Morphological and molecular diagnoses of Polydora brevipalpa Zachs,1933(Annelida: Spionidae) from the shellfish along the coast of China

Shell-boring species Polydora brevipalpa Zachs, 1933 is redescribed based on morphological observations and molecular approach for future unambiguous identification. Genetic distance analyses showed that the interspecific polydorid variation(16.7%–25.6%) was at least 15 times higher than the intraspecific one(0.2%–0.9%) based on the cytochrome c oxidase subunit I(CO1) gene sequences of polydorids. However, 18 S rDNA variation pattern demonstrated a rather narrow barcoding gap, with the interspecific polydorid variation(0.5%–5.6%) being very close to the intraspecific one(0.0%–0.4%). As such, the CO1 gene exhibited better DNA barcode for identification of polydorids than the 18 S rDNA gene because of the su ciently large barcoding gaps. Analysis of molecular variance results based on CO1 gene sequences showed that most variations in sequences(97.79%) lay within groups of adult worms and egg capsules rather than between them. This indicated that egg capsules from Crassostrea gigas(Thunberg,1793) in Ningbo and Nantong were related to the adult worms from Patinopecten yessoensis(Jay, 1857) in Dalian, and both of them belonged to P. brevipalpa. This result was further supported by parsimony network analysis, which showed that egg capsules collected from dif ferent localities and adult worms shared a single haplotype. This study was the first to report both P. brevipalpa infestation on C. gigas and to utilise the known CO1 sequences of the adult polydorids to validate morphologically unidentified egg capsules or early larvae. P. brevipalpa was most possibly brought to Chinese waters through transportation of Pa. yessoensis brood stock from Japan.     

Sequence and phylogenetic analyses of the chloroplast 16S rRNA, tufA, and rbcL genes from Bryopsis hypnoides

Using shotgun sequencing data, the complete sequences of chloroplast 16S rRNA and tufA genes were acquired from native specimens of Bryopsis hypnoides (Qingdao, China). There are two group I introns in the 16S rRNA gene, which is structurally similar to that of Caulerpa sertularioides (Bryopsidales, Chlorophyta). The chloroplast-encoded tufA gene sequence is 1 230 bp long, very AT-rich (61.5%), and is similar to previously published 16S rRNA sequences of bryopsidinean algae. Phylogenetic analyses based on chloroplast 16S rRNA and tufA gene sequence data support previous hypotheses that the Bryopsidineae, Halimedineae, and Ostreobidineae are three distinct lineages. These results also confirmed the exclusion of Avrainvillea from the family Udoteaceae. Phylogenetic analyses inferred that the genus Bryopsis as sister to Derbesia; however, this clade lacked robust nodal support. Moreover, the phylogenetic tree inferred from rbcL GenBank sequences, combined with the geographical distributions of Bryopsis species, identified a strongly supportive clade for three differently distributed Asian Bryopsis species. The preliminary results suggesting that these organisms are of distinct regional endemism.     

Establishment and characterization of a testicular Sertoli cell line from olive flounder Paralichthys olivaceus

The culture of Sertoli cells has become an indispensable resource in studying spermatogenesis.A new Sertoli cell line(POSC) that consisted predominantly of fibroblast-like cells was derived from the testis of the olive flounder Paralichthys olivaceus and sub-cultured for 48 passages.Analysis of the mtDNA COI gene partial sequence confirmed that the cell line was from P.olivaceus.Cells were optimally maintained at 25℃ in DMEM/F12 medium supplemented with fetal bovine serum,basic fibroblast growth factor,and epidermal growth factor.The growth curve of POSC showed a typical "S" shape.Chromosome analysis revealed that the cell line possessed the normal P.olivaceus diploid karyotype of 2n=48t.POSC expressed dmrt1 but not vasa,which was detected using RT-PCR and sequencing.Immunocytochemistry revealed that the cells exhibited the testicular Sertoli cell marker FasL.Therefore,POSC appeared to consist of testicular Sertoli cells.Bright fluorescent signals were observed after the cells were transfected with pEGFP-N3 plasmid,with the transfection efficiency reaching 10%.This research not only offers an ideal model for further gene expression and regulation studies on P.olivaceus,but also serves as valuable material in studying fish spermatogenesis,Sertoli cell-germ cell interactions,and the mechanism of growth and development of testis.     

