Development and application of fluorescence in situ hybridization (FISH) method for simple and rapid identification of the toxic dinoflagellates Alexandrium tamarense and Alexandrium catenella in cultured and natural seawater

ABSTRACT:   The toxic dinoflagellates Alexandrium tamarense (Lebour) Balech and A. catenella (Whedon and Kofoid) Balech produce potent neurotoxins, such as saxitoxin and gonyautoxin and have been mainly responsible for paralytic shellfish poisoning (PSP) in Japan. To prevent a negative effect on the fishery industry, it is necessary to identify these toxic species precisely and rapidly before and during the bloom. In this paper, a rapid and simple protocol of a fluorescence in situ hybridization (FISH) method using ribosomal RNA (rRNA)-targeted probes has been established for identifying the cultured strains and natural cells of A. tamarense and A. catenella . Using the FISH method established in this study, it was possible to identify these toxic species species-specifically and rapidly, within 30 min. The procedure of detection constituted three steps: (i) fixation/dehydration; (ii) hybridization; and (iii) washing; this made the identification simple. Moreover, this method did not require either special techniques or equipment, and the cost for detection was low. The specificity, rapidity, and simplicity of the developed method suggest that it might be useful for routine monitoring of these toxic microalgae.     

Isolation and culture of rabbit primordial germ cells

Primordial germ cells (PGCs) are embryonic precursors of the gametes of adult animals and are considered stem cells of the germline. Since their proliferation in vitro correlates well with the schedule of developmental changes in vivo, they might be interesting research tools for genomic imprinting, germ-cell tumors and fertility. Furthermore, once primordial germ cells are separated and placed on a feeder layer with cytokines, they become cultured pluripotent cell lines called embryonic germ (EG) cells. EG cells share several important characteristics with embryonic stem (ES) cells as they can also contribute to the germ line of chimeras. To investigate the characteristics of PGCs and establish rabbit EG (rEG) cells, we cultured rabbit PGCs (rPGCs) in vitro with various combinations of leukemia inhibitory factor (LIF), basic fibroblast growth factor (bFGF) and forskolin on inactivated mouse embryonic fibroblast (MEF) feeder layers. The present study found PGC proliferation in early cultures and induction of rEG-like colonies. These cells expressed pluripotent markers, such as alkaline phosphatase activity, OCT-4, Sox-2 and SSEA-1, in the undifferentiated state; however, the cells did not develop into a teratoma when injected into the kidney capsules of SCID mice, although the restricted differentiation potentials to neural cells were determined via embryoid body formation. From these characteristics and further characterization of the germ stem cell markers Vasa, SCP-1 and SCP-3, we suggested that these were hybrid cells with characteristics somewhere between PGC and EG cells.     

Molecular Characterization and Diagnosis of QoI Resistance in Cucumber and Eggplant Fungal Pathogens

ABSTRACT The molecular mechanism of QoI fungicide resistance was studied using isolates of cucumber Corynespora leaf spot fungus (Corynespora cassiicola) and the eggplant leaf mold (Mycovellosiella nattrassii). In both pathogens, a mutation at position 143 from glycine to alanine (G143A) was detected in the cytochrome b gene that encodes for the fungicide-targeted protein. Moreover, the nucleotide sequence at amino acid position 143 was converted from GGT or GGA in sensitive (wild-type) to GCT or GCA in resistant (mutant-type) isolates. The methods of polymerase chain reaction restriction fragment length polymorphism commonly used for QoI resistance monitoring were employed successfully, leading to the amplified gene fragment from resistant isolates being cut with the restriction enzyme ItaI. However, heteroplasmy (the coexistence of wild-type and mutated alleles) was found when the resistant isolates of C. cassiicola, M. nattrassii, and Colletotrichum gloeosporioides (strawberry anthracnose fungus) were subcultured in the presence or absence of QoI fungicides. QoI resistance of cucumber powdery and downy mildew isolates persisted for a few years following the removal of the selection pressure imposed by the fungicide under both laboratory and commercial greenhouse conditions. The proportion of mutated sequences in cytochrome b gene decreased over time in the pathogen population. The protective efficacy of the full dose of azoxystrobin decreased when the populations of powdery and downy mildews contained resistant isolates at 10%. Using FMBIO, a fluorescence bio-imaging analyzer, the mutant allele from the QoI-resistant isolates could be detected at the level of 1%, whereas the detection sensitivity of ethidium-bromide-stained gels was approximately 10 times lower.     

