Toxic effects of Karenia mikimotoi extracts on mammalian cells

Karenia is one of the most harmful and representative red tide genus in a temperate zone. Blooms caused by this genus have resulted in massive fish death in the South China Sea and the East China Sea. However, the potential effects of this dinoflagellate on human health through the transfer of toxins via marine food webs, and the mechanisms of toxicity, are still unknown. Therefore, we examined the toxic effects of a strain of K. mikimotoi (isolated from the South China Sea) on the proliferation and morphology of four mammalian cell lines (two normal cell lines and two cancer cell lines). In addition, we carried out a preliminary investigation on the mechanism of toxicity of the alga. The results show that the polar lipid-soluble component of K. mikimotoi significantly inhibited proliferation of the four cell lines, and resulted in the cells becoming spherical, swollen and damaged. The result of Annexin V and PI double-staining confirmed that cell membranes were disrupted. The malonaldehyde (MDA) contents in the medium of the four cell lines treated with the polar-lipid extracts all increased significantly, which indicates that the polar-lipid toxins produced by K. mikimotoi could adversely affect mammalian cells by inducing lipid peroxidation. We conclude that K. mikimotoi is a potential threat to human health, and the comprehensive effect of this dinoflagellate and its mechanisms should be investigated further.     

Preliminary Identification of Three New Isolates in Genus Alexandrium(Dinophyceae) from China Sea Area

1 Introduction Dinoflagellate Alexandrium has received intensive at-tention in recent years, as it is a valuable bioactive com-pound resource and related to harmful algal bloom (HAB)which has negative impact on marine ecology systemaquiculture, human health and tourism (Hallegraeff andBolch, 1992; Smayda, 1997; Vale and Antonia de MSampayo, 2001). However, not all of the species in genusAlexandrium produce toxin (Wendy et al., 2001), so rapididentification and clarification of the phyloge…     

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.     

Toxic effects of Karenia mikimotoi extracts on mammalian cells

Karenia is one of the most harmful and representative red tide genus in a temperate zone. Blooms caused by this genus have resulted in massive fish death in the South China Sea and the East China Sea. However, the potential effects of this dinoflagellate on human health through the transfer of toxins via marine food webs, and the mechanisms of toxicity, are still unknown. Therefore, we examined the toxic effects of a strain of K. mikimotoi (isolated from the South China Sea) on the proliferation and morphol...     

Preliminary identification of three new isolates in genus Alexandrium (Dinophyceae) from China sea area

The 5.8S ribosomal DNA sequences (5.8S rDNA) and their flanking regions, internal transcribed spacer 1 and spacer 2 (ITS1 and ITS2) of three new isolates in genus Alexandrium (Alexandrium sp. qd1, Alexandrium sp. qd2, Alexandrium sp. gz) from China were amplified, sequenced, and subjected to phylogenetic analysis. Alexandrium sp. gz and Alexandrium sp. qd1 were grouped with high bootstrap values with four strains/species, i.e., A. catenella South Korea strain, A. catenella Japan strain, and two from China, Alexandrium sp. AC03 and Alexandrium sp. AN01 being proposed to be A. catenalla in a previous study. Then Alexandrium sp. gz and Alexandrium sp. qd1 were identified as Alexandrium catenella. As A. catenella was isolated from Qingdao and Guangzhou sea areas, it supposedly distributed at least in these two areas and was genetically different. Alexandrium sp. qd2 differed greatly from species in Alexandrium. It clustered with Symbiodinium californium, Symbiodinium sp. G15 and Gymnodinium sp. Zhao 01 with 100% bootstrap value; so Alexandrium sp. qd2 affiliates to genus Symbiodinium, and is probably a free-living Symbiodinium species.     

Molecular identification based on ITS sequences for Kappaphycus and Eucheuma cultivated in China

The systematic classification of the Eucheumatoideae is difficult because of their variable morphology and interpretation of reproductive structures. Kappaphycus and Eucheuma specimens cultivated on the Hainan and Fujian coast of China were introduced from Vietnam, the Philippines and Indonesia. Combined with morphological characteristics, all Kappaphycus and Eucheuma cultivated strains were identified by internal transcribed spacer (ITS) sequences. The phylogenetic tree was constructed using neighbor-joining and maximum likelihood methods. The results indicate that different ITS sequence lengths occurred in the different genera and species. An obvious difference in morphology could be found in the protuberance shape between Kappaphycus and Eucheuma. The protuberance in Eucheuma was thorn-like and in Kappaphycus was wartlike or papillate. Their ITS sequence lengths differed significantly in nucleotide variation rates up to 58.55%-63.90%. All nucleotide variations occurred in the ITS1 and ITS2 regions except for five nucleotide transversions in the 5.8S rDNA region. In addition, the difference was at the branches among congeneric species. Kappaphycus sp. had branches with small buds, while K. alvarezii did not have such a feature. The nucleotide variation rates varied from 7.02% to 7.48% among species; within the same species of the clades it was <1.20%. Eucheumatoideae algae cultivated in China consisted of three clades, K. alvarezii, Kappaphycus sp., and E. denticulatum. The results indicate that ITS sequence analysis was an effective way for identification of interspecies and intraspecies phylogenetic relationships and might provide a clue for molecular identification of algal Eucheumatoideae.     

