Purification and Characterization of Isocitrate Lyase from Ethanol-grown Euglena gracilis

Isocitrate lyase was purified to homogeneity from ethanol-grown Euglena gracilis. The specific activity was 0.26 μmol/min/mg protein. The molecular mass of the enzyme was calculated to be 380 kDa by gel filtration on a Superose 6 column. The subunit molecular mass of the enzyme was 116 kDa as determined by SDS-polyacrylamide gel electrophoresis. These results showed that the native form of this enzyme was a trimer composed of three identical subunits. The pH optimum for cleavage and condensation reactions was 6.5 and 7.0, respectively. The K_m values for isocitrate, glyoxylate and succinate were 3.8, 1.3 and 7.7 mM, respectively. Isocitrate lyase absolutely required Mg for enzymatic activity. This is the first report of the purification of isocitrate lyase to homogeneity from Euglena gracilis.     

LABORATORY OBSERVATIONS ON THE EARLY LIFE STAGES OF THE DIAMOND SQUID THYSANOTEUTHIS RHOMBUS

The early life stages of the diamond squid Thysanoteuthis rhombusfrom early embryogenesis to post-hatching (0–7 days old)were observed through laboratory incubation using egg massescollected in the southern Sea of Japan. The egg diameter andmantle length increased during embryonic development throughhatching. Mantle-length growth was linear over time, and thegrowth rate was significantly higher at 25°C than at 20°C.The inner yolk was located on the dorsal side of the mantlecavity and increased in volume through hatching. Immediatelyafter they hatched, the hatchlings remained on the bottom ofculture plates with their ventral sides facing up, but 2–3days after hatching they began to swim with their dorsal sidesfacing up. Feeding experiments were conducted, but none of thehatchlings fed. Statolith growth increments were shown to formdaily. Ontogenetic changes that occur from fertilization throughpost-hatching are discussed. (Received 8 June 2005; accepted 2 November 2005)     

Amphibian chytridiomycosis in Japan: distribution,haplotypes and possible route of entry into Japan

A serious disease of amphibians caused by the chytrid fungus Batrachochytrium dendrobatidis was first found in Japan in December 2006 in imported pet frogs. This was the first report of chytridiomycosis in Asia. To assess the risk of pandemic chytridiomycosis to Japanese frogs, we surveyed the distribution of the fungus among captive and wild frog populations. We established a nested PCR assay that uses two pairs of PCR primers to amplify the internal transcribed spacer (ITS) region of a ribosomal RNA cassette to detect mild fungal infections from as little as 0.001 pg (1 fg) of B. dendrobatidis DNA. We collected swab samples from 265 amphibians sold at pet shops, 294 bred at institutes and 2103 collected at field sites from northern to southwestern Japan. We detected infections in native and exotic species, both in captivity and in the field. Sequencing of PCR products revealed 26 haplotypes of the B. dendrobatidis ITS region. Phylogenetic analysis showed that three of these haplotypes were specific to the Japanese giant salamander (Andrias japonicus) and appeared to have established a commensal relationship with this native amphibian. Many other haplotypes were carried by alien amphibians. The highest genetic diversity of B. dendrobatidis was found in the American bullfrog (Rana catesbeiana). Some strains of B. dendrobatidis appeared to be endemic to Japanese native amphibians, but many alien strains are being introduced into Japan via imported amphibians. To improve chytridiomycosis risk management, we must consider the risk of B. dendrobatidis changing hosts as a result of anthropogenic disturbance of the host‐specific distribution of the fungus.     

Breeding ecology and seasonal abundance of the giant water bug Appasus japonicus (Heteroptera, Belostomatidae)

Males of the giant water bug Appasus (= Diplonychus) japonicus Vuillefroy (Belostomatidae: Heteroptera) carry egg masses on their back, but little is known about the relationship between seasonal abundance and breeding ecology of the species. In the present study, therefore, a field survey based on a mark-and-recapture census was carried out at three survey points within a rice paddy area (0.3 km²) where A. japonicus forms a meta-population in northern Okayama, Japan. We investigated the body size, seasonal abundance, dispersion, egg mass size (number of eggs within one egg mass), number of egg masses and the total eggs carried on the back of each male as fundamental parameters of the population and breeding. Significant differences in egg mass size, number of egg masses, and total number of eggs that males carried was found among the survey points. The present results suggested the possibility that the differences in breeding parameters of A. japonicus were influenced by differences in environmental factors among the microhabitats. These results are discussed in conjunction with previous reports on seasonal abundance and breeding systems in Belostomatidae bugs.     

Accumulation of Trehalose as a Compatible Solute under Osmotic Stress in Euglena gracilis Z

ABSTRACT. The photosynthetic protozoon Euglena gracilis, accumulated a large amount of trehalose in the cells under salt or osmotic stresses. Radioactivity of [14C] paramylon, a β-1,3-polyglucan which was stored in the cells of E. gracilis. was degraded rapidly and this radioactivity was almost stoichiometrically incorporated into trehalose. The interconversion of trehalose from paramylon by salt or osmotic stresses was dependent on the concentrations or osmotic pressures, suggesting that E. gracilis accumulate trehalose as an osmoprotectant. After the removal of salt or osmotic stresses, trehalose was gradually degraded, however, it was not converted into paramylon.     

