The master sex-determination locus in threespine sticklebacks is on a nascent Y chromosome

BACKGROUND: Many different environmental and genetic sex-determination mechanisms are found in nature. Closely related species can use different master sex-determination switches, suggesting that these developmental pathways can evolve very rapidly. Previous cytological studies suggest that recently diverged species of stickleback fish have different sex chromosome complements. Here, we investigate the genetic and chromosomal mechanisms that underlie sex determination in the threespine stickleback (Gasterosteus aculeatus). RESULTS: Genome-wide linkage mapping identifies a single chromosome region at the distal end of linkage group (LG) 19, which controls male or female sexual development in threespine sticklebacks. Although sex chromosomes are not cytogenetically visible in this species, several lines of evidence suggest that LG 19 is an evolving sex chromosome system, similar to the XX female/XY male system in many other species: (1) males are consistently heterozygous for unique alleles in this region; (2) recombination between loci linked to the sex-determination region is reduced in male meiosis relative to female meiosis; (3) sequence analysis of X- and Y-specific bacterial artificial chromosome (BAC) clones from the sex-determination region reveals many sequence differences between the X- and Y-specific clones; and (4) the Y chromosome has accumulated transposable elements and local duplications. CONCLUSIONS: Taken together, our data suggest that threespine sticklebacks have a simple chromosomal mechanism for sex determination based on a nascent Y chromosome that is less than 10 million years old. Further analysis of the stickleback system will provide an exciting window into the evolution of sex-determination pathways and sex chromosomes in vertebrates.     

Changes in morphological characteristics of an introduced population of the threespine stickleback <Emphasis Type="Italic">Gasterosteus aculeatus</Emphasis> in Lake Towada,northern Japan

A freshwater population of the threespine stickleback Gasterosteus aculeatus occurs at an elevation of 400m in Lake Towada in the mountains of northern Japan. Morphological characteristics of each sex of this population were studied between 1985 and 1992. The 1985 fishes were larger in standard length than even anadromous populations, as well as all other freshwater populations, so the large body size of this population was unusual for the species. The population was also characterized by large ratios of head length, eye diameter, and gill raker length, and a higher ovary volume in 1992 than in 1985, but it was not readily characterized by meristic characters. The population may have been introduced within the last 20 years (probably in the early 1980s). The large body size of the population may not reflect a defense against predators because coexisting fishes were not important predators on threespine stickleback adults. It is supposed that the change of body size was induced by environmental changes in prey abundance in the lake and/or by the longevity of fishes.     

Myxoid leiomyosarcoma of the uterus. Report of a case with cytologic findings

BACKGROUND: Myxoid leiomyosarcoma is a rare variant of uterine sarcoma, exhibiting malignant biologic behavior despite the absence of cytologic atypia and of significant mitotic activity. CASE: A 20-year-old female was referred with a cystic pelvic mass. At laparotomy, the tumor, weighed 2,200 g and originating in the left lateral uterine wall, was removed. Microscopic examination revealed well-differentiated smooth muscle cells without atypia and with a few mitotic figures in the copious myxoid matrix, suggesting myxoid leiomyosarcoma. Three years following laparotomy, an irregular mass around the uterus was noted on sonographic examination, suggesting local recurrence. Two years and six months later, the second operation was performed, and a locally recurrent, multicystic tumor weighing 3,500 g was excised. The histopathology was similar to that of the primary tumor. Cytologic findings on imprint material from the tumor revealed a few isolated or sheet like small cells consisting of spindle and polygonal cells with round and oval nuclei. Cytologic atypia was also minimal. CONCLUSION: Myxoid leiomyosarcoma should be included in the differential diagnosis of smooth muscle neoplasia.     

Quantitative analysis of the lytic cycle of WO phages infecting Wolbachia

WO is a temperate bacteriophage that infects Wolbachia, a maternally inherited endosymbiont of arthropods. WO has lysogenic and lytic cycles, the latter of which is an important process for the spread of WO infection. In this study, we measured the lytic activities of two WO phages, WOCauB2 and WOCauB3, infecting a Wolbachia strain, wCauB. In the lytic cycle of WO, both ends of the prophage are ligated to create a junction sequence called attP in the phage genome. We performed real-time quantitative polymerase chain reaction to measure the amounts of attP sequences produced by WOCauB2 and WOCauB3 in wCauB-infected Ephestia kuehniella (Zeller) (Lepidoptera: Pyralidae) and wCauB-infected insect cell lines. WOCauB2 produced the phage genome more actively than WOCauB3 in E. kuehniella, whereas WOcauB3 was more active than WOCauB2 in the cell lines, suggesting that the environment of host cells in which Wolbachia is harbored affects the lytic activity of WO phages. The lytic activity was constantly very low: the amounts of attP relative to the prophages were lower than 1?×?10^?3 in all measurements, which was discussed in conjunction with the intracellular life of Wolbachia.     

