Yessotoxin detected in mussel (Mytilus californicus) and phytoplankton samples from the U.S. west coast

Yessotoxin (YTX) was detected in an algal sample and two mussel samples (0.07–0.10 μg g⁻¹) collected from Scripps Pier in La Jolla, California during a bloom of Lingulodinium polyedrum. Mussel samples collected from Monterey Bay, California also contained measurable YTX (levels up to 0.06 μg g⁻¹) in samples obtained during a 6-month (weekly) sampling period. Gonyaulax spinifera and L. polyedrum were identified in background concentrations in Monterey Bay during the time of contamination. An algal sample from Washington coastal waters collected during non-bloom conditions also contained YTX, possibly originating from Protoceratium reticulatum.Three strains of L. polyedrum (CCMP1931, CCMP1936, 104A) isolated from southern California coastal waters and one strain of G. spinifera (CCMP409) isolated from Maine were tested for YTX production using two methods, competitive ELISA and liquid chromatography–mass spectrometry (LC–MS). The ELISA method detected YTX in the particulate phase in two of three L. polyedrum strains. The LC–MS method did not detect YTX in the particulate or dissolved phase of any of the strains.To our knowledge, this is the first study to test and confirm YTX in environmental samples from California and Washington coastal waters. It is highly likely that L. polyedrum was responsible for the YTX contamination in the southern California samples. Future research needs to conclusively determine the biological origin(s) of YTX contamination in central California and Washington coastal waters.     

Species-specific probe, based on 18S rDNA sequence, could be used for identification of the mucilage producer microalga Gonyaulax fragilis (Dinophyta)

In the Adriatic Sea, the correlation between mucilage phenomena and the presence of Gonyaulax fragilis (Schütt) Kofoid (Dinophyta) has been recently demonstrated. The application of PCR-based methods and the development of species-specific molecular probes might represent powerful technologies for rapid and specific monitoring of microalgal species in seawater samples. Here, we report sequencing of the small subunit (SSU) ribosomal RNA gene (18S rDNA) of G. fragilis and its comparative analysis within the Dinophyta. Total DNAs were extracted and amplified from cultured cells of G. fragilis, which were isolated from natural phytoplanktonic association in the northern Adriatic Sea. Total 18S rDNA gene was amplified using 16S1N and 16S2N primers and sequenced using ad hoc designed internal primers. The primers amplified a product of expected size (length 1700/1800 bp). The phylogenetic analysis carried out by comparing G. fragilis sequence to homologous sequences of Lingulodinium polyedrum (Stein) Dodge, Gonyaulax spinifera (Claparède et Lachmann) Diesing, Protoceratium reticulatum (Claparède et Lachmann) Bütschli revealed a great nucleotide divergence of G. fragilis SSU sequence. Therefore, the SSU sequence could be used as species-specific marker for the identification of this mucilage producer microalga. In addition, such sequence could be used as target to design oligonucleotide probes for the construction of DNA microchips as diagnostic tool for the routine monitoring of harmful algae in seawater. An erratum to this article is available at .     

Effects of Protoceratium reticulatum yessotoxin on feeding rates of Acartia hudsonica: A bioassay using artificial particles coated with purified toxin

Paralytic shellfish toxins produced by dinoflagellates are known to deter copepod grazing. Dinoflagellate species, including Protoceratium reticulatum, also produce disulfated polyether yessotoxins that were previously referred to as diarrheic shellfish toxins. However, the role of yessotoxins in predator–prey relationships is not yet clear. In the present study, the effects of purified yessotoxin (YTX) on feeding activities of Acartia hudsonica (Copepoda, Calanoida) were experimentally investigated. Polystyrene fluorescent microspheres (10 μm in diameter) colored bright blue or yellow-green were coated with cell extracts of P. reticulatum that do not produce yessotoxins. The bright blue microspheres were further coated with YTX, and the yellow-green microspheres were used as the reference. The microspheres were then given to the copepods separately or in combination to measure clearance rates and feeding selectivity. A. hudsonica was found to feed on the yellow-green microspheres without YTX at twice the rate of the bright blue microspheres with YTX. We also confirmed that microsphere color per se did not affect the feeding rates. The bright blue microspheres adsorbed 1.8–43.3 pg of YTX per microsphere, which is similar to the cell-specific yessotoxin content of toxic P. reticulatum found in natural environments. These results suggest that production of yessotoxin is advantageous for P. reticulatum by deterring predation by copepods.     