‘Niigata S3’ is a new strawberry cultivar suitable for forcing culture under low temperature and insolation conditions

‘Niigata S3’ is a new strawberry (Fragaria × ananassa Duch.) cultivar that is early flowering and possesses high soluble solid content and good coloration. It was selected from a cross between Kei812 (seed parent) and ‘Asuka-Ruby’ (pollen parent). The first harvest date of ‘Niigata S3’ was December 27, 34 days earlier than ‘Echigohime’ and 9 days earlier than ‘Asuka-Ruby’ (means of 2007 and 2008). The marketable yield of ‘Niigata S3’ was 85% of ‘Echigohime’, 107% of ‘Asuka-Ruby’, while the early yield was 145% of ‘Echigohime’, 85% of ‘Asuka-Ruby’ (based on 2007 and 2008 means). The shape of the fruit is long conical, and its skin color medium-red. The fruit skin hardness of ‘Niigata S3’ was 31.5 g/mm², which was harder than ‘Echigohime’, and its average soluble solid content was 11.4%, which was higher than the values for ‘Echigohime’ and ‘Asuka-Ruby’ (2008). Furthermore, ‘Niigata S3’ did not bear apical overripe fruit. This new cultivar is adaptable to the climatic conditions of Niigata, as well as other regions that experience low winter temperatures and insolation.     

Interannual variability in stock abundance of the neon flying squid, Ommastrephes bartramii, in the North Pacific Ocean during 1979–1998: impact of driftnet fishing and oceanographic conditions

Variability in catch-per-unit-effort (CPUE) was examined for the autumn cohort of Ommastrephes bartramii collected with research driftnets during 1979–1998 along five longitudinal transects passing through the Subarctic, Transitional and Subtropical Domains in the North Pacific. CPUE was generally low during the period of intensive commercial driftnet fishing (1980–1992) and increased following the 1992 moratorium on the use of large-scale driftnets. However, CPUE levels were low for the cohorts hatched in 1992 and 1996 (captured in subsequent years owing to a one-year life of O. bartramii ) that experienced low sea surface temperatures from hatching to recruitment. Among similar-aged squid collected from 180° and 179°30'W in June, mantle lengths were significantly greater in 1997 than during 1995–96. These findings suggest that the driftnet fishery and sea surface temperatures from hatching to recruitment strongly affected stock abundance and possibly growth.     

Thermal behaviors and transitions of wood detected by temperature-modulated differential scanning calorimetry

The thermal properties and transitions of solid and ground wood samples conditioned at different humidity conditions were investigated by temperature-modulated differential scanning calorimetry. A time-dependent transition was detected as an endothermic peak in the total and non-reversing heat flows and as a step change in the reversing heat flow during the first heating run of samples with moisture contents above 5?%, but it disappeared in the second heating run. These different thermal behaviors indicate that the effect of heat and moisture on the thermal properties of wood is history dependent. This step change in the reversing heat flow is considered to be a glass transition of moist wood. Other relaxation processes (e.g., enthalpy relaxation) occur simultaneously with this glass transition. The temperature ranges of the transition and the relaxation decreased drastically as the moisture content increased up to 11?%, while they remained almost constant at higher moisture contents. In addition, the transitions of the ground wood occurred at lower temperatures than those of the solid wood at similar moisture contents. Kissinger plots revealed that the apparent activation energy for the glass transition of the solid wood with a moisture content of 11?% was about 600?kJ/mol, whereas that of the ground wood was 220?kJ/mol.     

Evaluation of biological activities of extracts from the fruiting body of <Emphasis Type="Italic">Pleurotus citrinopileatus</Emphasis> for skin cosmetics

Pleurotus citrinopileatus Singer has recently become a popular delicacy in East Asian countries. We prepared a methanol extract, soluble fractions from the methanol extract, and a hot water extract of the fruiting bodies of P. citrinopileatus. The biological activities such as melanin biosynthesis inhibition, tyrosinase inhibition, antioxidant activities [1,1-diphenyl-2-picrylhydrazyl (DPPH) radical scavenging, oxygen radical absorbance capacity (ORAC), and superoxide dismutase (SOD)-like activity], antibacterial activities, and antihyaluronidase activities of these extracts were evaluated. We found that the n-hexanesoluble, diethyl ether-soluble, and ethyl acetate-soluble fractions exhibited melanin biosynthesis inhibition in B16 melanoma cells, as well as antioxidant (ORAC) and antibacterial activities. However, the n-butanol-soluble and water-soluble fractions and the methanol and hot water extracts exhibited antioxidant (DPPH radical scavenging, SOD-like activity) and antihyaluronidase activities. These results indicate that the fruiting bodies of P. citrinopileatus have the potential to be used as an ingredient in skin cosmetics.     

