An insertion mutation in the Apoe gene associated with spontaneous hyperlipidemia in mice.

Introduction: Spontaneously hyperlipidemic (SHL) mice, a mouse strain derived from an inbred strain of Japanese wild (original)-type mice (KOR; Mus musculus molossinus), show high plasma cholesterol concentrations with disruption of the apolipoprotein E (Apoe) gene. However, the details of the Apoe gene of SHL mice have yet to be described. Material and methods: The DNA sequence of the Apoe gene of SHL mice was compared to that of control KOR mice in genomic DNA and cDNA analyses. Results: In the DNA analysis, a 4700-bp fragment was found to be inserted into exon 4 of the Apoe gene of SHL mice. The insertion contained two 365-bp repeats at each terminal and was flanked by a 6-bp target duplication at each side. The inserted fragment produced a frameshift of an early stop codon, resulting in a protein product that consisted of 87 amino acids in SHL mice compared to 311 amino acids in control KOR mice. Conclusions: These findings provide useful information about the molecular basis of SHL mice and related lipid disorders.

New species of Sargassum-boring Limnoria Leach, 1814 (Crustacea, Isopoda, Limnoriidae) from Japan.

A new marine Sargassum-boring species of Limnoria (Limnoriidae) is described on the basis of specimens obtained at Kamogawa-shi, Chiba Prefecture, Japan. Limnoria aspera sp. nov. shares a reduced mandibular palp to a seta, algal-feeding, and the clavate shaped epipod of the maxilliped with the other species of non-mandibular-palp group. L. aspera sp. nov. differs morphologically from the congeneric species by secondary unguis of pereopods and unique carinae of pleonite 5 and pleotelson. We describe the sequences of the mitochondrial COI gene and the nuclear 28S rDNA gene. L. aspera sp. nov. differs by 14.218.0% in p-distance based on COI sequences from other Japanese species, L. furca and L. nagatai.

Description of new species of algal-boring Limnoria (Crustacea, Isopoda, Limnoriidae) from Japan and redescription of Limnoria segnoides Menzies, 1957 and L. nagatai Nunomura, 2012.

Previous research on the mitochondrial COI gene sequences of several populations of Limnoria nagatai Nunomura suggested a cryptic species from the Sea of Japan. A new species of algal-boring limnoriid is here described. Limnoria furca sp. nov. was collected from the holdfasts of Eisenia bicyclis on the Oki Islands and the Sea of Japan coast of Honshu and Kyushu Islands, Japan. L. furca sp. nov. is diagnosed morphologically by lacking a mandibular palp, faint subparallel carinae on the pleotelson, faint Y-shaped carinae on pereonite 5, the bifid lacinia mobilis of right mandible and the shapes of the secondary unguis on the pereopods. Similar species, L. segnoides Menzies, 1957 and L. nagatai Nunomura, 2012, are redescribed on the basis of the holotypes.

Genetic structure and dispersal patterns in Limnoria nagatai (Limnoriidae, Isopoda) dwelling in non-buoyant kelps, Eisenia bicyclis and E. arborea, in Japan.

The marine isopod genus Limnoria contains algae-eating species. Previous phylogeographic studies have suggested that Limnoria species feeding on buoyant kelp underwent low genetic differentiation on a large spatial scale because rafting on floating host kelps promotes high levels of gene flow. In this paper, we survey the genetic structure of Limnoria nagatai, which bores into the non-buoyant kelps Eisenia bicyclis and E. arborea. We analyze the mitochondrial DNA (cytochrome oxidase subunit I [COI] gene) and morphological traits of L. nagatai, and the host kelps E. bicyclis and E. arborea from 14 populations along the Japanese archipelago of the Pacific Ocean and the Sea of Japan. Four major lineages are recognized within L. nagatai: three lineages in the Pacific Ocean, and one lineage in the Sea of Japan which might be a cryptic species. For L. nagatai, we show high genetic differentiation between geographically separated habitats in the Pacific Ocean, while low differentiation is found among continuous host kelps habitats in the Pacific Ocean as well as the Sea of Japan. L. nagatai in E. bicyclis in the Pacific Ocean has experienced large population expansion after the Last Glacial Maximum (LGM), whereas the lineage in E. bicyclis in the Sea of Japan has not. We suggest that Limnoria feeding on non-buoyant kelps, may attain low genetic differentiation because they might be able to disperse long distance if the habitat of host kelps is continuous. The historical events affecting Limnoria after the LGM may differ between the coasts of the Pacific Ocean and the Sea of Japan.