Influence of temperature, salinity and nutrient limitation on yessotoxin production and release by the dinoflagellate Protoceratium reticulatum in batch-cultures

The toxic dinoflagellate Protoceratium reticulatum (Claparède & Lachmann) Buetschli is recurrently present in the Adriatic sea. It is the producing organism of yessotoxin (YTX) and some of its analogues and thus its presence in seawater often results in shellfish farm closure for long periods. However, molluscs become highly toxic also at the presence of low cell concentrations, due to the high YTX content present in most algal strains. As no data were available on the environmental conditions favouring growth and YTX production by Adriatic P. reticulatum strains, in the present work, we investigated the effect of nutrient limitation, salinity and temperature on growth and YTX content in P. reticulatum cultures. Liquid chromatography–mass spectrometry (LC–MS) analyses were carried out to determine YTX production as well as the difference between the YTX amount retained in cells and that released in growth medium, in order to relate cell content to excretion mechanisms. The toxin content was determined in cells collected at the stationary phase, since both toxin production and release were found to be higher in this growth stage than in the exponential phase. As for nutrient-effect, a severe P-limitation strongly affected cell growth and favoured toxin accumulation, as consequences of both impaired cell division and lower toxin release. N-limited cultures, on the contrary, had a toxin content similar to controls and the highest percentage of release. P. reticulatum was confirmed to be tolerant towards salinity changes as it could grow at salinity values in the range of 22–42. The highest YTX production was observed at intermediate salinity values (32) whereas toxin release, expressed as percentage of the total amount produced, decreased as salinity increased. P. reticulatum growth was impaired in cultures kept at 26 °C in respect to those grown at 16 and 20 °C. YTX release decreased as temperature increased; however, cells kept at 26 °C displayed a very high YTX content. The environmental implications of these physiological behaviours highlight that farmed molluscs can become less toxic in colder waters at lower salinity values.     

Molecular identification of toxic Alexandrium tamiyavanichii (Dinophyceae) using two DNA probes

To improve labeling-intensity of whole-cell fluorescence in situ hybridization (FISH) in the molecular identification of toxic Alexandrium tamiyavanichii, two DNA probes (TAMID2 plus TAMIS1 designed from the LSU and SSU rDNA regions, respectively) were used to test the labeling intensity of targeted cultured A. tamiyavanichii cells. The cross-reactivity of the DNA probe to natural seawater samples and six Alexandrium species: A. affine, A. catenella, A. fraterculus, A. insuetum, A. pseudogonyaulax and A. tamarense, was also tested. The labeling intensity of the DNA probe TAMID2S1, a combination of two separate probes that target different regions of the rRNA, was 1.7–2.7 times higher than that of the single DNA probe TAMID2. With cultured A. tamiyavanichii cells in the dead growth phase at 30 days, the TAMID2S1 intensity was 1.9 times higher than that of TAMID2. During a 30-day culture, the labeling intensity of A. tamiyavanichii cells hybridized with TAMID2S1 decreased to 49.4% of the original intensity. No cross-reactivity to various microorganisms in natural seawater samples was found. The two DNA probes together, designated as TAMID2S1, readily detected A. tamiyavanichii added to natural seawater samples, even aged cultured cells.     