Monophyly or polyphyly? Possible conflict between morphological and molecular interpretations of the wellknown genus Zoothamnium(Ciliophora,Peritrichia)

In this paper,we explore possible conflict between morphological and molecular interpretations of phylogenetic relationships within the well-known peritrichous genus Zoothamnium. On the basis of morphological evidence,for a long time this genus has been believed to be a well-defined monophyletic taxon. Nonetheless,Z oothamnium exhibits higher genetic diversity than the gross morphology of its species. Here,we used all available genetic information for the small subunit of ribosomal RNA(SSU r RNA) and internal transcribed spacer region(ITS1-5.8S-ITS2) for this genus to reconstruct phylogenies for four datasets(SSU rRNA,ITS1,ITS2,and ITS1-5.8S-ITS2) and a combined dataset(SSU rRNA+ITS1-5.8SITS2) using different phylogenetic methods and with consideration of the secondary structure of the genes. Confidence in phylogenetic tree selection was assessed with the approximately unbiased test. The molecular results showed both that Zoothamnium is more likely to be polyphyletic,and morphologically similar genera Zoothamnopsis and Myoschiston were always nested among Zoothamnium species. Accordingly,as with some other groups of ciliates,to understand more fully the correct phylogeny of Zoothamnium there remains a need for additional data from both morphological and molecular studies,covering additional Zoothamnium spp. and members of closely related genera(e.g. Zoothamnopsis,Myoschiston,and Epistylis).     

Morphological and toxicity characteristics of Pseudo-nitzschia pungens strain PP0201-01 isolated from the East China Sea

The morphology and toxicity of Pseudo-nitzschia strain PP0201-01, isolated near the estuary of Changjiang （Yangtze） River in Year 2002 and tentatively identified as Pseudo-nitzschia pungens, were studied. The strain was identified morphologically as P. pungens using scanning electronic microscope （SEM）. The analytical results of LC-PDA （liquid chromatography with photodiode array detector）, LC-MS （liquid chromatography with mass detector）, and the toxicity data from mouse bioassay, showed that no domoic acid was produced by the strain. However, the mechanism of domoic acid production by Pseudo-nitzschia species is complicated. More works are needed to assess the toxic species and to evaluate the potential risk of amnesic shellfish poisoning （ASP） in China.     

Identification of two Skeletonema costatum-like diatoms by fluorescence in situ hybridization

A harmful algae bloom (HAB) is a dense aggregation of algae in a marine or aquatic environment that can result in significant environmental problems. To forecast the occurrence of HAB, development of a rapid and precise detection method is urgently required. In this study, two Skeletonema costatum-like diatoms (SK-1 and SK-2), were identified morphologically under a light microscope, and detected using fluorescent in situ hybridization (FISH). Strain SK-1 was isolated from a frequently HAB affected area of the East China Sea, and strain SK-2 from an aquatic farm in Qingdao, China. Fluorescent DNA probes were designed that were complementary to the ITS sequence (including 5.8S rDNA) of strain SK-1. After hybridization, strong green fluorescence was observed in cells of strain SK-1 under an epifluorescence microscope; however, no such fluorescence was observed with strain SK-2, which indicates that probes hybridized only the DNA of the target strain, SK-1, in species-specific manner, and that the two strains do not belong to a same species. This finding was confirmed by ITS sequence analysis. The FISH technique used in this study was sensitive, simple, and rapid, and is a promising tool for detecting target HAB species in natural environments.     

鱼干曲霉菌分离株鉴定及产毒特性

【目的】探明湛江市售鱼干中曲霉菌(Aspergillus spp.)分离株产AFB₁的特性。【方法】采用马铃薯-葡萄糖琼脂(PDA)对鱼干中的曲霉菌进行分离纯化,根据菌落形态、显微形态观察和ITS序列分析对分离株进行鉴定,采用液相色谱-串联质谱(LC-MS/MS)法分析菌株的AFB₁合成量。【结果】鱼干中曲霉菌的分离率高达47.66%,经形态学鉴定发现以黄曲霉(Aspergillus flavus)A03、烟曲霉(Aspergillus fumigatus)H32、杂色曲霉(Aspergillus versicolor)M23、欧式曲霉(Aspergillus europaeus)M11、棘孢曲霉(Aspergillus aculeatus)B21等为主,其中红笛鲷干(Lutjanus sanguinaus)中黄曲霉菌的含量高达1200 CFU/g。黄曲霉A03在红笛鲷干培养基中常温(27~30℃)培养21 d,产AFB₁能力达到3.8 ng/mL。【结论】鱼干中存在产AFB₁的黄曲霉菌,警示鱼干中存在AFB₁污染的风险。     

