Maki N. Inoue

Activation of mitogen-activated protein kinase during meiotic maturation in porcine oocytes

To investigate the involvement of mitogen-activated protein kinase (MAP kinase) in meiotic maturation of porcine oocytes, we assayed MAP kinase activity using myelin basic protein (MBP) as a substrate. MAP kinase activity was low during the germinal vesicle stage, 0-20 h of culture. An abrupt increase was observed at metaphase I (30 h of culture), and activity remained significantly higher than that at 0 h until 50 h of culture, with a transient slight decrease at the time of first polar body extrusion (40 h). Detection of the kinase activity by an in-gel phosphorylation assay confirmed that the 42 and 44 kDa MAP kinases were significantly activated in 45 h cultured oocytes but not in 0 h oocytes, and just slightly in 20 h oocytes. In immunoblotting, however, the 42 and 44 kDa bands were detected in 0, 20 and 45 h cultured oocytes. Furthermore, the signal strength of the two bands did not change during the period of culture, but shifted up to 45 h, indicating that the activation of MAP kinase depended not on the synthesis but on the phosphorylation of this enzyme. These results suggest that the activation of MAP kinase is involved in the regulation of meiotic maturation of porcine oocytes, and especially in the regulation after germinal vesicle breakdown.

Morphological variation in relation to flower use in bumblebees

To understand resource partitioning in a bumblebee community, we analyzed various morphological characters. A total of 1269 individuals of six bumblebee species, Bombus ardens, B. hypocrita, B. diversus, B. ignitus, B. honshuensis and B. beaticola, were examined and principal component analysis showed that the bumblebee species were clearly differentiated. Glossa, prementa and head lengths were positively correlated with the second component, and a longer proboscis was associated with a narrower body, which may help bees to intrude into and access deep‐lying nectar sources. Bombus diversus, with a long proboscis and narrow body, preferred flowers with a long corolla tube, whereas B. hypocrita and B. ignitus, which have short proboscises and wide bodies, visited flowers with short corollas or dish‐shaped flowers. Two pairs of consubgeneric species that have similar morphological characteristics, B. ardens and B. beaticola, and B. hypocrita and B. ignitus, divided flower resources by habitat selection and seasonal partitioning. For resource partitioning among bumblebee species, not only morphology but also other factors, such as habitat and seasonal preference, flower use, foraging behavior, and interspecific interactions, are responsible.

Colony growth and reproductive ability of feral nests of the introduced bumblebee Bombus terrestris in northern Japan

The introduced Bombus terrestris has been blamed for the decline of native bumblebee populations in Japan. To control this invasive species, it is necessary to understand its ecological traits in its newly established range. Here, we investigated the colony growth pattern and reproductive ability of feral nests of B. terrestris in northern Japan from 2003 to 2006. Nests collected at various times showed initially slow growth followed by rapid development. This pattern is consistent with findings in previous studies using laboratory-raised colonies. Our results also suggested that protandrous colonies tend to specialize in male production, whereas protogynous colonies specialize in gyne production, producing a split sex ratio in feral colonies of B. terrestris. The numerical population sex ratio was 1.40 gynes per male, calculated from the numbers of pre-emergence cocoons for gynes and males, showing a female-biased sex ratio at the population level. Mature colonies produced a mean of 376.5 cocoons and 90.2 gyne cocoons (22.1% of the total). The proportion of gynes produced by B. terrestris nests exceeded both those of conspecifics observed in other countries and those of consubgeneric native Japanese species. The propagule pressure hypothesis appears to explain the probability of establishment of this invasive species. Suitable nest sites for B. terrestris queens appeared to be in short supply, and B. terrestris may win usurpation contests against native species due to its large size, resulting in the decline of native bumblebee species.

A Switch in the Dynamics of Intra-Platelet VEGF-A from Cancer to the Later Phase of Liver Regeneration after Partial Hepatectomy in Humans

