Sterols of the syndinian dinoflagellate Amoebophrya sp., a parasite of the dinoflagellate Alexandrium tamarense (Dinophyceae)

Several harmful photosynthetic dinoflagellates have been examined over past decades for unique chemical biomarker sterols. Little emphasis has been placed on important heterotrophic genera, such as Amoebophrya, an obligate, intracellular parasite of other, often harmful, dinoflagellates with the ability to control host populations naturally. Therefore, the sterol composition of Amoebophrya was examined throughout the course of an infective cycle within its host dinoflagellate, Alexandrium tamarense, with the primary intent of identifying potential sterol biomarkers. Amoebophrya possessed two primary C(27) sterols, cholesterol and cholesta-5,22Z-dien-3beta-ol (cis-22-dehydrocholesterol), which are not unique to this genus, but were found in high relative percentages that are uncommon to other genera of dinoflagellates. Because the host also possesses cholesterol as one of its major sterols, carbon-stable isotope ratio characterization of cholesterol was performed in order to determine whether it was produced by Amoebophrya or derived intact from the host. Results indicated that cholesterol was not derived intact from the host. A comparison of the sterol profile of Amoebophrya to published sterol profiles of phylogenetic relatives revealed that its sterol profile most closely resembles that of the (proto)dinoflagellate Oxyrrhis marina rather than other extant genera.     

Isolation and alga-inhibiting characterization of Vibrio sp. BS02 against Alexandrium tamarense

The bacterium BS02 which is closely related to the genus Vibrio sp. and capable of inhibiting the toxic dinoflagellate Alexandrium tamarense was isolated from a mangrove area in Zhangjiangkou, Fujian Province, China. The bacterium was not species-specific since it displayed varying degrees of lysing activities against eight of the eighteen algae tested. There was a close interaction between initial bacterial and A. tamarense cell densities, indicating that algal growth was prompted at low bacterial concentrations, while the number of the alga cells was reduced at high concentrations. Alga-lysing characterization of Vibrio sp. BS02 suggested that the alga-lysing substance was extracellularly produced, less than 500 in molecular weight, as well as non proteinaceous, stable under wide range of temperature and pH conditions, UV radiation, repeated freezing and thawing and heavy metal treatments. These findings suggested that BS02 could play an important role in controlling harmful algal blooms.     

Genomic Insights into the Saccharomyces sensu stricto Complex

The Saccharomyces sensu stricto group encompasses species ranging from the industrially ubiquitous yeast Saccharomyces cerevisiae to those that are confined to geographically limited environmental niches. The wealth of genomic data that are now available for the Saccharomyces genus is providing unprecedented insights into the genomic processes that can drive speciation and evolution, both in the natural environment and in response to human-driven selective forces during the historical “domestication” of these yeasts for baking, brewing, and winemaking.     

Genomic Insights into the Emerging Human Pathogen Mycobacterium massiliense

Mycobacterium massiliense (Mycobacterium abscessus group) is an emerging pathogen causing pulmonary disease and skin and soft tissue infections. We report the genome sequence of the type strain CCUG 48898.     

Ultrastructure of Amoebophrya sp. and its changes during the course of infection

Amoebophrya is a syndinian parasite that kills harmful bloom forming algae. Previously uncharacterized ultrastructural aspects of infection and development were elucidated. The biflagellate dinospore has two mitochondria, electron-dense bodies, striated strips, trichocysts, and a nucleus with peripherally condensed chromatin. After finding an Akashiwo sanguinea host and adhering to its surface, the parasite penetrates the host surface, apparently using a microfilament based motility and electron-dense bodies within a microtubular basket in the process of parasitophorous vacuole membrane formation. After entering the host nucleus, possibly by a similar mechanism used to enter the host cell, the parasite cytosol expanded substantially prior to mitosis. From 12-36 hours mitochondria were inconspicuous but present. Chromatin condensation was variable. By 36 hours post-infection, parasites had multiple nuclei, a microtubule-supported cytopharynx, and were beginning to form a fully internal mastigocoel. By 48 hours, the characteristic "beehive" appearance was apparent with flagella projecting into a fully developed mastigocoel. The cytoplasm contained trichocysts, elongated mitochondria, and nuclei with peripherally condensed chromatin. Although Amoebophrya lacks an apical complex, its electron-dense bodies show functional similarities to apicomplexan rhoptries. Its lack of permanently condensed chromosomes, but compact dinospore chromatin, supports the idea that dinoflagellate permanently condensed chromosomes may be a remnant of a parasitic ancestor with a compact dispersal stage.     