Dynamics of soil carbon following destruction of tropical rainforest and the subsequent establishment of Imperata grassland in Indonesian Borneo using stable carbon isotopes

Southeast Asia has the highest rate of tropical rainforest deforestation worldwide, and large deforested areas have been replaced ultimately by the highly invasive grass Imperata cylindrica. However, information on the carbon (C) budget with such land transition is very scarce. This study presents the dynamics of soil C following rainforest destruction and the subsequent establishment of Imperata grassland in the lowland humid tropics of Indonesian Borneo using stable C isotopes. To evaluate the relative contribution of organic matter originating from primary forest (C₃) and grasslands (C₄), we compared soil C stock and natural ¹³C abundance from six sites to a depth of 100 cm using samples with a wide range of soil textures. Twelve years after the first soil sampling in the grasslands, we re‐sampled to examine temporal changes in soil organic matter. The grassland topsoil (0–5 cm) is an active layer with rapid decomposition and incorporation of fresh C (mean residence time: 7.5 year) and a substantial proportion of the stable C pool (37%). The decline in forest‐derived C was slight, even at 5–10 cm depths, and subsoil (20–100 cm depth) forest‐derived C did not change along the forest‐to‐grassland chronosequence. Grassland‐derived C stock increased significantly in the subsurface and subsoils (5–100 cm). Simulation indicated that total soil C stock (0–100 cm) increased by 18.6 Mg ha^?1 from initial primary forest (58.0 Mg ha^?1) to a new equilibrium state of the grassland (76.6 Mg ha^?1) after 30–50 years of grassland establishment. This research indicates that the soil did not function as a CO₂ source when the deforested area was replaced by Imperata grassland on the Ultisols of the Asian humid tropics. Instead, increased soil C stocks offset CO₂ emissions, with the C offset accounting for 6.6–7.4% of the loss of biomass C stock.     

Fungal farming in a non-social beetle

Culturing of microbes for food production, called cultivation mutualism, has been well-documented from eusocial and subsocial insects such as ants, termites and ambrosia beetles, but poorly described from solitary, non-social insects. Here we report a fungal farming in a non-social lizard beetle Doubledaya bucculenta (Coleoptera: Erotylidae: Languriinae), which entails development of a special female structure for fungal storage/inoculation, so-called mycangium, and also obligate dependence of the insect on the fungal associate. Adult females of D. bucculenta bore a hole on a recently-dead bamboo culm with their specialized mandibles, lay an egg into the internode cavity, and plug the hole with bamboo fibres. We found that the inner wall of the bamboo internode harboring a larva is always covered with a white fungal layer. A specific Saccharomycetes yeast, Wickerhamomyces anomalus ( = Pichia anomala), was consistently isolated from the inner wall of the bamboo internodes and also from the body surface of the larvae. Histological examination of the ovipositor of adult females revealed an exoskeletal pocket on the eighth abdominal segment. The putative mycangium contained yeast cells, and W. anomalus was repeatedly detected from the symbiotic organ. When first instar larvae were placed on culture media inoculated with W. anomalus, they grew and developed normally to adulthood. By contrast, first instar larvae placed on either sterile culture media or autoclaved strips of bamboo inner wall exhibited arrested growth at the second instar, and addition of W. anomalus to the media resumed growth and development of the larvae. These results strongly suggest a mutualistic nature of the D. bucculenta-W. anomalus association with morphological specialization and physiological dependence. Based on these results, we compare the fungal farming of D. bucculenta with those of social and subsocial insects, and discuss ecological factors relevant to the evolution of fungal farming in a non-social insect.     