Effect of soil physical properties on soybean nodulation and N2 fixation at the early growth stage in heavy soil field in Hachirougata Polder,Japan

We studied the effect of the soil physical properties on soybean nodulation and N₂ fixation in the heavy soil of an upland field (UF) and an upland field converted from a paddy field (UCPF) in the Hachirougata polder, Japan. Seeds of the soybean cultivar Ryuho were sown in each field with or without inoculation of Bradyrhizobium japonicum A1017. The soybean plants were sampled at 35 (V3) and 65 (Rl) d after sowing (DAS), and then nodulation and the percentage of N derived from N₂ fixation in the xylem sap were determined. The soil physical properties were different between UF and UCPF, especially the air permeability and soil water regime. Nodule growth was restricted in UCPF irrespective of rhizobial inoculation, though rhizobial infection was not inhibited by the unfavorable soil physical conditions. Soybean plant growth was closely related to the nodule mass and N₂ fixation activity, and the inoculation of a superior rhizobium strain was effective only at 35 DAS. These results indicate that soybean nodulation and N₂ fixation was considerably affected by the physical properties of heavy soil, and that it is important to maintain the N₂ fixation activity and inoculate the soybean plants with a superior rhizobium strain at a later growth stage in order to increase soybean production in heavy soil fields.     

Levels of active oxygen species are controlled by ascorbic acid and anthocyanin in Arabidopsis

Stabilization of the levels of active oxygen species (AOS) is important to the survival of organisms. To clarify the system controlling levels of AOS in plants, this study used an electron spin resonance (ESR) method to directly measure superoxide radical (O(2)(.-)) scavenging activities in the wild-type Arabidopsis thaliana (Col and Ler ecotypes), two anthocyanin mutants (tt3 and ttg1), and an ascorbic acid mutant (vtc1). Under ordinary growth conditions, Arabidopsis contained superoxide-scavenging activity (SOSA) of approximately 300-500 SOD units/g of fresh weight. The ESR pattern indicated that most (40-50%) of this activity was due to ascorbic acid. For the analysis of SOSA under conditions of oxidative stress, synthesis of AOS was induced by gamma-irradiation. The radical scavenging activity in irradiated plants increased approximately 10-fold following an associated increase in the accumulation of ascorbic acid and anthocyanin. The accumulation of ascorbic acid and anthocyanin was suppressed by treatment with an antioxidant before irradiation and was induced by treatment with a radical-generating reagent. The contributions of ascorbic acid and anthocyanin to the total superoxide radical scavenging activity differed among ecotypes. In the Ler ecotype, ascorbic acid accumulated at twice the level of that in the Col ecotype, and induction of anthocyanin was half that in Col. To confirm the activity of ascorbic acid and anthocyanin against AOS stress, the viability of the wild type and mutants (tt2, tt3,tt5, ttg1, and vtc1) was examined after gamma-irradiation. Only the plants in which ascorbic acid and anthocyanin were induced had the ability to grow and flower.     

The functional effect of Gly209 and Ile213 substitutions on lysozyme activity of family 19 chitinase encoded by cyanophage Ma-LMM01

ORF69 in the cyanophage infecting Microcystis aeruginosa, Ma-LMM01, shows homology to the family 19 chitinases where the catalytic domain has structural similarity to lysozyme. Chitinases hydrolyze chitin, a β-1, 4-linked monopolymer of N-acetylglucosamine (GlcNAc); whereas lysozymes hydrolyzes peptidoglycan, alternating β-1, 4-linked copolymers of N-acetylmuramic acid (MurNAc) and GlcNAc. Using amino acid sequence comparison to ORF69, the putative sugar binding residues, Gln162 and Lys165, from the barley chitinase (the model enzyme for the family 19 chitinases) corresponding to subsites −4 and −3 were found to be replaced with Gly209 and Ile213, respectively, in ORF69. To analyze their contribution to substrate binding affinity, ORF69 was cloned into Escherichia coli; and two mutant proteins G209Q and I213K were prepared using site-directed mutagenesis. The wild-type gene product (gp69) showed both lysozyme and chitinase activities. In contrast, the I213K mutant showed a decrease (70%) in lysozyme activity and a significant increase (50%) in chitinase activity; whereas, the G209Q mutant almost completely abolished both enzyme activities. The data suggest the Ile213 residue is involved in recognizing the substrate MurNAc; and Gly209 has significant contribution in chitinase and lysozyme activities for the wild-type gp69.