Production of C2 toxin by Alexandrium tamarense CI01 using different culture methods

The dinoflagellate Alexandrium tamarense CI01 wasgrown in three types of cultures: batch culture, semi-continuous culture andtemporary culture, to investigate the effects of different culture methods oncell growth and the productivity of C2 toxin (C2, a paralytic shellfish toxin).In the batch cultures, cells grew in three phases: a short lag phase, anexponential phase with a specific growth rate () of 0.78day^–1 and a relatively long stationary phase. Themaximum toxin productivity was 1.2 mol L^–1 or77 fmol cell^–1 in 9 days. In the semi-continuouscultures, cells grewin response to the dilution cycles, with values being 0.64, 0.32 and 0.35day^–1 for one-day, two-day and three-day cycles,respectively. The toxin yield was about one half of that of the batch cultures.A "temporary" culture method was used to maintain the metabolically activecellsremoved from the semi-continuous cultures, in a nutrient-depleted condition, toachieve a high toxin productivity of 1.0 molL^–1 in 4 days. Thus,the semi-continuous culture method provided an efficient means to generateamounts of metabolically active algal cells. The temporary culture offered aneffective way to produce C2. The highest yields of C2 were obtained in3–4days when the temporary cultures were aerated.     

Improvement in growth and toxin production of Alexandrium tamarense by two-step culture method

The growth and toxin content of the dinoflagellate Alexandrium tamarense ATHK was markedly affected by culture methods. In early growth phase at lower cell density static or mild agitation methods were beneficial to growth, but continuous agitation or aeration, to some extent, had an adverse effect on cell growth. Static culture in 2 L Erlenmeyer flasks had the highest growth rate (0.38 d⁻¹) but smaller cell size compared with other culture conditions. Cells grown under aerated conditions possessed low nitrogen and phosphorus cell yields, namely high N and P cell-quota. At day 18, cells grown in continuous agitated and 1 h aerated culture entered the late stationary phase and their cellular toxin contents were higher (0.67 and 0.54 pg cell⁻¹) compared with cells grown by other culture methods (0.27–0.49 pg cell⁻¹). The highest cell density and cellular toxin content were 17190 cells mL⁻¹ and 1.26 pg cell⁻¹ respectively in an airlift photobioreactor with two-step culture. The results indicate that A. tamarense could be grown successfully in airlift photobioreactor by a two-step culture method, which involved cultivating the cells statically for 4 days and then aerating the medium. This provides an efficient way to enhance cell and toxin yield of A. tamarense.     

Bacterial Populations in Subgingival Plaque Under Healthy and Diseased Conditions: Genomic Insights into Oral Adaptation Strategies by Lactobacillus sp. Strain DISK7

Indian Journal of Microbiology - Human oral cavity is a complex habitat comprising about 700 microbial species and represents the most complex microbiota after gastrointestinal tract. In fact, oral...     

