棕囊藻属的分类现状

棕囊藻属藻类生活史复杂、地理差异显著、游动单细胞个体微小,至今尚无明确的分类标准。在目前已报道的9个种中,Sournia(1988)认为只有两个种比较可靠,即形成胶群体的P.pouchetii(sensu lato)(包括P.globosa)和不形成胶群体的P.scrobiculata;一些学者将P.puchetii(sensu lato)进一步细分成P.pouchetii(sensu stricto)、P.globosa,P.antarctica等3种,为方便起见,多数学者将形成胶群体的棕囊藻定名为P.pouchetii,或写成未定种的形式Phaeocystis sp.棕囊藻属分类的混乱制约了相关研究的深入,解决棕囊藻分类混乱问题有赖于新的技术和方法的使用。     

棕囊藻属(Phaeocystis)的分类与生活史(综述)

棕囊藻属Phaeocystis（定鞭藻纲Prymnesiophyceae)的分类问题目前还有争论。其种的分类标准是以初始的群体形态、地理分布、细胞特征等以及分子生物学特征,如染色体倍性,基因组大小等为依据。基于以上各种分类特征,目前比较确定的棕囊藻属藻类有四种：一种是只观察到单细胞形态的凹孔棕囊藻（P.scrobiculata),另外三种是能够形成群体的波切棕囊藻（P.pouchetii)、球形棕囊藻（P.globosa)和南极棕囊藻（P.antarctica)。棕囊藻具有一个复杂的异形生活史,介于几种游离的单细胞（不动的细胞,具有鞭毛的动细胞,小游动孢子以及可能存在的大游动孢子）和群体之间的形态交替。但其生活史中仍有许多不确定的问题。     

MOLECULAR EVIDENCE IDENTIFIES BLOOM-FORMING PHAEOCYSTIS (PRYMNESIOPHYTA) FROM COASTAL WATERS OF SOUTHEAST CHINA AS PHAEOCYSTIS GLOBOSA

Chen, Y. Q., Zhou, H. & Qu, L. H. Key Laboratory of Gene Engineering of Education Ministry, Biotechnology Research Center, Zhongshan University, Guangzhou 510275, P. R. China Sequence data from the 18S small subunit ribosomal RNA gene and rDNA ITS regions have been used to identify the species of a Phaeocystis (Prymnesiophyta) that caused harmful algae blooms in the coastal waters of southeast China. This Phaeocystis has morphological and physiological features that differ from those previously described for either P. globosa Scherf- fel or P. pouchetii (Hariot)Lagerheim. However, the sequence comparison of the Phaeocystis 18S rDNA and rDNA ITS clearly showed that it was remarkably similar to several isolates of P. globosa. Thus, the species isolated from the southeast coast of China is identified as P. globosa rather than P. cf. pouchetii or another species. Our results also demonstrate that phenotypes of different members of the genus Phaeocystis are variable, apparently changing in response to environmental conditions. It is concluded that, on the basis of this phylogenetic analysis, the bloom forming southeast China coast species of Phaeocystis most likely originated from an endemic warm-water, rather than a foreign source.     

A Comparison of Digoxigenin and Biotin Labelled DNA and RNA Probes for in Situ Hybridization

A number of in situ hybridization protocols using digoxigenin or biotin labelled probes were assessed for viral nucleic acid detection in formalin fixed, paraffin embedded tissue. Single-step detection protocols for biotin labelled probes produced low sensitivity; however, enzyme based one-step detection protocols for digoxigenin probes produced high sensitivity for both RNA and DNA systems. For both probe types, multistep detection protocols produced equally high sensitivity. Use of an enhanced APAAP procedure for digoxigenin labelled probes acheived maximal sensitivity without use of biotin-streptavidin reactions. The sensitivity of nucleic acid detection obtained with a digoxigenin labelled probe is comparable to that obtained using biotin. Digoxigenin labelled probes for nucleic acid detection are recommended for tissues with endogenous biotin.     

Evaluation of the specificity of five oligoprobes for identification of cyathostomin species from horses

Here, we report evaluation of five oligoprobes designed from intergenic spacer (IGS) region sequences for identification of cyathostomin species. Oligoprobes were designed for identification of Cylicocyclus ashworthi, Cylicocyclus nassatus, Cylicostephanus longibursatus, Cylicostephanus goldi and a fifth probe designed to identify all members of this tribe. PCR amplification of IGS DNA from 16 cyathostomin species allowed sequence comparison and identification of four putative species-specific probes. Southern blotting of amplified products from 16 species showed that all probes were species-specific. The fifth probe recognised all 16 cyathostomin species but did not bind to members of the genus Strongylus. Furthermore, these probes were used to identify individual infective L3, eggs and L4 indicating that they will be invaluable to furthering the study of the epidemiology and pathogenesis of these important equine nematodes.     

