Cytoskeletal and mitochondrial properties of bovine oocytes obtained by Ovum Pick‐Up: the effects of follicle stimulation and in vitro maturation

Follicle stimulation by follicular stimulating hormone (FSH) is known to improve developmental competence of bovine oocytes obtained by Ovum Pick‐Up (OPU); however, the exact factors in oocytes affected by this treatment have remained unclear. We compared in vitro matured (IVM) oocytes obtained at the immature stage from cows by OPU either without or with stimulation with FSH (non‐stimulated and stimulated OPU, respectively) to those obtained by superstimulation and in vivo maturation in terms of cytoskeleton morphology, mitochondrial distribution, intracellular adenosine triphosphate (ATP) content and H₂O₂ levels at the metaphase‐II stage and intracellular Ca²⁺ levels after in vitro fertilization (IVF). Confocal microscopy after immunostaining revealed reduced size of the meiotic spindle, associated with increased tendencies of microfilament degradation and insufficient mitochondrial re‐distribution in non‐stimulated OPU‐derived IVM oocytes compared with those collected by stimulated OPU, which in turn resembled in vivo matured oocytes. However, there was no difference in mitochondrial functions between oocytes obtained by stimulated or non‐stimulated OPU in terms of ATP content, cytoplasmic H₂O₂ levels, base Ca²⁺ levels and the frequencies and amplitudes of Ca²⁺ oscillations after IVF. Larger size of metaphase spindles in oocytes obtained by stimulated OPU may reflect and potentially contribute to their high developmental competence.     

Genetic basis of multiple resistance to the brown planthopper (Nilaparvata lugens St?l) and the green rice leafhopper (Nephotettix cincticeps Uhler) in the rice cultivar ‘ASD7’ (Oryza sativa L. ssp. indica)

The rice cultivar ASD7 (Oryza sativa L. ssp. indica) is resistant to the brown planthopper (BPH; Nilaparvata lugens Stål) and the green leafhopper (Nephotettix virescens Distant). Here, we analyzed multiple genetic resistance to BPH and the green rice leafhopper (GRH; Nephotettix cincticeps Uhler). Using two independent F₂ populations derived from a cross between ASD7 and Taichung 65 (Oryza sativa ssp. japonica), we detected two QTLs (qBPH6 and qBPH12) for resistance to BPH and one QTL (qGRH5) for resistance to GRH. Linkage analysis in BC₂F₃ populations revealed that qBPH12 controlled resistance to BPH and co-segregated with SSR markers RM28466 and RM7376 in plants homozygous for the ASD7 allele at qBPH6. Plants homozygous for the ASD7 alleles at both QTLs showed a much faster antibiosis response to BPH than plants homozygous at only one of these QTLs. It revealed that epistatic interaction between qBPH6 and qBPH12 is the basis of resistance to BPH in ASD7. In addition, qGRH5 controlled resistance to GRH and co-segregated with SSR markers RM6082 and RM3381. qGRH5 is identical to GRH1. Thus, we clarified the genetic basis of multiple resistance of ASD7 to BPH and GRH.     

Effects of flooding on downstream processes of glycolysis and fermentation in roots of Melaleuca cajuputi seedlings

We investigated the energy metabolism in roots of flooded Melaleuca cajuputi Powell, a tropical flood-tolerant tree species, by measuring adenylate concentrations and activities of glycolytic and fermentative enzymes under flooded conditions. Adenylate energy charge (AEC) decreased slightly to 0.72 on the second day of flooding and recovered to around 0.8 by the fourth day of flooding. Activities of pyruvate decarboxylase (EC 4.1.1.1) and alcohol dehydrogenase (EC 1.1.1.1) increased initially and then decreased to the control level after 14 days of flooding. On the other hand, activities of pyruvate kinase (EC 2.7.1.40), phosphoenolpyruvate phosphatase (EC 3.1.3.2), and a series of phosphoenolpyruvate carboxylase (EC 4.1.1.31), malate dehydrogenase (EC 1.1.1.37), and NADP dependent malic enzyme (EC 1.1.1.40), which can convert PEP into pyruvate, were not induced in flooded roots throughout the experiment. These results suggest that neither the downstream reactions of glycolysis nor ATP production via glycolysis was enhanced by flooding, whereas alcohol fermentation was enhanced. With the low ATP yield of the glycolysis–alcohol fermentation pathway and no induction of glycolytic enzymes, the glycolysis–alcohol fermentation pathway itself contributes little to ATP production in flooded roots of M. cajuputi. These physiological responses of M. cajuputi to flooding may have the advantages of surviving flooded conditions because they can avoid exhaustion of sugar and accumulation of ethanol, a toxic end product of alcohol fermentation.     

