was not defined as an NMS homologue. Protein structure analysis indicated it had a 26aa signal peptide and was a secretory pathway protein. RT-PCR demonstrated that the expression of PoIR2 was quickly induced and drastically increased in liver, kidney, spleen, gills, intestine, heart, and skeletal muscle after infected with V. anguillarum. These results indicated that the PoIR2 might play some important role in Japanese flounder immune response system. This gene was named PoIR2 (P.olivaceus immune-related gene 2, GenBank accession number: EU224372). The mature PoIR2 peptide was expressed in BL21(DE3) pLysS using pET-32a(+) vector and a great part of the recombinant mature peptide existed as soluble type.     

In vitro acute cytotoxicity of neonicotinoid insecticide imidacloprid to gill cell line of flounder Paralichthy olivaceus

In vitro acute cytotoxicity of neonicotinoid insecticide imidacloprid (IMI) to the gill cell line of flounder (FG) that collected in the gill of Paralichthys olivaceus, was examined by 3 widely used endpoint bioassays: NR (neutral red), MTT (3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide) and TCP (total cell protein). The result shows that the IMI increased at concentrations ≥0.5 μg/ml. The IC50 value of NR, MTT, and TCP was 41.86, 38.46, and 39.08 μg/ml, respectively. The ultrastructural observation revealed that the mitochondria of the cells exposed to 60 μg/ml IMI for 48 h were severely damaged, swollen or disrupted, while their nuclei and rough endoplasmic reticulum (RER) remained normal. This would suggest that the mitochondria are probably the primary target of IMI.     

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)     

Screening of eye-position related genes with DD-RT-PCR and RDA in the hybrids between Japanese flounder <Emphasis Type="Italic">Paralichthys olivaceus</Emphasis> and stone flounder <Emphasis Type="Italic">Kareius bicoloratus</Emphasis>

Flatfish or flounder moves one eye to change body proportion into vertebral asymmetry during metamorphosis, during which some become sinistral while others dextral. However, the mechanism behinds the eye-position has not been well understood. In this research, hybrids between Japanese flounder(♀) and stone flounder (♂) show mixed eye-location in both dextral type and sinistral type, and thus become good samples for studying the eye-migration. mRNAs from pro-metamorphosis sinistral and dextral hybrids larvae were screened with classical differential display RT-PCR (DD-RT-PCR) and representational difference analysis of cDNA (cDNA-RDA); 30 and 47 putative fragments were isolated, respectively. The cDNA fragments of creatine kinase and trypsinogen 2 precursor genes isolated by cDNA-RDA exhibited eye-position expression patterns during metamorphosis. However, none of the fragments was proved to be related to flatfishes’ eye-position specifically. Therefore, further studies and more sensitive gene isolated methods are needed to solve the problems. Supported by National Natural Science Foundation of China (No. 30600455) and the National Basic Research Program of China (973 Program, No.2004CB117402)     

Antioxidant and isozyme features of two strains of Laminaria japonica （Phaeophyceae）

Healthy sporophytes of two gametophyte mutants of Laminaria japonica with different heat resistances: kelp 901 (901, with comparatively stronger heat-resistance) and Rongcheng No.1 (RC, sensi- tive to heat stress), were respectively collected during October to December 2002 from Yantai and Rong- cheng Sea Farm in the Shandong Peninsula of China. The contents of some biochemical materials and antioxidant capacity were analyzed under controlled laboratory conditions to identify if there is any rela- tion between the overall antioxidant capacity and the heat-resistance in L. japonica and to understand possible mechanism of heat-resistance. Results show that: (1) the overall antioxidant capacity in healthy sporophyte of 901, such as vitamin E, polyphenol, and ascorbic acid contents and the enzymatic activity of SOD, POD, CAT, Gpx, PPO, and PAL, were not always higher than that of RC under controlled labo- ratory conditions, and no significance (P>0.05) was shown in total antioxidant capacity (T-AOC) in 901 and RC. Result suggested that the difference in antioxidant capacity was not a decisive factor for different heat-resistances in L. japonica; (2) the simultaneous assay on isozymes was carried out using vertical polyacrylamide gel electrophoresis (PAGE). Considerable differences in peroxide (PRX), malate dehy- drogenase (MDH), malic enzyme (ME), polyphenol oxidase (PPO) and glutamate dehydrogenase (GDH) were obtained in 901 and RC from either the band number, relative mobility (Rf), or staining intensity, and ME could be used as an indicator to distinguish healthy sporophyte of 901 and RC under controlled labo- ratory conditions.     