Background Liver regeneration (LR) involves an early inductive phase characterized by the proliferation of hepatocytes, and a delayed angiogenic phase distinguished by the expansion of non-parenchymal compartment. The interest in understanding the mechanism of LR has lately shifted from the proliferation and growth of parenchymal cells to vascular remodeling during LR. Angiogenesis accompanied by LR exerts a pivotal role to accomplish the process. Vascular endothelial growth factor (VEGF) has been elucidated as the most dynamic regulator of angiogenesis. From this perspective, platelet derived/Intra-platelet (IP) VEGF-A should be associated with LR. Material and Methods Thirty-seven patients diagnosed with hepatocellular carcinoma and undergoing partial hepatectomy (PH) were enrolled in the study. Serum and IP VEGF-A was monitored preoperatively and at four weeks of PH. Liver volumetry was determined on computer models derived from computed tomography (CT) scan. Results Serum and IP VEGF-A was significantly elevated at four weeks of PH. Preoperative IP VEGF-A was higher in patients with advanced cancer and vascular invasion. Postoperative IP VEGF-A was higher after major liver resection. There was a statistically significant correlation between postoperative IP VEGF-A and the future remnant liver volume. Moreover, the soluble vascular endothelial growth factor receptor-1 (sVEGFR1) was distinctly down-regulated suggesting a fine-tuned angiogenesis at the later phase of LR. Conclusion IP VEGF-A is overexpressed during later phase of LR suggesting its implications in inducing angiogenesis during LR.

Midgut-based resistance to oral infection by a nucleopolyhedrovirus in the laboratory-selected strain of the smaller tea tortrix, Adoxophyes honmai (Lepidoptera: Tortricidae)

A strain of Adoxophyes honmai resistant to Adoxophyes honmai nucleopolyhedrovirus (AdhoNPV) was established from a field-collected colony by repeated selection. Fifth-instar larvae of this resistant strain (R-strain) had over 66 666-fold greater resistance in terms of 50 % lethal concentration values to oral infection of AdhoNPV than non-selected strain larvae (susceptible for AdhoNPV; S2-strain). In this study, the mechanism of resistance to AdhoNPV was determined in R-strain larvae. An assessment of viral genome replication in AdhoNPV-infected S2- and R-strain larvae by quantitative PCR showed no viral genome replication occurring in R-strain larvae. Transcription of AdhoNPV ie-1, vp39 and polyhedrin genes was also not detected in R-strain midgut cells. Besides, a fluorescent brightener had no effect on AdhoNPV infection in either S2- or R-strain. However, binding and fusion of occlusion-derived virus with R-strain were significantly lower than those of S2-strain. These findings suggest that R-strain Adoxophyeshonmai larvae possess a midgut-based resistance to oral infection by AdhoNPV in which midgut epithelial cells are infected less efficiently.

Genetic Structure and Potential Environmental Determinants of Local Genetic Diversity in Japanese Honeybees (Apis cerana japonica)

Declines in honeybee populations have been a recent concern. Although causes of the declines remain unclear, environmental factors may be responsible. We focused on the potential environmental determinants of local populations of wild honeybees, Apis cerana japonica, in Japan. This subspecies has little genetic variation in terms of its mitochondrial DNA sequences, and genetic variations at nuclear loci are as yet unknown. We estimated the genetic structure and environmental determinants of local genetic diversity in nuclear microsatellite genotypes of fathers and mothers, inferred from workers collected at 139 sites. The genotypes of fathers and mothers showed weak isolation by distance and negligible genetic structure. The local genetic diversity was high in central Japan, decreasing toward the peripheries, and depended on the climate and land use characteristics of the sites. The local genetic diversity decreased as the annual precipitation increased, and increased as the proportion of urban and paddy field areas increased. Positive effects of natural forest area, which have also been observed in terms of forager abundance in farms, were not detected with respect to the local genetic diversity. The findings suggest that A. cerana japonica forms a single population connected by gene flow in its main distributional range, and that climate and landscape properties potentially affect its local genetic diversity.

Juvenile hormone (JH) esterase activity but not JH epoxide hydrolase activity is downregulated in larval Adoxophyes honmai following nucleopolyhedroviruses infection.

Juvenile hormones (JHs) and ecdysteroids are critical insect developmental hormones. JH esterase (JHE) and JH epoxide hydrolase (JHEH) are JH-selective enzymes that metabolize JH and thus regulate the titer of JH. Baculoviruses are known to alter host endocrine regulation. The nucleopolyhedroviruses, AdhoNPV and AdorNPV, are known to have slow and fast killing activity against Adoxophyes honmai (Lepidoptera: Tortricidae), respectively. Here we found that when penultimate (4th) instar A. honmai are inoculated with AdhoNPV or AdorNPV, the mean survival time is 9.7 and 8.2 days, respectively. The larvae molted once but did not pupate. The AdhoNPV- or AdorNPV-infected larvae did not show a dramatic increase in JHE activity as was found in mock-infected larvae, instead they showed a marked decrease in JHE activity. In contrast, both viral infections had no effect on JHEH activity. In order to further characterize the JHE activity, the JHE-coding sequence of A. honmai (ahjhe) was cloned and confirmed to encode a biologically active JHE. Quantitative real-time PCR analysis of ahjhe expression in 4th and 5th instar A. honmai revealed that AdhoNPV and AdorNPV are able to reduce ahjhe expression levels.

