Sequence and polymerase chain reaction–restriction fragment length polymorphism analysis of the large subunit rRNA gene of bivalve: Simple and widely applicable technique for multiple species identification of bivalve larva

ABSTRACT:   One of the biggest and long-standing difficulties in investigation of larval ecology in the field is species-level identification. In the present study, we developed polymerase chain reaction–restriction fragment length polymorphism (PCR-RFLP) analysis based on the large subunit (LSU) rRNA gene (rDNA) D1/D2/D3 region for identification of multiple species of bivalve larvae using 14 species of bivalve collected from Maizuru Bay. The LSU rDNA D1/D2/D3 region of all analyzed species could be amplified by PCR using bvD1f/bvD3r primers, and RFLP analysis by Hae III digestion on the PCR products showed species-specific fragment patterns. Furthermore, this analysis applied to single bivalve larvae in Maizuru Bay revealed efficient amplification of the target region and the species-specific pattern from 80% of the larvae, 75% of which showed a pattern that matched a certain pattern of the adult bivalves. In addition, the analysis of inter- and intraspecies variation of the LSU rDNA D1/D2/D3 region using the sequence data of the genus Crassostrea from the DDBJ database showed high applicability of this RFLP analysis on closely related species. Because of the wide applicability and technical simplicity, this method can become the standard for the identification of bivalve larvae species once the sequence data of the LSU rDNA D1/D2/D3 region of many bivalve species have been accumulated.     

Functional degeneration of the resistance gene nsv against Melon necrotic spot virus at low temperature

The single recessive gene, nsv, which confers resistance against Melon necrotic spot virus (MNSV), has recently been used to develop virus-resistant melon cultivars in Japan. However, the Chiba isolate of MNSV, a common isolate in Japan, infected resistant cultivars when inoculated melon plants were grown at 15°C. Viral RNAs accumulated in protoplasts from resistant cultivars at both 15 and 20°C. Mechanical inoculation of the cotyledons caused MNSV to spread throughout the leaves at 15°C, but not at 20°C. These results support our novel hypothesis that a temperature-sensitive inactivation of disease resistance genes occurs at the nsv locus in melon cultivars with the resistance gene grown at temperatures below 20°C. The first and second authors contributed equally to this research.     

Physical and mechanical properties of wood after moisture conditioning

Some properties of wood (hinoki:Chamaecyparis obtusa) moisture-conditioned by an adsorption process from a dry state and by two desorption processes (from a water-saturated state and from a state with a moisture content slightly below the fiber saturation point) were investigated. The moisture contents of wood conditioned by the adsorption process and by the desorption process continued to approach to one another for the moisture-conditioning period of over 50 weeks. Accordingly, sorption hysteresis should be regarded as a transitional phenomenon that occurs during the process of approaching the true equilibrium, which requires a long time. The wood conditioned by the desorption process beginning from a water-saturated state showed slightly smaller dimensions than those conditioned by the adsorption process with the same moisture content; however, the wood conditioned by the desorption process from a moisture content below the fiber saturation point showed slightly larger dimensions than those conditioned by the adsorption process. The wood conditioned by the adsorption process from a dry state showed a higher modulus of elasticity and modulus of rupture than did the wood conditioned from a water-saturated state with the same moisture content. The mechanical properties of the wood also varied based on the states at which the desorption process was started. This is a notable characteristic of the relation between the drying condition and the mechanical properties of wood.     

Genetic population structure of the deep-sea whelk Buccinum tsubai in the Japan Sea

ABSTRACT:   The purpose of the present paper was to examine the genetic population structure of Buccinum tsubai in the Japan Sea. Mitochondrial 16SrRNA gene sequence analysis was performed with specimens from various stations in the Japan Sea. Mitochondrial lineages were clearly patterned geographically in four separate areas: the Hokkaido area, the Yamagata–Toyama area, the Yamato Bank area, and the San'in area. The main distribution depth range of B. tsubai is between 200 m and 1000 m isodepths, and the horizontal distance between the 200 m and 1000 m isodepth lines represents the specific spatial scale of the habitat (SSSH). These four areas were separated either by the complete discontinuity of the SSSH area or by its narrow spatial extension. Genetic distances between the main haplotypes of each area were calculated as Jukes–Cantor distances, the value of which ranged between 0.012 and 0.017. This value seemed to be unrelated to the geographic distance. There was no tendency for clustering according to depth. In future, the morphological characters of the four lineages of B. tsubai should be compared in detail in order to elucidate significant genetic differences among them.     