PCR-derived ssDNA probes for fluorescent in situ hybridization to HIV-1 RNA.

We developed a simple and rapid technique to synthesize single-stranded DNA (ssDNA) probes for fluorescent in situ hybridization (ISH) to human immunodeficiency virus 1 (HIV-1) RNA. The target HIV-1 regions were amplified by the polymerase chain reaction (PCR) and were simultaneously labeled with dUTP. This product served as template for an optimized asymmetric PCR (one-primer PCR) that incorporated digoxigenin (dig)-labeled dUTP. The input DNA was subsequently digested by uracil DNA glycosylase, leaving intact, single-stranded, digoxigenin-labeled DNA probe. A cocktail of ssDNA probes representing 55% of the HIV-1 genome was hybridized to HIV-1-infected 8E5 T-cells and uninfected H9 T-cells. For comparison, parallel hybridizations were done with a plasmid-derived RNA probe mix covering 85% of the genome and a PCR-derived RNA probe mix covering 63% of the genome. All three probe types produced bright signals, but the best signal-to-noise ratios and the highest sensitivities were obtained with the ssDNA probe. In addition, the ssDNA probe syntheses generated large amounts of probe (0.5 to 1 microg ssDNA probe per synthesis) and were easier to perform than the RNA probe syntheses. These results suggest that ssDNA probes may be preferable to RNA probes for fluorescent ISH. (J Histochem Cytochem 48:285-293, 2000)     

Haplorchis pumilio and H. taichui in Vietnam discriminated using ITS-2 DNA sequence data from adults and larvae

Samples of Haplorchis taichui and Haplorchis pumilio of different life-stages (cercariae, metacercariae and adults) and from different host species (snail, fish, dog, cat and human) were collected in Nghe An and Nam Dinh Provinces in Vietnam. Samples from Thailand were available for comparison. All adults and metacercariae were initially identified using morphological criteria. Polymerase chain reaction (PCR) assays were developed for discriminating between the species. The complete sequence for the nuclear ribosomal internal transcribed spacer-2 (ITS-2) was obtained from one adult and one metacercaria of H. taichui and three adults and three metacercariae of H. pumilio from Vietnam. Sequences from cercariae from three different snails clustered with those of H. pumilio. Intra-individual variation in the ITS-2 region was detected by sequencing of cloned PCR products. These are the first sequences from Vietnamese Haplorchis spp. to be reported and demonstrate that H. taichui and H. pumilio can be identified unambiguously from any life-stage, including the cercarial stage that is difficult to identify using morphology. Discrepancies in the literature are discussed and examples of apparent misidentification highlighted. The data provide a resource to assist in taxonomic studies on heterophyids, in the design of probes for diagnosis and for field surveys to identify infection in snails.     

Phylogenetic analysis of members of the Phycodnaviridae virus family, using amplified fragments of the major capsid protein gene

Algal viruses are considered ecologically important by affecting host population dynamics and nutrient flow in aquatic food webs. Members of the family Phycodnaviridae are also interesting due to their extraordinary genome size. Few algal viruses in the Phycodnaviridae family have been sequenced, and those that have been have few genes in common and low gene homology. It has hence been difficult to design general PCR primers that allow further studies of their ecology and diversity. In this study, we screened the nine type I core genes of the nucleocytoplasmic large DNA viruses for sequences suitable for designing a general set of primers. Sequence comparison between members of the Phycodnaviridae family, including three partly sequenced viruses infecting the prymnesiophyte Pyramimonas orientalis and the haptophytes Phaeocystis pouchetii and Chrysochromulina ericina (Pyramimonas orientalis virus 01B [PoV-01B], Phaeocystis pouchetii virus 01 [PpV-01], and Chrysochromulina ericina virus 01B [CeV-01B], respectively), revealed eight conserved regions in the major capsid protein (MCP). Two of these regions also showed conservation at the nucleotide level, and this allowed us to design degenerate PCR primers. The primers produced 347- to 518-bp amplicons when applied to lysates from algal viruses kept in culture and from natural viral communities. The aim of this work was to use the MCP as a proxy to infer phylogenetic relationships and genetic diversity among members of the Phycodnaviridae family and to determine the occurrence and diversity of this gene in natural viral communities. The results support the current legitimate genera in the Phycodnaviridae based on alga host species. However, while placing the mimivirus in close proximity to the type species, PBCV-1, of Phycodnaviridae along with the three new viruses assigned to the family (PoV-01B, PpV-01, and CeV-01B), the results also indicate that the coccolithoviruses and phaeoviruses are more diverged from this group. Phylogenetic analysis of amplicons from virus assemblages from Norwegian coastal waters as well as from isolated algal viruses revealed a cluster of viruses infecting members of the prymnesiophyte and prasinophyte alga divisions. Other distinct clusters were also identified, containing amplicons from this study as well as sequences retrieved from the Sargasso Sea metagenome. This shows that closely related sequences of this family are present at geographically distant locations within the marine environment.     