Diversity of nodule-endophytic agrobacteria-like strains associated with different grain legumes in Tunisia

This study represents the first report describing the genetic diversity of nodule-endophytic agrobacteria isolated from diverse legumes and their phylogenetic relationships with the valid species of agrobacteria, as well as the non-recognized genomospecies of the former Agrobacterium tumefaciens (Rhizobium radiobacter). The genetic diversity of a collection of 18 non-nodulating agrobacteria-like strains, previously isolated from root nodules of Vicia faba, Cicer arietinum and Phaseolus vulgaris from different geographical regions of Tunisia, was studied by REP-PCR and PCR-RFLP of the 16S-23S rDNA IGS, as well as by sequence analysis of the 16S rDNA and the housekeeping genes recA and atpD. The aim of the work was to study the genetic diversity of the different isolates and to check for any host-specificity. The results from the different techniques were congruent and suggested a specific interaction for P. vulgaris, whereas no specific endophytic interaction was observed for V. faba and C. arietinum. The phylogenetic analysis clearly indicated that some isolates were affiliated to R. radiobacter or to its non-recognized genomic species (genomovars G2, G4 and G9). However, the other isolates probably constitute new species within Rhizobium (Agrobacterium) and Shinella.     

Development of a qualitative PCR method for the Alexandrium spp. (Dinophyceae) detection in contaminated mussels (Mytilus galloprovincialis)

Paralytic shellfish poisoning (PSP) is a syndrome caused by the consumption of shellfish contaminated with neurotoxins produced by organisms of the marine dinoflagellate genus Alexandrium. A. minutum is the most widespread species responsible for PSP in the Western Mediterranean basin. The standard monitoring of shellfish farms for the presence of harmful algae and related toxins usually requires the microscopic examination of phytoplankton populations, bioassays and toxin determination by HPLC. These procedures are time-consuming and require remarkable experience, thus limiting the number of specimens that can be analyzed by a single laboratory unit. Molecular biology techniques may be helpful in the detection of target microorganisms in field samples. In this study, we developed a qualitative PCR assay for the rapid detection of all potentially toxic species belonging to the Alexandrium genus and specifically A. minutum, in contaminated mussels. Alexandrium genus-specific primers were designed to target the 5.8S rDNA region, while an A. minutum species-specific primer was designed to bind in the ITS1 region. The assay was validated using several fixed seawater samples from the Mediterranean basin, which were analyzed using PCR along with standard microscopy procedures. The assay provided a rapid method for monitoring the presence of Alexandrium spp. in mussel tissues, as well as in seawater samples. The results showed that PCR is a valid, rapid alternative procedure for the detection of target phytoplankton species either in seawater or directly in mussels, where microalgae can accumulate.     

Flavobacterium frigidimaris sp. nov., isolated from Antarctic seawater

We described the polyphasic characterization of the psychrotolerant isolated from Antarctic seawater. The strain was closely related to Flavobacterium hydatis, F. pectinovorum, and F. saccharophilum on the basis of the 16S rDNA sequence analysis. However, DNA–DNA hybridization experiments showed that the DNA-similarities between strain KUC-1^T and the reference strains of Flavobacterium were less than 30%. Therefore, we can definite a new species of Flavobacterium phylogenetically, and strain KUC-1^T can be considered to be a new species of Flavobacterium. i.e. F. frigidimaris (KUC-1^T: JCM 12218^T and DSM 15937^T; mol% G+C of DNA of the type strain is 34.5 mol%). Useful phenotypical features for discrimination of F. frigidimaris from other Flavobacterium species, such as a resistance to NaCl, optimum growth temperature, and cellular fatty acid composition, were also determined.     

Phylogenetic analysis of the <Emphasis Type="Italic">lux</Emphasis> operon distinguishes two evolutionarily distinct clades of <Emphasis Type="Italic">Photobacterium leiognathi</Emphasis>