The phylogeography of the cycad genus Dioon (Zamiaceae) clarifies its Cenozoic expansion and diversification in the Mexican transition zone.

Background and Aims: Biogeographic transition zones are promising areas to study processes of biogeographic evolution and its influence on biological groups. The Mexican transition zone originated due to the overlap of Nearctic and Neotropical biota, which promoted great biological diversification. However, since most previous studies in this area were focused on revealing the phylogeography of Nearctic plants, how historical biogeographic configuration influenced the expansion and diversification of the Neotropical flora remains almost unknown. Using the cycad genus Dioon (Zamiaceae), this study aimed to test whether the biogeographic provinciality of the Mexican transition zone reflects the history of diversification of Neotropical plants. Methods: Two chloroplast DNA (cpDNA) regions were analysed from 101 specimens of 15 Dioon species to reveal the distribution of haplogroups. In addition, genome-wide single nucleotide polymorphisms (SNPs) from 84 specimens were used to test the concordance between phylogenetic clusters and the biogeographic provinces. An ultrametric tree was constructed from the sequences containing SNPs to reconstruct the biogeographic events of vicariance and dispersal of Dioon across the Neotropical biogeographic provinces. Key Results: Four Dioon lineages with strong phylogeographic structures were recognized using both cpDNA and SNP data. The lineages correspond to two clades that originated from a common ancestor in Eastern Mexico. One clade expanded and diversified in South-east Mexico and Central America. Another clade diversified into three lineages that dispersed to North-east, South and North-west Mexico. Each lineage was biogeographically delimitated. Biogeographic provinces might have provided disparate ecological conditions that facilitated speciation in Dioon since the Miocene. Conclusions: The current genetic structure and species diversity of Dioon depict the history of expansion and diversification of the northernmost Neotropical provinces. Past biogeographic connectivities were favoured by elevated topographies, since mountain systems served as corridors for the migration of Dioon and as refugia of tropical communities that diversified during the formation of modern Neotropical forests.

A new species of seagrass-boring Limnoria (Limnoriidae, Isopoda, Crustacea) from Japan.

The marine seagrass-boring isopod, Limnoria rhombipunctata sp. nov. (Limnoriidae) is described from the rhizome of Phyllospadix iwatensis seagrass, in shallow coastal waters off Chiba Prefecture, Japan. L. rhombipunctata sp. nov. is distinguished from other Limnoria species by the unique carinae of pleonite 5 and pleotelson, two branched lacinia mobilis of the right mandible, 3 flagellar articles of antenna 1, and triangular epipod of the maxilliped. Specimens of L. magadanensis, a species similar to L. rhombipunctata sp. nov., are re-examined and compared with L. rhombipunctata sp. nov.

Field Experiments of Pollination Ecology: The Case of Lycoris sanguinea var. sanguinea.

Plant-pollinator interactions have been studied for approximately one hundred years. During that time, many field methods have been developed to clarify the pollination effectiveness of each pollinator for visited flowers. Pollinator observations have been one of the most common methods to identify pollinators, and bagging and cage experiments have been conducted to show the effectiveness of specific pollinators. In a previous study of Lycoris sanguinea var. sanguinea, its effective pollinators, the visitation frequencies of each floral visitor, and its reproductive strategies were not identified. This study reports the observation that small bees visited flowers that were partially opened (breaking buds). To the best of our knowledge, this phenomenon has not been reported previously. Further, this study investigates the hypothesis that small bees can pollinate at that flowering stage. This study demonstrates the basic methods of field experiments in pollination ecology using L. sanguinea var. sanguinea. Pollinator observations and digital video showed the visitation frequencies of each floral visitor. Bagging and cage experiments revealed that these flowers could be pollinated fully and that breaking-bud pollination could be important for the pollination of this plant species. The advantages and disadvantages of each method are discussed, and recent developments, including laboratory experiments, are described.