The nuclear rDNA region of Gyrodactylus arcuatus and G. branchicus (Monogenea: Gyrodactylidae)

The primary structure of the ribosomal DNA internal transcribed spacers (ITS-1 and ITS-2) and 5.8S rRNA gene were used to characterize and identify 2 monogenean species of Gyrodacrylus living externally on the threespine stickleback (Gasterosteus aculeatus). The ITS region was amplified by PCR from freshwater, brackish, and marine isolates of Gyrodactylus arcuatus and G. branchicus, and the ends of the coding regions were identified by comparative alignment. No intraspecific and very low interspecific variation were observed in the 5.8S rRNA gene; high inter- and low intraspecific variation were revealed in the ITS-1 and ITS-2 regions. The morphological species identification was in all cases confirmed by the molecular identification. Intraspecifically, samples from 2 locations in the North Sea could be differentiated, but the Baltic sample resembled North Sea genotypes. Our approach offers perspectives for a multimetric genetical, morphometrical, and ecological taxonomy of the genus Gyrodactylus.     

Characterization of Fasciola samples by ITS of rDNA sequences revealed the existence of Fasciola hepatica and Fasciola gigantica in Yunnan Province, China

On mainland China, liver flukes of Fasciola spp. (Digenea: Fasciolidae) can cause serious acute and chronic morbidity in numerous species of mammals such as sheep, goats, cattle, and humans. The objective of the present study was to examine the taxonomic identity of Fasciola species in Yunnan province by sequences of the first and second internal transcribed spacers (ITS-1 and ITS-2) of nuclear ribosomal DNA (rDNA). The ITS rDNA was amplified from 10 samples representing Fasciola species in cattle from 2 geographical locations in Yunnan Province, by polymerase chain reaction (PCR), and the products were sequenced directly. The lengths of the ITS-1 and ITS-2 sequences were 422 and 361-362 base pairs, respectively, for all samples sequenced. Using ITS sequences, 2 Fasciola species were revealed, namely Fasciola hepatica and Fasciola gigantica. This is the first demonstration of F. gigantica in cattle in Yunnan Province, China using a molecular approach; our findings have implications for studying the population genetic characterization of the Chinese Fasciola species and for the prevention and control of Fasciola spp. in this province.     

Colony size, cell number, carbon and nitrogen contents of Phaeocystis pouchetii from western Norway

Phaeocystis pouchetii is an ecologically important colony-formingmarine phytoplankton species in northern hemisphere cold waters.It plays a central role in the processing of biogeochemicallyimportant elements in the upper ocean during spring blooms.Here, we report highly significant quantitative relationshipsamong colony size, cell number and particulate carbon and nitrogencontents of field populations of P. pouchetii, which providethe means to quantitatively convert its biological expressioninto units of biogeochemical significance. Populations weresampled from mesocosms incubated in situ in western Norway andeither fertilized with nitrate and phosphate or left unamended.Phaeocystis colony blooms developed in both scenarios, but weremuch greater in fertilized treatments. Colonies from the lattertreatments were larger, contained higher concentrations of cellsand more particulate carbon and nitrogen than those in the unfertilizedmesocosm. Considering all data, log cell number increased linearlywith log colony volume with a slope of 0.54. Log carbon andnitrogen increased with log colony volume, with respective slopesof 0.92 and 1.22, indicating a significant component of non-cellularcarbon and nitrogen within the colonies. Carbon and nitrogencontents of colonies were linearly related, and fertilized coloniescontained more nitrogen relative to carbon than unfertilizedcolonies. These equations are particularly applicable to contemporarystudies of P. pouchetii because they represent colonies growingin environments that mimic a continuum from natural to eutrophicatedecosystems.     