The luminous marine bacterium Photobacterium mandapamensis was synonymized several years ago with Photobacterium leiognathi based on a high degree of phenotypic and genetic similarity. To test the possibility that P. leiognathi as now formulated, however, actually contains two distinct bacterial groups reflecting the earlier identification of P. mandapamensis and P. leiognathi as separate species, we compared P. leiognathi strains isolated from light-organ symbiosis with leiognathid fishes (i.e., ATCC 25521^T, ATCC 25587, lequu.1.1 and lleuc.1.1) with strains from seawater originally described as P. mandapamensis and later synonymized as P. leiognathi (i.e., ATCC 27561^T and ATCC 33981) and certain strains initially identified as P. leiognathi (i.e., PL-721, PL-741, 554). Analysis of the 16S rRNA and gyrB genes did not resolve distinct clades, affirming a close relationship among these strains. However, strains ATCC 27561^T, ATCC 33981, PL-721, PL-741 and 554 were found to bear a luxF gene in the lux operon (luxABFE), whereas ATCC 25521^T, ATCC 25587, lequu.1.1 and lleuc.1.1 lack this gene (luxABE). Phylogenetic analysis of the luxAB(F)E region confirmed this distinction. Furthermore, ATCC 27561^T, ATCC 33981, PL-721, PL-741 and 554 all produced a higher level of luminescence on high-salt medium, as previously described for PL-721, whereas ATCC 25521^T, ATCC 25587, lequu.1.1 and lleuc.1.1 all produced a higher level of luminescence on low-salt medium, a characteristic of P. leiognathi from leiognathid fish light organs. These results demonstrate that P. leiognathi contains two evolutionarily and phenotypically distinct clades, P. leiognathi subsp. leiognathi (strains ATCC 25521^T, ATCC 25587, lequu.1.1 and lleuc.1.1), and P. leiognathi subsp. mandapamensis (strains ATCC 27561^T, ATCC 33981, PL-721, PL-741 and 554).Electronic Supplementary Material Supplementary material is available for this article at .     

Development of Molecular Identification Method for Genus Alexandrium (Dinophyceae) Using Whole-Cell FISH

We have developed a method to identify species in the genus Alexandrium using whole-cell fluorescent in situ hybridization with FITC-labeled oligonucleotide probes that target large subunit ribosomal rRNA molecules. The probes were designed based on the sequence of the rDNA D1-D2 region of Alexandrium species. DNA probes specific for toxic A. tamarense and A. catenella and nontoxic A. affine, A. fraterculus, A. insuetum, and A. pseudogonyaulax, respectively, were applied to vegetative cells of all above Alexandrium species to test the sensitivity of the probes. Each DNA probe hybridized specifically with vegetative cells of the corresponding Alexandrium species and showed no cross-reactivity to noncorresponding Alexandrium species. In addition, no cross-reactivity of the probes was observed in experiments using concentrated natural seawater samples. The TAMAD2 probe, which is highly specific to A. tamarense, a common toxic species in Korean coastal waters, provides a simple and reliable molecular tool for identification of toxic Alexandrium species.     

一株产虫草素的冬虫夏草内生真菌 Bionectria ochroleuca OS17

虫草素是一种核苷类似物,具有多种药理作用。利用虫草素产生菌发酵的方式获得虫草素,可能是产生虫草素的一种新方法。为筛选能产虫草素的内生真菌,从中国传统的中药冬虫夏草子实体中共分离得到42株真菌,经PDB液体培养后,采用高效液相色谱（HPLC）鉴定其代谢产物。结果表明：菌株OS17的代谢产物中含有虫草素,且虫草素产量为40.16 mg/L。基于形态学特征和ITS序列分析,菌株OS17被鉴定为淡色生赤壳菌(Bionectria ochroleuca)。这是首次从冬虫夏草中分离得到能产虫草素的生赤壳属,表明了内生真菌具有产生多种生物活性物质的潜力。     

Isolation of microbial pathogens of subclinical mastitis from raw sheep's milk of Epirus (Greece) and their role in its hygiene