First record of Biecheleria cincta (Dinophyceae) from Chinese coasts, with morphological and molecular characterization of the strains

The presence of Biecheleria cincta (=Woloszynskia cincta) in the Chinese coasts is reported for the first time. In scanning electron microscope, three to five series of vesicles and an elongated apical vesicle (EAV) were visible in the epicone, and both the hypocone and the cingulum had three series of vesicles each. Thin sections revealed that B. cincta possesses stalked pyrenoids and an unusual eyespot consisting of a stack of cisternae with brick-like materials (type E), thus supporting its transfer from Woloszynskia to Biecheleria. Spiny cysts formed spontaneously in culture, with an encystment rate of around 20%. Both large subunit ribosomal DNA (LSU rDNA) and internal transcribed spacer region (ITS) sequences in 12 strains from the Chinese coasts were determined. Phylogenetic analyses based on LSU rDNA and ITS sequences using Bayesian inference and maximum likelihood revealed two distinct ribotypes (referred to as ribotype A and B) in B. cincta. ITS region pairwise distances within B. cincta ranged from 0.024 to 0.072, suggesting the existence of a complex of cryptic species.     

Relationship between morphospecies and microcystin-producing genotypes of Microcystis species in Chinese freshwaters

Twenty water bodies in China were sampled,and 186 strains of different Microcystis species were isolated,from which eight morpho species were identified and 43 stains containing the mcyB gene were detected.Phylogenetic analysis based on the mcyB gene indicated that the microcystin(MC)-producing Microcystis in China could be divided into two groups(Ⅰ and Ⅱ) and showed significant differences between the two groups.The maximum sequence similarity was 69.1%.Microcystins(MCs) were measured by high-performance liquid chromatography(HPLC) analysis,and no microcystin-RR(MC-RR) was detected in some strains belonging to Group Ⅱ.Compared to other regions of the world,the proportion of Chinese MC-producing was different,and the regional differences were more obvious.A whole-cell polymerase chain reactio(PCR) assay was conducted to analyze the proportion of the mcyB gene in the laboratory cultured and field cultured Microcystis.The proportion of four morphospecies(M.vividis,M.ichthyoblabe,M.novacekii,and M.aeruginosa) that contained the mcyB gene exceeded 50% in the field cultured sample s.Compared with former studies,M. aeruginosa was the mo st likely morphotype that can produce MCs in the world.This study provided new insight of Microcystis hazard assessment and field monitoring.     

Identification of Cytochrome P450 (CYP) genes in Zhikong scallop (Chlamys farreri)

Cytochrome P450 (CYP) superfamily is one of the membership largest and function most diverse protein superfamily recogniozed among living beings. Members of this superfamily were further assigned to different families and subfamilies based on their amino acid similarities. According to their phylogenetic relationships, the CYP genes which likely diverged from common ancestor gene and may share common functions were grouped into one clan. Widely distributing scallops are a group of the most conspicuous bivalve; however the studies on their CYP is acarce. In this study, we searched the genome and expressed sequence tags of Zhikong scallop (Chlamys farreri) for CYP genes. In total, 88 non-redundant CYP were identified, which were homed in 13 CYPs gene families. Phylogenetic analysis divided these genes into 4 CYP clans. As in deuterostomes, Clan 2 was the largest, which contained 33 genes belonging to CYP1, CYP2, CYP17 and CYP356 families. Clan 3 contgained 19 genes belonging to CYP3, CYP5 and CYP30 families. Clan 4 contained 23 genes, all belonging to CYP4 family. The mitochondrial CYP clan contained 9 genes belonging to CYP10 and CYP24 families. In comparison, protostomes (C. farreri, D. pluex, D. melanogaster) contained more CYP genes than deuterostomes (S. purpuratus and vertebrates) in Clan 2 but less genes in Clan 3 and Clan 4. Our findings will aid to deciphering CYP function and evolution in scallops and bivalves.     