Relationships between two Pseudoalteromonas strains isolated from the Canada Basin and the Southern Ocean using a polyphasic approach

The evolutionary relationships and taxonomic position of two marine planktonic bacterial strains BSw20211 and BSw10014, isolated from the Canada Basin and from the Southern Ocean, respectively, were determined using a polyphasic taxonomic approach. There was a close phylogenetic relationship between the two strains and most phenotypic properties were shared. Nonetheless, they were found to belong to different species of the genus Pseudoalteromonas on the basis of genotypic analyses. Findings were consistent with the suggestion that gyrB gene sequence comparison and DNA-DNA relatedness might better define phylogenetic relationships of bacteria at the species level. However, a cut-off value of 90% gyrB gene sequence similarity was not reliable for the differentiation of species within the genus Pseudoalteromonas.     

Morphological and genetic comparison of two strains of a Prorocentrum species isolated from Zhejiang coastal water of China and Masan Bay of Korea

In this paper, we examined the detailed morphology of two strains of Prorocentrum isolated from the coastal waters of Zhejiang (Wenling area), China, and Masan Bay of Korea. A taxonomic comparison was made among strains on the basis of morphological and molecular data. The cellular dimensions of the Chinese Wenling strain (LAMB090508) and Korean strain (PDKS0206) were similar and the cells of both strains were of asymmetric and elongated shape. The posterior end of most cells was rounded. Megacytic zones of aged cells were broader with dense tiny knobs. The roundish nucleus was located in the posterior part of the cell. A few irregular shaped chloroplasts were distributed within the cell. The nucleotide similarity of the two strains, determined from the 5.8S rDNA-ITS sequences, was 99.83%. The comparative results of morphology and molecular analysis suggest that both strains isolated from China and Korea were the identical species, Prorocentrum donghaiense Lu.     

Molecular Phylogenetic Lineage of Plagiopogon and Askenasia (Protozoa,Ciliophora) Revealed by Their Gene Sequences

Prostomates and haptorians are two basal groups of ciliates with limited morphological characteristics available for taxonomy. Morphologically, the structures used to identify prostomates and haptorians are similar or even identical, which generate heavy taxonomic and phylogenetic confusion. In present work, phylogenetic positions lineage of two rare genera, Plagiopogon and Askenasia, were investigated. Three genes including small subunit ribosomal RNA gene(hereafter SSU r DNA), internal transcribed spacer region(ITS region), and large subunit ribosomal RNA gene(LSU r DNA) were analyzed, 10 new sequences five species each. Our findings included 1) class Prostomatea and order Haptorida are multiphyletic; 2) it may not be appropriate to place order Cyclotrichiida in subclass Haptoria, and the systematic lineage of order Cyclotrichiida needs to be verified further; 3) genus Plagiopogon branches consistently within a clade covering most prostomes and is basal of clade Colepidae, implying its close lineage to Prostomatea; and 4) Askenasia is phylogenetically distant from the subclass Haptoria but close to classes Prostomatea, Plagiopylea and Oligohymenophorea. We supposed that the toxicyst of Askenasia may be close to taxa of prostomes instead of haptorians, and the dorsal brush is a more typical morphological characteristics of haptorians than toxicysts.     

Morphological and toxicity characteristics of Pseudo-nitzschia pungens strain PP0201-01 isolated from the East China Sea

The morphology and toxicity ofPseudo-nitzschia strain PP0201-01, isolated near the estuary of Changjiang (Yangtze) River in Year 2002 and tentatively identified asPseudo-nitzschia pungens, were studied. The strain was identified morphologically asP. pungens using scanning electronic microscope (SEM). The analytical results of LC-PDA (liquid chromatography with photodiode array detector), LC-MS (liquid chromatography with mass detector), and the toxicity data from mouse bioassay, showed that no domoic acid was produced by the strain. However, the mechanism of domoic acid production byPseudo-nitzschia species is complicated. More works are needed to assess the toxic species and to evaluate the potential risk of amnesic shellfish poisoning (ASP) in China. Supported by the projects of Ministry of Science and Technology (No. 2001CB409704 and No. 2001BA804A20), and the innovation project of the Institute of Oceanology, CAS (No. 200223107).     