Effect of oral administration of colostrum on inflammation in the udders of dairy cows suffering from mastitis

The present study was undertaken to examine whether oral administration of colostrum to mastitic cows reduced inflammation in the udder. Fifty milliliters of a colostrum whey product was administered orally daily for 3 days to cows suffering from mastitis. Milk was collected on day 0 and 7 of colostrum administration. For Experiment 1, milk from 11 udder quarters with high somatic cell counts (SCC) in four cows was used. SCC in milk decreased significantly after colostrum administration, whereas colostrum administration increased sodium and IgA concentrations significantly compared with those before administration. In Experiment 2, cows with clinical mastitis were divided into two groups, with and without colostrum administration, whereas all cows with subclinical mastitis were administered colostrum. Antibiotics were infused into the mammary gland from the first day of colostrum administration for 2–4 days. There was no significant decrease in SCC after colostrum administration in any group. However, udder firmness in both clinical mastitis groups was reduced after administration regardless of colostrum administration. IgA concentration in both clinical mastitis groups was significantly increased after colostrum administration compared to that before administration, although there was no significant difference between them. These results suggest the possibility that oral administration of colostrum attenuates inflammation of the mammary gland. Further studies are required to examine the effect of colostrum more precisely using cows with subclinical and chronic mastitis and longer duration of colostrum administration.     

Genetic diversity of Central Asian and north Caucasian Aegilops species as revealed by RAPD markers

RAPD analysis of 112 accessions of Aegilops tauschii Coss. (genome DD), Ae. cylindrica Host (CCDD), Ae. crassa Boiss. (DDMM), Ae. biuncialis Vis. (UUMM) and Ae. triuncialis L. (UUCC) collected in the Central Asia and north Caucasia was conducted. Aegilops accessions were divided into two major groups, corresponding to the D genome species and the U genome species. These groups were also separated into sub-groups according to species, except for the Ae. tauschii-cylindrica complex of accessions from Central Asia. Aegilops tauschii from north Caucasia was divided into two varietal groups, tauschii and meyeri. The Central Asian accessions of Aegilops species were more diverse than the accessions from north Caucasia. Aegilops tauschii and Ae. cylindrica accessions from north Caucasia were genetically uniform. Associations between altitudal variation of Aegilops species and variability of RAPD markers were not found.     

Healing process of the wounds of the branches of the Japanese persimmon that were caused by girdling,scoring, and strangulation

Girdling is widely used for many crops, mainly in order to improve the fruit set, size, and quality and to increase the yield of flower buds. However, girdling may damage trees, and permanent injury can occur if callus bridges are not formed across the ring. We determined the regeneration of vascular bundles and the healing process of wounds caused by surgical treatments. It occurred as follows: necrosis of some cell layers on the edge of the cut, callus formation and dedifferentiation of the parenchyma cells, callus proliferation, contact between callus pads, callus bridge formation, and differentiation into the mature vascular bundle; however, the innermost callus cells remained undifferentiated. At 3 days after treatment (DAT), several phloem parenchyma cell layers showed symptoms of necrosis. At 10 DAT, callus formation started in the phloem tissue below the periderm. Initially, the callus cells were uniform; however, the callus tissue pad was distinguished into 2 layers based on color and firmness. After callus bridge formation, the inner callus differentiated into the mature xylem and the outer callus, into mature phloem. In strangulation, xylem elements were formed from the inner callus pad before callus bridge formation. The callus bridge formed within 6 weeks, 20–25 days, and 14 weeks after girdling, scoring, and strangulation, respectively.     

Heavy metal accumulations in a semi-enclosed hypereutrophic system: Lake Teganuma,Japan

Heavy metal concentrations and other physical and chemical properties were studied so as to assess the heavy metal accumulation process in the sediments of a hypereutrophic lake, Lake Teganuma, Japan. The accumulations were most evident near the inflow inlets of two main inflow rivers in Kaminuma (the west part of Lake Teganuma) with the significant Zn accumulation. Except for Fe, the vertical distributions of heavy metals showed the highest concentrations in the surface 10 cm and decreased gradually with the sediment depth. The high concentrations in the upper layer was caused by an increase in the non-residual fractions. The large influx of heavy metal to Lake Teganuma seems to be due to the domestic effluents via the two rivers in last decades.     

Isolation of a Protein Bound to Canker-forming Factor from Citrus Plant

Interaction analysis using affinity analysis (Affinity Sensors, Cambridge, UK) indicated the presence of proteins bound to Apl1, a virulence factor of Xanthomonas campestris pv. citri required for the formation of canker symptom on citrus, in the fraction 25–50% ammonium sulfate of citrus crude extracts. Three proteins of 25, 50 and 110-kD were eluted from an Apl1-affinity column. Western analysis revealed that Apl1 binds specifically to the 25-kD and 110-kD but not to the 50-kD protein. When crude extracts of soybean and of tobacco were applied to the Apl1-affinity column, only faint bands were detected. This result suggests that Apl1 targets exist specifically in citrus plants. The amino acid sequence of the N-terminal of the 25-kD protein was determined, and homology search analysis revealed that this sequence was almost identical to those commonly present in S-adenosyl-l-methionine : trans-caffeoyl-coenzyme A 3-O-methyltransferase (CCoAMT) from several plants. This enzyme is specific to the substrate trans -caffeoyl-CoA and catalyzes the synthesis of trans-feruloyl-CoA for lignin formation. Received 4 November 1999/ Accepted in revised form 26 November 1999