Insights into the phylogeny of sporadotrichid ciliates (Protozoa, Ciliophora: Hypotricha) based on genealogical analyses of multiple molecular markers

The sporadotrichid ciliates are an especially diverse group. A number of investigators have studied the morphological, morphogenetic, and molecular relationships among members of this group. Despite this, a consistent classification is still lacking and several important questions about the phylogenetic relationships within this group remain unsolved. To improve our understanding of these relationships, we constructed phylogenetic trees using the nucleotide sequences of the small-subunit rRNA (SSrRNA) gene and amino acid sequences of actin I and α-tubulin. Analyses of SSrRNA gene sequences indicated that: 1) the Sporadotrichida sensu Lynn (2008) and the Oxytrichidae are polyphyletic; 2) the Uroleptus species, which are classified to urostylids, formed a sister group with the oxytrichids; 3) Halteria grandinella, which is grouped morphologically with oligotrich species, clustered within the oxytrichids. These results are congruent with previous studies based on SSrRNA gene sequences. However, the amino acid sequences of actin I and α-tubulin yielded different topologies. The main results are: 1) in all phylogenetic trees, the genus Oxytricha was paraphyletic; 2) Uroleptus was sister to a subset of Urostyla and Holosticha, albeit with low supporting values; 3) Halteria grandinella was separated distantly from the Oxytrichidae in trees inferred from actin I amino acid sequences but clustered with oligotrichids in the α-tubulin analysis. The inconsistency among the trees inferred from these different molecular markers may be caused by rapidly accumulated genetic characterizations of ciliates. Further studies with additional molecular markers and sampling of more taxa are expected to better address the relationships among sporadotrichids.     

Phylogenetic analysis of cultivable bacteria isolated from Arctic sea-ice

Phylogenetic analysis based on 16S rDNA of 8 strains of cultivable bacteria isolated from Arctic sea-ice was studied.The results showed that strain BJ1 belonged to genus Planococcus,which was a genus of low mole percent G C gram-positive bacteria;strain BJ6 belonged to genus Burkholderia of β-proteobacteria and the rest 6 strain all belonged to γ-proteobacteria,of which strain BJ8 was a species of Pseudoalteromonas,strain BJ2-BJ5 and BJ7 were members of genus Psychrobacter.Phylogenetic analysis also indicated that bacteria of genus Psychrobacter of the isolates formed a relatively independent phylogenetic cluster in comparison with other bacteria belonged to genus Psychrobacter.     

Comparative Analysis of Mitochondrial Control Region Sequence from Three Flatfish Species （Pleuronectidae）

The 5‘-end of the mitochondrial control region sequences of three flatfishes (Pleuronectiformes: Pleuronectidae) were amplified and sequenced. These sequences were compared with those of other three Pleuronectids species retrieved from GenBank. A phylogenetic tree was constructed based on the partial control region sequences. The results of phylogenetic analysis are consistent with those of conventional systematics. Compared to previous studies, the structure of the 5‘-end of mitochondrial control region was analyzed. The terminal associated sequence motif and its complementary motif were identified at the 5‘-end of the sequences. A conserved sequence block, named as CM5‘ d, was identified in the 5‘-end of control region sequences in all Pleuronectids. Another central conserved sequence block, named as CSB-F, was detected in the central conserved blocks.     

两个奥利亚罗非鱼群体热休克蛋白70基因序列比对分析

采用RACE技术,对两个奥利亚罗非鱼群体(Oreochromis aurea)(美国奥利亚和中国奥利亚)热休克蛋白Hsp70基因完整编码区(code sequences,CDS)cDNA的进行克隆测序。序列分析结果表明:两个奥利亚罗非鱼群体热休克蛋白Hsp70基因CDS序列完全相同,全长1 923 bp,编码640个氨基酸,相对分子质量为70.29×103,理论等电点5.462,均具有Hsp70家族的3个签名序列:IDLGTTYS、IFDLGGGTFD、VVLVGGSTRIPKIQK;核定位信号标签KRKHKKDISQNKRALRR;Dank特征基序DLGTT-S-V;胞质Hsp70特征基序EEVD;靠近C端的GGMP4肽序列;2个糖基化位点NKSI和NVSA。对所得基因序列与已发表的青锵(Oryzias latipes)等物种Hsp70基因的氨基酸序列进行同源性比较,发现两个奥利亚罗非鱼群体与莫桑比克罗非鱼最高99.4%,与牙鲆最低83.9%;系统进化树分析表明奥利亚罗非鱼的Hsp70 cDNA序列与青鳉等物种的Hsp70基因聚在一支,而与牙鲆的Hsp70基因相分离。     