Effects of urea and trimethylamine-N-oxide on ATPase of requiem shark myofibril and its constituents

ABSTRACT: The effects of trimethylamine- N -oxide (TMAO) on the urea-resistibility of requiem shark myofibrils were investigated, using Ca²⁺- and Mg²⁺-ATPase activities as a parameter. Both activities were hardly changed or activated up to 0.6 M urea. In contrast, the two activities both decreased to less than 50% in the presence of TMAO up to 0.5 M. When measured at a 2 : 1 molar ratio of urea and TMAO, Ca²⁺- and Mg²⁺-ATPase activities were similar to those in the presence of TMAO alone, indicating that TMAO reduced the urea-resistibility of myofibrils. Myosin, the most abundant protein in myofibrils, from requiem shark exhibited the effects of urea and TMAO on its Ca²⁺-ATPase activity, which was primarily similar to those of myofibrils. However, Ca²⁺-ATPase activities in the coexistence of urea and TMAO for actomyosin reconstituted from requiem shark myosin and chicken F-actin were approximately average of those measured independently in the presence of either urea or TMAO alone. Carp myofibrils, reconstituted actomyosin and myosin, which were used as teleost references, all showed a tendency in the effects of urea and TMAO on Ca²⁺-ATPase activities that was similar to those of requiem shark counterparts.     

Detection of Culicoides brevitarsis activity in Kyushu

Culicoides brevitarsis transmits important ruminant arboviruses, such as Akabane, Aino and bluetongue viruses. The presence of this species has so far been recognized primarily in Okinawa, the southernmost prefecture of Japan. In entomological surveys in 2008 and 2009, C. brevitarsis was collected at 8 sites throughout Nagasaki, Kumamoto and Kagoshima Prefectures. The collection sites are all located near pastures, where the larvae of C. brevitarsis can grow in cattle dung left in the field. C. brevitarsis was confirmed at the same sites in two consecutive years, suggesting that it overwinters in Kyushu. Given the risk of arbovirus transmission, the ecology of C. brevitarsis in Japan, such as its distribution range, seasonal abundance and larval breeding sites, should be investigated in more detail.     

Changes in microbial biomass after continuous application of azolla and rice straw in soil

A model experiment was conducted under tropical conditions with a view to evaluating the changes in microbial biomass and nutrient dynamics in upland soil through the continuous application of azolla and rice straw (2 g C kg^-1 soil per each application). Flush decomposition of C was observed immediately after each application and the rate of mineralization did not change appreciably during this period. After flush decomposition, the rate of C mineralization from azolla was higher than that from rice straw until 9 to 13 weeks after each application and thereafter the mineralization rate was similar. The amount of inorganic N released from azolla increased following each application, whereas inorganic N in rice straw plot was immediately immobilized and the rate of immobilization increased until the 3rd application and did not increase further after the 4th application. The amounts of biomass C and N increased immediately after residue incorporation, reached the maximum level one week after each application and declined thereafter. Maximum biomass formation increased until the 2nd application and then the level remained constant. Maximum biomass N formation was higher in azolla than in rice straw after the 1st application, but after repeated applications, the difference became less pronounced: Continuous increase in biomass in a certain week after each application was observed, probably because of the cumulative effects of the previous applications. The increase suggests that continuous application of organic materials may enable to improve the amount of soil microbial biomass.     

Application of rRNA-based probe for detection of a bacterium in rhizoplane

Recently, in situ hybridization technique targeted to ribosomal RNA (Delong et al. 1989) has been widely used to detect specific microorganisms in various environments such as marine environment (Nishimura 1995), sediments (Spring et al. 1993), inside of organisms (Amann et al. 1991), and soil (Hahn et al. 1992). This technique provides information not only on microscale localization of the microorganisms but also on their metabolic activities in situ. So far, detection by in situ hybridization targeted to ribosomes in soil has been scarcely reported because two problems arise when this method is applied to soil. The first is that cells in natural soils cannot be easily stained because the cellular ribosome content is low in an oligotrophic soil environment. The second is that non-specific binding of the probes to soil particles and autofluorescence of soil components such as organic matter and mineral particles interfere with the signal from the probe specifically hybridized.     

Effect of anion composition of simulated acid rain on nutrient behavior in reclaimed saline soils

Abstract

Field experiments with the “Taikichi” taro cultivar were conducted in volcanic ash soil of Kagoshima Prefecture, Japan, in order to determine the effects of potassium applied with fertilizers and manures on the growth and yield of taro. The experimental design was accord ing to the L27 (3¹³) orthogonal factorial experiment, the three factors selected being the manures, nitrogen fertilizers and amount of potassium application.

“Metsubure” corm formation was infrequent in hog manured plots without potassium application, but was significantly increased by the application of potassium fertilizer. Furthermore, the occurrence of “Metsubure” corms was significantly lower in fields prepared with hog manure than in those prepared with plant residue and cattle manure.

Chemical analysis of the taro plants and manures revealed that the plants absorbed much more calcium when planted in hog manure than in other manures. The CaO/K₂O ratio was also higher. The main reason for this was assumed to be the lower content of potassium in the hog manure (0.37% K₂O on a dry basis).

The analytic results suggest that potassium disturbs the calcium uptake ability of the taro plant causing “Metsubure” corm formation.

The authors also attempted to determine the effects of several kinds of nitrogen fertilizers on “Metsubure” corm formation, but no clear results were obtained in this experiment.