FLOW CYTOMETRIC ANALYSES OF VIRAL INFECTION IN TWO MARINE PHYTOPLANKTON SPECIES, MICROMONAS PUSILLA (PRASINOPHYCEAE) AND PHAEOCYSTIS POUCHETII (PRYMNESIOPHYCEAE)

Cell characteristics of two axenic marine phytoplankton species, Micromonas pusilla (Butscher) Manton et Parke and Phaeocystis pouchetii (Hariot) Lagerheim, were followed during viral infection using flow cytometry. Distinct differences between noninfected and infected cultures were detected in the forward scatter intensities for both algal species. Changes in side scatter signals on viral infection were found only for P. pouchetii. Chlorophyll red fluorescence intensity per cell decreased gradually over time in the infected cultures. DNA analyses were performed using the nucleic acid–specific fluorescent dye SYBR Green I. Shortly after infection the fraction of algal cells with more than one genome equivalent increased for both species because of the replication of viral DNA in the infected cells. Over time, a population of algal cells with low red autofluorescence and low DNA fluorescence developed, likely representing algal cells just prior to viral lysis. The present study provides insight into basic virus–algal host cell interactions. It shows that flow cytometry can be a useful tool to discriminate between virus infected and noninfected phytoplankton cells.     

Isolation of Leishmania (Viannia) braziliensis from Lutzomyia spinicrassa (species group Verrucarum) Morales Osorno Mesa, Osorno and Hoyos 1969, in the Venezuelan Andean region

Natural infection with Leishmania spp. in phlebotomine sandflies was searched for during a longitudinal study carried out from July 1997 to July 1998 in the village Catarnica, Municipality Independencia, Táchira State. This hamlet is an old endemic focus of cutaneous leishmaniasis in the Venezuelan Andean region, which lies close to the Colombian border at 1,300 m a.s.l., in an agricultural area mainly used for cultivating coffee. Phlebotomine sandflies were collected using Shannon traps placed in the peridomestic habitat from 19:00 to 21:00 hs. Males were stored in alcohol 70 % while females were kept in Nunc vials with 10 % DMSO and cryopreserved in liquid nitrogen for subsequent dissection and identification. The most abundant anthropophilic species was Lutzomyia spinicrassa with 3,032 males and 4,290 females (85.4%). Among 1,633 (38%) females of Lu. spinicrassa dissected, 26 11.6%) were infected with promastigotes, while no natural infection was found in 209 females of other species. The flagellates were identified as Leishmania braziliensis braziliensis using PCR with species specific primers derived from nuclear DNA and hybridization using species specific probe labelled with digoxigenin. This parasite had been previously isolated from patients with cutaneous leishmaniasis from the same area. These results show Lu. spinicrassa as a new proven vector of Leishmania braziliensis in the Andean region of Venezuela.     

Identification of repetitive, genome-specific probes in crucifer oilseed species.

Direct amplification of minisatellite DNA by PCR (DAMD PCR) was used to amplify and subsequently clone several fragments of DNA from crucifer species. The PCR-derived fragments of DNA were generated using known minisatellite core sequences as PCR primers. Southern hybridization of these putative minisatellite DNA fragments revealed that many were genome-specific; they hybridized with high affinity only to the genomic DNA of the species from which they were cloned. The DNA fragments were believed to be dispersed in the genome, based on smear-like hybridization signals on EcoRI-, BamHI-, and HindIII-digested genomic DNA. Genome-specific probes were specifically isolated from Brassica rapa (A genome), Brassica nigra (B genome), and Sinapis alba in addition to several other crucifer species. The sequence of a B. rapa specific probe (pBr17.1.3A) contained a minisatellite region that could be divided into three tandem repeats; each repeat contained between two and five subrepeats and each subrepeat shared a highly conserved core region of 29 bp. This minisatellite sequence also hybridized with high affinity to the A genome species B. napus and B. juncea. This research showed that dispersed, genome-specific probes can be isolated using DAMD PCR and that these probes could be used to detect and quantify alien DNA present in progeny from intergeneric or interspecific crosses.     

Specificity of six gene sequences for the detection of the genus Brucella by DNA amplification

M. DA COSTA, J.-P. GUILLOU, B. GARIN-BASTUJI, M. THIÉBAUD AND G. DUBRAY. 1996. DNA extracted from all Brucella species, reference and vaccine strains were amplified by PCR using primers specific for the genes encoding a 31-kDa Brucella protein, the heat shock proteins (DnaJ, DnaK, HtrA and GroEL) and 16S RNA. No difference was found between Brucella species and biovars with all primer pairs used, even after restriction enzyme analysis of the amplified fragments. The specificity of the amplified products was confirmed by hybridization with a digoxigenin 3'-labelled specific probe and by PCR using 98 non- Brucella micro-organisms' DNA. Only Ochrobactrum anthropi and Phyllobacterium spp. yielded a PCR product by using 31-kDa DnaK, DnaJ, GroEL and 16S RNA primers. After hybridization and restriction analysis, 16S RNA fragments of 3301 and 3331 O. anthropi strains showed a total similarity to those from Brucella. A similar result was shown with DnaJ fragments obtained with 3301 strain of O. anthropi after Eco RI digestion.     