The natural raw milk microflora is a factor that expresses its sensorial characteristics. The microbial charge into the mammary gland of healthy animal is low and the application of right and healthy conditions during milking and cheese making procedure, prevents from contaminating as well as maintains the natural microflora in order to lend the particular characteristics of milk.The purpose of the present project was the study of the Total Viable Count (T.V.C.) and the count of total psychrotropic bacteria of raw sheep milk from Boutsiko and Karamaniko breeds, collected from healthy animals, as well as the isolation, identification and enumeration of pathogenic bacteria related with the hygiene and the quality of raw sheep milk (with a particular interest in bacteria that may cause human infection).During the experiment we examined two hundred forty (240) samples of raw sheep milk. In these samples a) Staphylococcus aureus, Salmonella sp., Escherichia coli, Clostridium perfringens (vegetative cells and spores) and Bacillus sp. were isolated and identified b) the Total Viable Count and the total number of psychrotropic bacteria were also specified. The sampling, the preparation of samples and decimal dilutions were based on international methods. The Total viable count was determined using the standard methods of the American Public Health Association, 2002. The total number of psychrotropic bacteria was determined using APHA 1976, 1978 rules. The identification of the bacteria was carried out according to the Bergey’s manual. Microscopic examination of Gram stained cells, catalase, oxidase and biochemical tests were performed when necessary to further identify.From the 240 milk samples tested, only 5% were E. coli positive, with mean counts ranged from 2 × 10³ to 2.4 × 10⁴ cfu/ml. S. aureus was isolated from 24% of the samples and the mean count per ml was ranged from <10 to 3.4 × 10². Meanwhile, Bacillus spp. was also detected in 29% samples. Vegetative forms and spores of C. perfringens were detected in 13% and 63% of the samples respectively. However, microbiological analyses revealed the presence of a small number of selected pathogens in milk samples such as Salmonella, which was only detected in 5% of the samples. Listeria sp., Pseudomonas sp. and Vibrio cholerae were never found.From the experimental results, the Total Viable Count from raw sheep milk samples, fulfils the microbiological criteria of EU Legislation in a percentage of approximately 97%.     

Identification and characterization of lactic acid bacteria in ragi tape

One hundred and eighteen lactic acid bacteria (LAB) were isolated from five different types of ragi tape, a traditional dry-starter of Balinese rice wine. The isolates could be classified into three groups based on the cell shape and capability to produce gas from glucose. Group I contained 66 homofermentative cocci, group II contained seven homofermentative rods, and group III contained 45 heterofermentative rods. Among these 118 isolates, 21 isolates representing these groups were selected and were first identified using phenotypic characters. The identification performed phenotypically was confirmed by sequencing of variable region 8 (V8) of the 16S rDNA. The comparative studies led to the identification of Pediococcus pentosaceus, Enterococcus faecium, Lactobacillus curvatus, Weissella confusa, and W. paramesenteroides from the ragi tape examined.     

Population dynamics of alien gammarid species in the River Odra estuary

During the period of 1988–2000, three alien gammarid species, i.e. Gammarus tigrinus Sexton, 1939, Pontogammarus robustoides G. O. Sars, 1894, and Dikerogammarus haemobaphes (Eichwald, 1841) were recorded in the Odra estuary. This study, the first record of G. tigrinus and P. robustoides in Poland, focuses on the distribution, abundance, and biomass as well as on population structure of the three new species found in the coastal zone of the estuary. G. tigrinus and P. robustoides were first sampled in the Szczecin Lagoon (mid-part of the estuary) in 1988. The densities of both species were high from the time they were first recorded and exceeded 10 000 ind. m^–2, while the biomass was 25 and 140 g m^–2, respectively. G.tigrinus occurred at higher densities in northern and eastern parts of the Lagoon, while P. robustoides was particularly abundant in the south-eastern part, most heavily affected by River Odra discharge. In 1999, the third gammarid, D. haemobaphes, was recorded in the southern part of the estuary. Both in 1999 and 2000, the species was found in qualitative samples (dredge collections of Dreissena polymorpha) taken from the western branch of the river. As estimated from quantitative samples, densities and biomass in the eastern branch were rather low and did not exceed 150 ind. m^–2 and 2.7 g m^–2, respectively. The Szczecin Lagoon population of G. tigrinus produces at least two generations within a year: a summer one and an overwintering one. The latter is dominated by older and larger specimens. The species may reproduce in the Lagoon from April until November.     