Genetic variation in two sea cucumber （Apostichopus japonicus） stocks revealed by ISSR markers

Sea cucumber Apostichopus japonicus samples were collected in Changdao, Penglai (PL), 27 individuals, and Lingshandao, Qingdao (QD), 30 individuals, in the Shandong Peninsula, China. Ten SSR primers were used to assess the genetic variation and relationship between and within the two stocks. Respectively, for each stock, the percentage of polymorphic bands was 85.2% and 86.9%; the gene diver- sity was 0.360 5 and 0.342 8; and the Shannon’s information index was 0.515 0 and 0.499 0. At species level, the percentage of polymorphic bands was 92.2%, the total gene diversity was 0.378 9 and the Shannon’s information index was 0.550 4. The coefficient of overall genetic differentiation and the ge- netic distances between the stocks were also calculated to be 0.073 0 and 0.079 6 using the POPGENE program. Results show that the genetic diversity of the two stocks is still large but the genetic distance between the two stocks is close. A dendrogram was constructed for the 57 individuals from the two stocks, showing that the genetic structure was unitary for PL stock but complex for QD stock.     

Uncertainties in Stock Assessment and the Precautionary Management Suggestions for the Anchovy （Engraulisjaponicus） Stock in the Yellow Sea

Anchovy(Engraulis japonicus) is an abundant fish species in the Yellow Sea,and its natural stock is decreasing rapidly in recent years. Based on the stock-recruitment(SR) data from 1987 to 2002 published in Zhao et al.(2003),the criterion BIC(Bayesian Information Criterion) is applied to selecting a suitable model from six normal and lognormal error structured SR statisti-cal models,the age-structured model is used to calculate the biological reference points(BRPs),and the precision of the SR parame-ters and BRPs are calculated using bootstrap method. The results indicate that the anchovy fishery resource in the Yellow Sea is in an over-fished state. The precaution management principle requires that the fishery should be closed immediately.     

Isozymes Analysis of the Golden Cuttlefish Sepia esculenta（Cephalopoda ： Sepiidae）

Thirty-nine isozymes in four tissues (mantle muscle, buccal bulb muscle, eye and liver) of Sepia esculenta were screened for enzymatic analysis using starch gel electrophoretic technique. Eighteen enzymes (G3PDH, LDH, MDH, MEP, IDHP, PGDH, GRS, NP, AAT, CK, AK, EST, ALP, ACP, FBP, MPI, GPI and PGM) show strong activities and good convergence in zymogram. They are proved to be suitable genetic markers in Sepia esculenta. Among the tissues used, mantle muscle is the best for electrophoretic analysis of isozymes. Eye and liver are fairly good for some special enzymes, such as LDH, EST, MPI, etc. Twenty-six loci are detected. The proportion of polymorphic loci is 0.115 in the Qingdao sample and 0.153 in the Rizhao sample (P<0.99). The mean values of the observed and expected heterozygosity per locus of Qingdao sample are 0.016 and 0.017, while those of the Rizhao sample are 0.023 and 0.025 respectively.     

Genetic variation of natural and cultured stocks of Paralichthys olivaceus by allozyme and RAPD

Population genetics of the left-eyed flounder, Paralichthys olivaceus, including natural and cultured stocks distributed in the coastal waters near Qingdao of eastern maritime China, was analyzed in allozyme and RAPD. The results showed that among total 29 gene loci of 15 isozymes, 9 and 7 were po- lymorphic in natural and cultured stocks, respectively. The status of genetic diversity in P. olivaceus is low in terms of polymorphic loci in chi-square test and genetic departure index of Hardy-Weinberg equi- librium. More alleles in IDHP, CAT, GDH and Ldh-C allozymes were found in the fish, which could be used as markers in assortive breeding and distinguishing stock, population or species evolution. Total 88 and 86 RAPD bands ranging from 200 to 2 500 bp were recognized individually in average of 7.8–8.0 bands per primer. The genetic diversity in cultured stock is lower than that in natural ones showing an ob- viously decreasing genetic divergence. Therefore, effective countermeasures must be taken to protect ge- netic resources of marine cultured fishes. The 2 markers have their own pros and cons. Combining the 2 markers to investigate the genetic variation of populations is suggested. The results provide basic data of this flounder and they are useful for studying genetic improvement and genetic resources of the fish.