一株H_5亚型禽流感病毒血凝素基因的克隆与序列分析

根据GenBank中发表的H5亚型禽流感病毒HA基因序列设计2对引物,用RT-PCR方法从禽流感病毒广东分离病毒株(A/Chicken/Guangdong/1997)中扩增HA基因cDNA片段,并将其克隆至pMD-18T载体进行核苷酸序列测定。结果表明:用2对引物所扩增的片段大小分别约为1300 bp和800 bp,经序列拼接获得的HA基因cDNA长度约为1601 bp,编码533个氨基酸,与国内己发表的11个代表株的核苷酸和氨基酸序列同源性为分别为96.9%~99.9%和86.5%~93.0%;HA基因编码的氨基酸序列的系统进化树也表明A/Chicken/Guang-dong/1997、A/Goose/Huadong/01/2000、A/Ck/Hk/37.4/2002、A/Chicken/Zhoukou/2/02、A/Duck/Guangxi/53/2002、A/Duck/Fujian/01/2002等毒株处于同一进化枝,亲缘关系较近;而与A/Silly/Chicken/Hongkong/SF189/01株处于不同进化枝,亲缘关系较远。     

Genotype and Phylogenetic Diversity of <Emphasis Type="Italic">Symbiodinium</Emphasis> ITS2 Sequences Within Clade C in Three Typical Coral Species from Luhuitou Fringing Reef of the South China Sea

Dinoflagellates in the genus Symbiodinium, including nine clades (A–I), mainly form mutualistic symbioses with corals. More than 100 Symbiodinium molecular types have been identified by the ITS2-based genotype method within any given clade, and specifically within Symbiodinium clade C. However, the genotype identification method using the ITS2 sequence is likely to lead to high diversity estimates due to the intra-genomic variations in the ITS2 space; thus, further validation is essential for a correct identification. In this study, the molecular diversity of Symbiodinium ITS2 sequences cloned from two stone corals, Acropora sp. SY-01 and Pocillopora sp. SY-05, and one soft coral, Sarcophyton sp. SY-07, living in the northern part of South China Sea (SCS), were analyzed and compared using the ITS2-based genotype identification method, coupled with ITS2-based secondary structural and phylogenetic analyses. As the result, 12 Symbiodinium ITS2 genotypes were identified, while only six and three Symbiodinium ITS2 genotypes were supported by ITS2-based secondary structural and phylogenetic analyses, respectively. In addition, no shared Symbiodinium ITS2 genotypes were observed among the three coral species, suggesting coral species-dependent Symbiodinium genotypes were within clade C. In summary, the present study provides a theoretical basis for validating the molecular diversity of Symbiodinium ITS2 genotypes in corals.     

Occurrence of Bothriocephalus acheilognathi (Cestoda, Bothriocephallidea) in grass carp Ctenopharyngodon idella in the Changjiang River drainage

Bothriocephalus acheilognathi is a potentially serious pathogen in wild or cultured fish in worldwide distribution. We examined 58-farmed grass carp from Nanchang in the Changjiang (Yangtze) River drainage, from which 20.7% were found to harbor the parasite with an infection intensity of 36.9±54.7. The parasites were identified based on morphology and rDNA ITS sequence analysis. The present report represents the first record of the parasite in grass carp Ctenopharyngodon idella in the river drainage.     

Application of rRNA probes and fluorescence in situ hybridization for rapid detection of the toxic dinoflagellate Alexandrium minutum

The dinoflagellate Alexandrium minutum is often associated with harmful algal blooms (HABs). This species consists of many strains that differ in their ability to produce toxins but have similar morphology, making identification difficult. In this study, species-specific rRNA probes were designed for whole-cell fluorescence in situ hybridization (FISH) to distinguish A. minutum from two phylogenetic clades. We acquired the complete SSU to LSU rDNA sequences (GenBank accession numbers JF906989-JF906999) of 11 Alexandrium strains and used these to design rRNA targeted oligonucleotide probes. Three ribotype-specific probes, M-GC-1, M-PC-2, and M-PC-3, were designed. The former is specific for the GC clade ("Global clade") of A. minutum, the majority of which have been found non-toxic, and the latter two are specific for the PSP (paralytic shellfish poisoning)-producing PC clade ("Pacific clade"). The specificity of these three probes was confirmed by FISH. All cells in observed fields of view were fluorescently labeled when probes and target species were incubated under optimized FISH conditions. However, the accessibility of rRNA molecules in ribosomes varied among the probe binding positions. Thus, there was variation in the distribution of positive signals in labeled cells within nucleolus and cytosol (M-GC-1, M-PC-3), or just nucleolus (M-PC-2). Our results provide a methodological basis for studying the biogeography and population dynamics of A. minutum, and providing an early warning of toxic HABs.