Occurrence and distribution of virulent strains of Vibrio parahaemolyticus in seafoods marketed from Cochin (India)

This study was aimed for the detection of Vibrio parahaemolyticus by biochemical and molecular methods in seafood samples collected from the markets of Cochin located at the southwest coast of India. A total of seventy-two V. parahaemolyticus cultures were isolated by selecting sucrose and cellobiose non-fermenting colonies. All the biochemically confirmed strains were found to have 368-bp toxR gene fragment, while an additional 24% of the samples were confirmed as V. parahaemolyticus by toxR based polymerase chain reaction (PCR) from enrichment broths. PCR based methods are used to detect tdh, trh, and orf8 genes for the identification of pathogenic and pandemic V. parahaemolyticus. Only one out of two urease positive isolates amplified the trh (500bp) gene. About 10% of the isolates showed weak haemolysis and none were found to amplify tdh (269 bp) and orf8 (746 bp) genes, thus indicating the meager incidence of pandemic strains from this area. The incidence of trh positive isolates from market samples signals towards the adoption of stringent seafood safety measures for the products meant for human consumption.     

Identification and quantification of indole-3-acetic acid in the kelp Laminaria japonica Areschoug and its effect on growth of marine microalgae

A method was established for the identification and quantification of indole-3-acetic acid (IAA) in extracts of the kelp Laminaria japonica. An IAA content of 90–95 μg kg⁻¹ fresh weight in kelp extract was determined by high performance liquid chromatography (HPLC). IAA identification was based on a combination of co-chromatography and comparative chromatography with a standard, analysis of UV spectra, and atmospheric pressure electrospray mass spectrometry (APESI-MS). IAA was isolated by silica gel chromatography and HPLC. The effect on the growth of four marine microalgae of the pure IAA isolated from kelp extract was investigated. Exogenously added IAA from kelp enhanced the growth of Chlorella sp., Dunaliella salina and Porphyridium cruentum, but not that of Chaetoceros muelleri. IAA from kelp significantly inhibited the accumulation of soluble cellular proteins in Chlorella sp. and P. cruentum, and had a very significant effect on chlorophyll biosynthesis in Chlorella sp. However, there was no obvious effect of IAA on the regulation of biosynthesis of cellular polysaccharides in these four marine microalgae.     

北细辛不同部位内生真菌的多样性与抑菌活性

为探讨北细辛(Asarum heterotropoides)不同部位可培养内生真菌种群结构的差异,采用植物组织平板分离法分离北细辛根、根状茎、越冬芽三个部位内生真菌,结合形态学和分子生物学方法鉴定菌株,并系统分析和比较其种类和多样性。从北细辛三个部位共分离得到117株内生真菌,其中97株归属于6纲6目8科9属17种。相似性和多样性分析表明,北细辛不同部位内生真菌的组成结构上存在差异,根部内生真菌的多样性指数最高,越冬芽最低。采用菌饼法检测其抑菌活性,G 3-3、G 4-10-1、G 2-2-1、GZJ 2-12-2对金黄色葡萄球菌(Staphylococcus aureus)有抑制作用,G 3-3、G 2-2-1、GZJ 2-12-2、GZJ 1-6、G 3-8对枯草芽胞杆菌(Bacillus subtilis)有抑制作用。GZJ 2-12-2对金黄色葡萄球菌、大肠埃希菌(Escherichia coli)、枯草芽胞杆菌均有抑制作用。     

Theileria ovis discovered in China

A polymerase chain reaction (PCR) using 989/990 primers was conducted to identify a newly isolated Theileria sp. in Xinjiang Province of China. The target DNA fragments of the complete 18S rRNA gene were cloned and sequenced. The phylogenetic relationship of newly isolated Theileria spp. was inferred based on the 18S rRNA gene. The results showed that the new Theileria sp. belonged to the cluster of Theileria ovis. Moreover, the findings were confirmed by T. ovis species-specific PCR. An expected 520 bp fragment of T. ovis DNA was obtained from 25 out of 320 (8%) field blood samples, and blood of an experimental sheep infested by Hyalomma anatolicum anatolicum collected in Xinjiang. The infection rate of T. ovis was 78% (25/32) in Xinjiang province. The investigation did not find T. ovis positive samples from the field samples collected from the other twelve provinces. This study indicates that T. ovis is prevalent in Xinjiang province of China and its transmission vector is H. anatolicum anatolicum.