Accurate measurement of optical properties of narrow leaves and conifer needles with a typical integrating sphere and spectroradiometer

Accurate information on the optical properties (reflectance and transmittance spectra) of single leaves is important for an ecophysiological understanding of light use by leaves, radiative transfer models and remote sensing of terrestrial ecosystems. In general, leaf optical properties are measured with an integrating sphere and a spectroradiometer. However, this method is usually difficult to use with grass leaves and conifer needles because they are too narrow to cover the sample port of a typical integrating sphere. Although ways to measure the optical properties of narrow leaves have been suggested, they have problems. We propose a new measurement protocol and calculation algorithms. The protocol does not damage sample leaves and is valid for various types of leaves, including green and senescent. We tested our technique with leaves of Aucuba japonica, an evergreen broadleaved shrub, and compared the spectral data of whole leaves and narrow strips of the leaves. The reflectance and transmittance of the strips matched those of the whole leaves, indicating that our technique can accurately estimate the optical properties of narrow leaves. Tests of conifer needles confirmed the applicability.     

Meat‐eating enhances larval development of Anthracophora rusticola Burmeister (Coleoptera: Scarabaeidae), which breeds in bird nests

The scarab beetle Anthracophora rusticola Burmeister breeds in the nests of carnivorous birds including the honey buzzard Pernis apivorus (Linnaeus), Chinese goshawk Accipiter soloensis (Horsfield), oriental stork Ciconia boyciana Swinhoe and great cormorant Phalacrocorax carbo (Linnaeus). Although larvae of the beetle probably feed on animal matter (e.g. remnants of birds' food) in the nests of these birds, the effect of eating flesh on the growth of larvae has not been investigated. Laboratory experiments clearly showed that the development was faster, survival rate higher and body heavier just before pupation in larvae fed dried chicken meat with humus soil than in those fed only humus soil. We also investigated feeding behaviors of larvae of the beetle. A piece of chicken meat placed on the surface of humus soil was pulled under the soil by the larva, mostly at night. Video recordings made during the night showed that larvae came to the humus soil surface and ate the dried chicken meat or tried to take the meat under the soil. Our laboratory observations suggest that larvae in nature stay under the humus accumulated in bird nests during the daytime, and then come up to the surface mostly at night to actively seek and eat remnants of the birds' food.     

Fighting, dispersing, and sneaking: body-size dependent mating tactics by male Librodor japonicus beetles

Abstract.  1. Scaling relations between weapons and body size depart from linearity in many male beetles. In many previous studies, these males have been divided into major and minor males with a switch point, that is male dimorphism. Major and minor males adopt strikingly different reproductive tactics.
2. We found three size-dependent behaviours, i.e. fighting, dispersing, and sneaking, however, among Librodor japonicus males with dimorphic mandibles. We statistically classified males into large, medium, and small (L-, M-, and S-males) sizes and then compared the dispersal of males from a foraging site, behaviours to gain access to females, and sizes of mandibles, wings, and testes.
3. M-males dispersed earlier than L- and S-males from a territory in a field, but no difference in the frequency of dispersal was observed between L- and S-males. Observations of male–male interactions in the laboratory showed that L-males frequently fought with other males in a fighting arena, while S-males often showed sneaking behaviour without fighting.
4. On the basis of the morphological analysis, we concluded that S-males invested their available resources more in sperm (= testes), M-males more in wings, and L-males more in mandibles in L. japonicus .
5. Even though a morphological male dimorphism was detected, it might be possible to classify the males of the armed beetles into more than two behavioural tactics if we examine their behaviours.     

Wood-fall associations from Late Cretaceous deep-water sediments of Hokkaido, Japan

Sunken wood (wood-fall) in the deep sea today is colonized and consumed by diverse invertebrate communities that show phylogenetic relationships to the chemotrophic fauna on whale carcasses, hydrothermal vents and hydrocarbon seeps. Here we document Late Cretaceous examples of wood-fall communities from deep-water sediments of the Yezo Group on Hokkaido, Japan, consisting of provannid, skeneiform, and patelliform gastropods, and thyasirid and nuculanid bivalves. These species are similar or identical to those found on plesiosaur bones and at hydrocarbon seeps in the same sediments, showing that many members of the modern chemotrophic deep-sea fauna colonized this range of habitats at least since Late Cretaceous time. We also document for the first time faecal chimneys in boreholes in these wood fragments, which were presumably built by xylophagain bivalves.     

Effect of Cobalamin Deficiency on the Biosynthesis of Phosphatidylcholine in Euglena gracilis

Euglena gracilis requires cobalamin (Cbl) as an essential growth factor. Phosphatidylcholine (PC) synthesis was greatly reduced by Cbl deficiency. Rapid cell division occurred after Cbl was replenished, and PC was actively synthesized during the cell divisions. When the deficient cells were given methionine (a precursor for the choline moiety), active synthesis of PC occurred even without the Cbl supplement, although cell division was not induced. As methionine synthase in Euglena requires methylcobalamin as a coenzyme, decrease in methionine synthesis may account for reduced PC synthesis under Cbl-deficient conditions. Phosphatidyleth-anolamine and phosphatidylserine synthesis were also suppressed, commensurate with decrease of PC synthesis, under Cbl deficiency, even though Cbl is not thought to participate in their synthesis. In contrast, a lot of triglyceride and wax ester accumulated in Cbl-deficient cells. Moreover, Cbl depletion altered fatty acid composition, notably due to increased proportion of odd-numbered fatty acids