Single-cell analysis of paralytic shellfish toxins in Alexandrium tamarense by HPLC with post-column fluorescent derivatization

We developed a methodology for analyzing the C-toxin (C2) content in single Alexandrium tamarense cells; this method was based on high performance liquid chromatography (HPLC). C2 is the main paralytic shellfish toxin (PST) detected in a clonal culture of A. tamarense, which is a common causative organism in cases of paralytic shellfish poisoning in Japan. This HPLC method employs post-column fluorescent derivatization (FL). Mobile phase, column size, flow rate, reagent concentrations, and lamp type for the fluorescent detector were all optimized for the detection of C2. With this improved methodology, we could measure 1 fmol of C2 with a signal to noise ratio (S/N) = 2. Clonal heterogeneity within the toxic strain, which was maintained for 13 years after re-isolation from the original clonal culture, ranged from <1 fmol to 700 fmol cell⁻¹. This report is the first to demonstrate definitively that PST content varies on a cell-by-cell basis in a clonal culture of a dinoflagellate that causes paralytic shellfish poisoning.     

Implications of life-history transitions on the population genetic structure of the toxigenic marine dinoflagellate Alexandrium tamarense

Genotypic or phenotypic markers for characterization of natural populations of marine microalgae have typically addressed questions regarding differentiation among populations, usually with reference to a single or few clonal isolates. Based upon a large number of contemporaneous isolates from the same geographical population of the toxigenic species Alexandrium tamarense from the North Sea, we uncovered significant genetic substructure and low but significant multilocus linkage disequilibrium (LD) within the planktonic population. Between the alternative molecular genotyping approaches, only amplified fragment length polymorphism (AFLP) revealed cryptic genetic population substructure by Bayesian clustering, whereas microsatellite markers failed to yield concordant patterns. Both markers, however, gave evidence for genetic differentiation of population subgroups as defined by AFLP. A considerable portion of multilocus LD could be attributed to population subdivision. The remaining LD within population subgroups is interpreted as an indicator of frequency shifts of clonal lineages during vegetative growth of planktonic populations. Phenotypic characters such as cellular content and composition of neurotoxins associated with paralytic shellfish poisoning (PSP) and allelochemical properties may contribute to intra- or inter-annual differentiation of planktonic populations, if clonal lineages that express these characters are selectively favoured. Nevertheless, significant phenotypic differentiation for these characters among the genetically differentiated subgroups was only detected for PSP toxin content in two of the four population subgroups. By integrating the analysis of phenotypic and genotypic characteristics, we developed a conceptual population genetic model to explain the importance of life-cycle dynamics and transitions in the evolutionary ecology of these dinoflagellates.     

Genomic Insights into the Evolutionary Origin of Xanthomonas axonopodis pv. citri and Its Ecological Relatives

Xanthomonas axonopodis pv. citri (Xac) is the causal agent of citrus bacterial canker (CBC) and is a serious problem worldwide. Like CBC, several important diseases in other fruits, such as mango, pomegranate, and grape, are also caused by Xanthomonas pathovars that display remarkable specificity toward their hosts. While citrus and mango diseases were documented more than 100 years ago, the pomegranate and grape diseases have been known only since the 1950s and 1970s, respectively. Interestingly, diseases caused by all these pathovars were noted first in India. Our genome-based phylogenetic studies suggest that these diverse pathogens belong to a single species and these pathovars may be just a group of rapidly evolving strains. Furthermore, the recently reported pathovars, such as those infecting grape and pomegranate, form independent clonal lineages, while the citrus and mango pathovars that have been known for a long time form one clonal lineage. Such an understanding of their phylogenomic relationship has further allowed us to understand major and unique variations in the lineages that give rise to these pathovars. Whole-genome sequencing studies including ecological relatives from their putative country of origin has allowed us to understand the evolutionary history of Xac and other pathovars that infect fruits.     

Species-Specific Detection and Quantification of Toxic Marine Dinoflagellates Alexandrium tamarense and A. catenella byReal-Time PCR Assay

A Real-time polymerase chain reaction (PCR) assay was designed and evaluated for rapid detection and quantification of the toxic dinoflagellates Alexandrium catenella and A. tamarense, which cause paralytic shellfish poisoning. Two sets of PCR primers and fluorogenic probes targeting these two species were derived from the sequence of 28S ribosomal DNA. PCR specificity was examined in closely related Alexandrium spp. and many other microalgae. A. catenella–specific primers and probe detected the PCR amplification only from A. catenella strains, and nonspecific signals were not detected from any microalgae. Also, A. tamarense–specific primers and probe also detected the targeted species, suggesting the strict species specificity of each PCR. This assay could detect one cell of each species, showing its high sensitivity. Moreover, using the developed standard curves, A. tamarense and A. catenella could be quantified in agreement with the quantification by optical microscopy. The performance characteristics of species specificity, sensitivity, and rapidity suggest that this method is applicable to the monitoring of the toxic A. tamarense and A. catenella.     