The invasion of alien ants across continents with special reference to Argentine Ants and Red Imported Fire Ants

Abstract Alien ants are considered to be among the more damaging of invasive insects. Five ant species are ranked among the 100 worlds worst invaders by the IUCN. Within the introduced regions, they displace or disrupt the local arthropod fauna, cause agricultural damage by protecting plant pests, and even affect human health. The Argentine Ant, Linepithema humile, has successfully spread from its native range in South America across much of the globe. This species is highly polygynous and possesses a social structure, called unicoloniality, whereby individuals mix freely among separated nests. The introduced populations of the Argentine Ant are characterized by the formation of very large supercolonies across tens or thousands of kilometers, whereas supercolonies in the native ranges are generally smaller, varying from just a few meters in diameter to many hundreds of meters long. In Japan the Argentine Ant, first noted in 1993, is now found in several regions. Early detection and rapid response systems as well as control measures are required to prevent further expansion of the species. A vital component of this prevention is the identification of pathways of introduction into new locations. The recent advances in molecular markers have allowed us to retrospectively construct the invasion history as well as that of the Red Imported Fire Ant, Solenopsis invicta. More recently, many studies on invasion biology have focussed on the ecological and economic links. We first review the previously published studies inferring historical introduction pathways of the alien ants. We then combine our genetic studies with human activity history to describe global dispersal pathways of the alien ants. Finally, we discuss the interactions between human activity and biodiversity through understanding the dispersal history of the alien ants.

An ascovirus isolated from Spodoptera litura (Noctuidae: Lepidoptera) transmitted by the generalist endoparasitoid Meteorus pulchricornis (Braconidae: Hymenoptera)

The family Ascoviridae is a recently described virus family whose members are transmitted by parasitoids and cause chronic and lethal infections in lepidopteran insects. Little is known about the biology and ecology of ascoviruses, and few isolates have been found outside the United States. We report here the isolation of a new ascovirus variant from Spodoptera litura in Japan. Full genome sequence and phylogenetic analyses showed that this virus was closely related to variants in Heliothis virescens ascovirus-3a, and it was named HvAV-3j. HvAV-3j has a DNA genome of 191 718 bp, with 189 putative ORFs and a GC content of 45.6 %, and is highly similar to HvAV-3h, which was isolated in China. In a field survey, the endoparasitoid Meteorus pulchricornis caused a high percentage of parasitization in populations of S. litura larvae, and under laboratory conditions M. pulchricornis was able to transmit HvAV-3j from infected to uninfected larvae by oviposition. Meteorus pulchricornis is thus likely to be a major vector for HvAV-3j transmission in Japan. This species is recognized here for the first time as a vector of ascoviruses that parasitizes a range of host species that extends across families.

Identification of an Early Male-Killing Agent in the Oriental Tea Tortrix, Homona magnanima

Arthropods are frequently infected with inherited symbionts, which sometimes confer fitness benefits on female hosts or manipulate host reproduction. Early male killing, in which infected males die during embryogenesis, is induced by some bacteria, such as Wolbachia and Spiroplasma. A female-biased sex ratio has been found in Homona magnanima, collected from a tea plantation in Japan. Here, we examined the male-killing trait in H. magnanima and identified the agent that induces early male killing. The sex ratio distortion (SR) strain produced only females and no males, and its egg hatch rate was significantly lower than that of the normal (N) strain. The N strain was infected with only Wolbachia, whereas the SR strain was infected with both Wolbachia and Spiroplasma. Antibiotic treatment with 0.10% tetracycline restored the 1:1 sex ratio in the SR strain. Females treated with 0.05% tetracycline were positive for Spiroplasma but not for Wolbachia and showed a female-biased sex ratio, whereas Wolbachia-positive females did not revert to male killing. When inoculated with a homogenate of the SR strain female, females infected with only Spiroplasma produced female-biased offspring. Sequence analysis of the 16S rRNA gene revealed that Spiroplasma sp. of H. magnanima belonged to the ixodetis clade. These results indicate that Spiroplasma was responsible for male killing in H. magnanima. Late male killing is induced in H. magnanima by an RNA-like virus, and therefore this is the first case in which different male-killing agents expressed at different times in the life cycle have been found within one host species.