Electron microscope evidence of virus infection in cultured marine fish

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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 matemal 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.     

淋巴囊肿病毒中国分离株基因组序列和结构分析

LCDV-cn是分离自中国养殖牙鲆的淋巴囊肿病病原,给中国牙鲆养殖业带来了严重危害。通过PCR、克隆和测序,获得了LCDV-cn部分基因的序列,证明LCDV-cn与LCDV-C的基因组序列是一致的。比较基因组学分析发现,LCDV-cn基因组中有137个ORF是其独有的、与LCDV-1及其它虹彩病毒无同源区域。对LCDV-cn基因组进行生物信息学分析发现,在LCDV-cn独有的ORF中,有的编码与宿主同源的蛋白,有的编码与DNA复制相关的蛋白,某些ORF编码的蛋白含有跨膜区等。LCDV-cn的独有基因或许对其毒力和宿主范围有关。     

黄喉拟水龟体表溃疡病原菌SG_(24)的分类鉴定

从患溃疡的黄喉拟水龟病灶中分离到一株病原菌SG24。对菌株SG24进行了常规生理生化测定和ATBExpression半自动细菌鉴定仪鉴定,并测定16S rRNA序列,分析其与相关细菌相应序列的同源性,构建了系统进化树。普通细菌学方法结果显示菌株SG24为黏质沙雷氏菌(Serratia marcescens)。以沙雷氏菌属的16S rRNA基因序列设计一对引物进行PCR扩增,获得了菌株SG24大小约950 bp的16S rRNA部分基因片段,测序结果显示菌株SG24与黏质沙雷氏菌同类,与已登录的黏质沙雷氏菌(S.marcescensDQ207558)的16S rRNA同源性大于99%。综合以上分类鉴定结果,确定菌株SG24属于沙雷氏菌属的黏质沙雷氏菌。药物敏感试验结果表明:菌株SG24对链霉素、庆大霉素、壮观霉素、强力霉素、卡那霉素、阿米卡星、诺氟沙星、氧氟沙星和复方新诺明敏感。     

Computational identification and characterization of microRNAs and their targets in Penaeus monodon

Study on shrimp miRNAs was limited and just 7 mature miRNA sequences of Marsupenaeus japonicus are deposited in mir Base database. In this study, miRNAs and their target gene candidates were computationally identified from shrimp Penaeu s monodon and then experimentally validated. Using 39 908 expressed sequence tags(ESTs) and 21 124 genome survey sequences(GSSs) of P. monodon(pmo) as reference dataset, a comprehensive approach based on inter-species homolog search was employed to investigate the candidate miRNAs(i.e. pmo-miRNA). A total of eight miRNAs belonging to 7 families were computationally identified and five out of them were subsequently validated by PCR and sequencing. Of these, pmo-miR-4961a, pmo-miR-4961b, pmo-miR-4979 and pmo-miR-3819 were first identified from shrimps. Both the mature pmo-miRNAs and the corresponding precursors were conserved among different species. Based on perfect or near-perfect match to the target region, the target gene candidates of pmomiRNAs were predicted from 10 331 mRNA sequences of P. monodon. A total of 20 genes were predicted as the targets of pmo-miR-4961a, pmo-miR-4961b, pmo-miR-4979 and pmo-miR-6492. Experimental validation by dual luciferase reporter assay confi rmed the targeting between 3 pmo-miRNAs and one or two of their target genes, especially the pmo-miR-4979 which could significantly down-regulate the expression of target gene(JR226772). This study updates the miRNAs and their targets in P. monodon and lays a solid foundation for future RNAi study.