Differential diagnosis of Trichostrongylus and hookworm eggs via PCR using ITS-1 sequence

Trichostrongylus eggs observed in cellophane-thick smears are difficult, in practice, to distinguish from hookworm eggs. In order to overcome these limitations, a molecular approach was conducted. A Trichostrongylus colubriformis adult worm was obtained from a human in Laos, which was identified morphologically. ITS-1 sequence of this worm was determined, and found to be most similar with that of T. colubriformis among the Trichostrongylus spp. reported so far. Then, this sequence was compared with those of human hookworm species, Ancylostoma duodenale and Necator americanus, and species-specific oligonucleotide primers were designed. Polymerase chain reaction (PCR) using these primers evidenced specifically amplified PCR products of Trichostrongylus sp., A. duodenale and N. americanus from the eggs of each (520 bp, 690 bp, and 870 bp, respectively). A species-specific PCR technique can be developed in order to study the epidemiology of Trichostrongylus spp. and hookworms in endemic areas.     

Direct molecular analysis of the fragile X syndrome in a sample of Egyptian and German patients using non-radioactive PCR and Southern blot followed by chemiluminescent detection

Molecular genetic analysis of individuals from 6 Egyptian and 33 German families with fragile X syndrome and 240 further patients with mental retardation was performed applying a completely non-radioactive system. The aim of our study was the development of a non-radioactive detection method and its implementation in molecular diagnosis of the fragile X syndrome. Furthermore, we wanted to assess differences in the mutation sizes between Egyptian and German patients and between Egyptian and German carriers of a premutation. Using non-radioactive polymerase chain reaction (PCR), agarose gel electrophoresis and blotting of the PCR products, followed by hybridisation with a digoxigenin-labelled oligonucleotide probe (CGG)₅ and chemiluminescent detection, we identified the fragile X full mutation (amplification of a CGG repeat in the FMR-1 gene ranging from several hundred to several thousand repeat units) in all patients. We observed no differences in the length of the CGG repeat between the Egyptian and German patients and carriers, respectively. However, in one prenatal diagnosis, we detected only one normal sized allele in a female fetus using the PCR-agarose assay, whereas Southern blot analysis with the digoxigenin labelled probe StB 12.3 revealed presence of a full mutation. Our newly established nonradioactive genomic blotting method is based on the conventional radioactive Southern blot analysis. Labelling of the probe StB 12.3 with digoxigenin via PCR allowed the detection of normal, premutated and fully mutated alleles. For exact sizing of small premutated or large normal alleles, we separated digoxigenin labelled PCR products through denaturing poly-acrylamide gelelectrophoresis (PAGE) and transfered them to a nylon membrane using a gel dryer. The blotted PCR-fragments can easily be detected with alkaline phosphate-labelled anti-digoxigenin antibody. The number of trinucleotide repeat units can be determined by scoring the detected bands against a digoxigenated M13 sequencing ladder. Our newly developed digoxigenin/chemiluminescence approach using PCR and Southern blot analysis provides reliable results for routine detection of full fragile X mutations and premutations.     

A low-density oligonucleotide array study for parallel detection of harmful algal species using hybridization of consensus PCR products of LSU rDNA D2 domain

A low-density oligonucleotide array approach based on the hybridization of consensus PCR products of LSU rDNA was developed in order to simultaneously detect various harmful algae. A set of oligonucleotide probes for the hybridization of specific LSU rDNA D2 regions was developed for the identification of 10 representative harmful microalgae. Each probe was spotted onto a streptoavidin-coated glass slide by pipetting. Universal primers were designed within the conserved regions adjacent to the D2 regions of all harmful algae and used to PCR amplify the complete D2 regions. The PCR products were hybridized to the oligonucleotides arrayed on the slide. The array produced unique hybridization patterns for each species of harmful algae and allowed us to differentiate the closely related species. Furthermore, we were able to simultaneously detect several predominant HAB species from a mixture of culture strains and from a natural sample. These results show that DNA microarray can be a new technical platform for parallel discrimination of harmful algae and has great potential to alter the manner in which researchers monitor these microorganisms.