CsFTL3, a chrysanthemum FLOWERING LOCUS T-like gene, is a key regulator of photoperiodic flowering in chrysanthemums

Chrysanthemum is a typical short-day (SD) plant that responds to shortening daylength during the transition from the vegetative to the reproductive phase. FLOWERING LOCUS T (FT)/Heading date 3a (Hd3a) plays a pivotal role in the induction of phase transition and is proposed to encode a florigen. Three FT-like genes were isolated from Chrysanthemum seticuspe (Maxim.) Hand.-Mazz. f. boreale (Makino) H. Ohashi & Yonek, a wild diploid chrysanthemum: CsFTL1, CsFTL2, and CsFTL3. The organ-specific expression patterns of the three genes were similar: they were all expressed mainly in the leaves. However, their response to daylength differed in that under SD (floral-inductive) conditions, the expression of CsFTL1 and CsFTL2 was down-regulated, whereas that of CsFTL3 was up-regulated. CsFTL3 had the potential to induce early flowering since its overexpression in chrysanthemum could induce flowering under non-inductive conditions. CsFTL3-dependent graft-transmissible signals partially substituted for SD stimuli in chrysanthemum. The CsFTL3 expression levels in the two C. seticuspe accessions that differed in their critical daylengths for flowering closely coincided with the flowering response. The CsFTL3 expression levels in the leaves were higher under floral-inductive photoperiods than under non-inductive conditions in both the accessions, with the induction of floral integrator and/or floral meristem identity genes occurring in the shoot apexes. Taken together, these results indicate that the gene product of CsFTL3 is a key regulator of photoperiodic flowering in chrysanthemums.     

Development of a repeated-dose liver micronucleus assay using adult rats: an investigation of diethylnitrosamine and 2,4-diaminotoluene

Various liver micronucleus assay methods, such as those involving partial hepatectomy, treatment with mitogens, and the use of juvenile animals, have been developed. These assays have been proven to be of high sensitivity and specificity to predict hepatocarcinogenicity of compounds that cannot be detected by bone marrow micronucleus assays. On the contrary, the existing assays have only been evaluated for their use in detecting micronucleus induction in the settings of relatively short-term cell proliferation. However, the integration of in vivo genotoxicity endpoints into routine toxicity studies is increasingly desired from the viewpoint of animal welfare to reduce the number of animals used. In the present study, the rodent hepatocarcinogens diethylnitrosamine (DEN) and 2,4-diaminotoluene (2,4-DAT) were repeatedly administered orally to male Crl:CD (SD) rats (6 weeks old at the beginning of administration) for 5, 14, and 28 days, and changes in the frequency of hepatocytes with micronuclei in liver tissues that had undergone no artificial treatment to accelerate cell proliferation were evaluated. At the same time, a new method of hepatocyte isolation involving the treatment of a portion of the liver with collagenase in a centrifuge tube, without the use of in situ perfusion, was established. The induction of micronucleated hepatocytes was achieved after the repeated administration of DEN for 5 days or longer and of 2,4-DAT for 14 days or longer. Micronucleus frequencies were increased depending on the number of administrations, indicating that micronucleated hepatocytes had possibly remained for a long period of time and accumulated additively. It therefore appears that even in adult rat liver with low mitotic activity, a repeated-dose of a chemical substance for 14 days or longer enables the detection of micronucleus induction. In addition, the establishment of a method to isolate hepatocytes without perfusion using only a part of the liver enables the integration of liver micronucleus assays into general toxicity studies.     

Contribution of FSH and triiodothyronine to the development of circadian clocks during granulosa cell maturation

The involvement of FSH and triiodothyronine (T(3)) in circadian clocks was investigated using immature granulosa cells of ovaries during the progress of cell maturation. Granulosa cells were prepared from preantral follicles of mouse Period2 (Per2)-dLuc reporter gene transgenic rats injected subcutaneously with the synthetic nonsteroidal estrogen diethylstilbestrol. Analysis of the cellular clock of the immature granulosa cells was performed partly using a serum-free culture system. Several bioluminescence oscillations of Per2-dLuc promoter activity were generated in the presence of FSH + fetal bovine serum, but not in the presence of either FSH or serum. As revealed by bioluminescence recording and analysis of clock gene expression, the granulosa cells lack the functional cellular clock at the immature stage, although Lhr was greatly expressed during the period of cell maturation. The granulosa cells gained a strong circadian rhythm of bioluminescence during stimulation with FSH, whereas LH reset the cellular clock of matured granulosa cells. During strong circadian rhythms of clock genes, the Star gene showed significant expression in matured granulosa cells. In contrast, T(3) showed an inhibitory effect on the development of the functional cellular clock during the period of cell maturation. These results indicate that FSH provides a cue for the development of the functional cellular clock of the immature granulosa cells, and T(3) blocks the development of the cellular clock.