Infection of marine copepods by Metschnikowia sp.

Summary It was observed that yeasts belonging to the genusMetschnikowia parasitize marine copepods both under natural and experimental conditions. The physiological activity ofMetschnikowia sp. was investigated under a variety of conditions and the importance of these organisms in the ecology of the Strait of Georgia has been discussed.     

Development of specific rRNA probes to distinguish between geographic clades of the Alexandrium tamarense species complex

The globally occurring Alexandrium tamarense/fundyense/catenellaspecies complex consists of toxic and non-toxic strains thatare morphologically difficult to distinguish. We developed fourspecific ribosomal RNA probes that can identify the entire speciescomplex, the strains of the toxic North American clade and thestrains of the two non-toxic clades from Western Europe andthe Mediterranean Sea by DNA dot blot and fluorescence in situhybridization. These probes are a first step for the developmentof an early warning system for the presence of A. tamarense.     

A Quantitative Assessment of the Role of the Parasite Amoebophrya in the Termination of Alexandrium fundyense Blooms within a Small Coastal Embayment

Parasitic dinoflagellates of the genus Amoebophrya infect free-living dinoflagellates, some of which can cause harmful algal blooms (HABs). High prevalence of Amoebophrya spp. has been linked to the decline of some HABs in marine systems. The objective of this study was to evaluate the impact of Amoebophrya spp. on the dynamics of dinoflagellate blooms in Salt Pond (MA, USA), particularly the harmful species Alexandrium fundyense. The abundance of Amoebophrya life stages was estimated 3–7 days per week through the full duration of an annual A. fundyense bloom using fluorescence in situ hybridization coupled with tyramide signal amplification (FISH- TSA). More than 20 potential hosts were recorded including Dinophysis spp., Protoperidinium spp. and Gonyaulax spp., but the only dinoflagellate cells infected by Amoebophrya spp. during the sampling period were A. fundyense. Maximum A. fundyense concentration co-occurred with an increase of infected hosts, followed by a massive release of Amoebophrya dinospores in the water column. On average, Amoebophrya spp. infected and killed ∼30% of the A. fundyense population per day in the end phase of the bloom. The decline of the host A. fundyense population coincided with a dramatic life-cycle transition from vegetative division to sexual fusion. This transition occurred after maximum infected host concentrations and before peak infection percentages were observed, suggesting that most A. fundyense escaped parasite infection through sexual fusion. The results of this work highlight the importance of high frequency sampling of both parasite and host populations to accurately assess the impact of parasites on natural plankton assemblages.     

Pan-Genomic Analysis Provides Insights into the Genomic Variation and Evolution of Salmonella Paratyphi A

Salmonella Paratyphi A (S. Paratyphi A) is a highly adapted, human-specific pathogen that causes paratyphoid fever. Cases of paratyphoid fever have recently been increasing, and the disease is becoming a major public health concern, especially in Eastern and Southern Asia. To investigate the genomic variation and evolution of S. Paratyphi A, a pan-genomic analysis was performed on five newly sequenced S. Paratyphi A strains and two other reference strains. A whole genome comparison revealed that the seven genomes are collinear and that their organization is highly conserved. The high rate of substitutions in part of the core genome indicates that there are frequent homologous recombination events. Based on the changes in the pan-genome size and cluster number (both in the core functional genes and core pseudogenes), it can be inferred that the sharply increasing number of pseudogene clusters may have strong correlation with the inactivation of functional genes, and indicates that the S. Paratyphi A genome is being degraded.