Cloning and characterization of carboxyl terminus of heat shock cognate 70-interacting protein gene from the silkworm, Bombyx mori.

Carboxyl terminus of heat shock cognate 70-interacting protein (CHIP) is an evolutionarily conserved E3 ubiquitin ligase across different eukaryotic species and is known to play a key role in protein quality control. CHIP has two distinct functional domains, an N-terminal tetratricopeptide repeat (TPR) and a C-terminal U-box domain, which are required for the ubiquitination of numerous labile client proteins that are chaperoned by heat shock proteins (HSPs) and heat shock cognate proteins (HSCs). During our screen for CHIP-like proteins in the Bombyx mori databases, we found a novel silkworm gene, Bombyx mori CHIP. Phylogenetic analysis showed that BmCHIP belongs to Lepidopteran lineages. Quantitative reverse transcription-PCR analysis indicated that BmCHIP was relatively highly expressed in the gonad and fat body. A pull-down experiment and auto-ubiquitination assay showed that BmCHIP interacted with BmHSC70 and had E3 ligase activity. Additionally, immunohistochemical analysis revealed that BmCHIP was partially co-localized with ubiquitin in BmN4 cells. These data support that BmCHIP plays an important role in the ubiquitin proteasome system as an E3 ubiquitin ligase in B. mori.

Breaking-bud pollination: a new pollination process in partially opened flowers by small bees.

Plant-pollinator interactions have usually been researched in flowers that have fully opened. However, some pollinators can visit flowers before full opening and contribute to fruit and seed sets. In this paper, we researched the pollination biology of flowers just starting to open in four field experiments. We observed the insect visitors to Lycoris sanguinea var. sanguinea for 3 years at five sites. These observations revealed that only small bees, Lasioglossum japonicum, often entered through tiny spaces between the tepals of 'breaking buds' (i.e. partially opened flowers) and collected pollen. We hypothesized that they can pollinate this species at the breaking-bud stage, when the stigma is located near the anthers. To measure the pollination effect of small bees at the breaking-bud stage, we bagged several breaking buds after small bees had visited them and examined whether these buds were pollinated. In bagging experiments, 30% of the breaking buds set fruit and seeds. Fruit-set ratios of the breaking buds did not differ significantly from those of the fully opened flowers, which had been visited by several insect species. We also counted the pollen grain numbers on the body of L. japonicum and on the anthers of randomly-selected and manipulated flowers. These experiments revealed that all of the captured bees had some pollen of target plants and that L. japonicum collected most of the pollen grains at the breaking-bud stage. Our results showed that the new pollination process, breaking-bud pollination, happened in breaking buds by L. japonicum, although there is no evidence to reveal that this is the most effective pollination method for L. sanguinea var. sanguinea. In principle, this new pollination process can occur in other flowering plants and our results are a major contribution to studies of plant-pollinator interactions.

Involvement of HSC70-4 and other inducible HSPs in Bombyx mori nucleopolyhedrovirus infection.

Heat shock proteins (HSPs) and heat shock cognate proteins (HSCs) function as molecular chaperones under normal cellular conditions. In this report, we describe the role of Bombyx mori heat shock cognate protein 70-4 (BmHSC70-4), which is a constitutively expressed member of the heat shock protein 70 (HSP70) family, in B. mori nucleopolyhedrovirus (BmNPV) infection. We first generated the BmHSC70-4 antibody, which can react specifically with an endogenous BmHSC70 from BmN cells. Immunohistochemistry has demonstrated that BmHSC70-4 was expressed at steady-state levels throughout the BmNPV infection and was accumulated in the nucleus of BmNPV-infected cells at a very late phase of infection. Western blot experiments have also shown that BmHSC70-4 is a novel component protein of budded virus (BV) and occlusion-derived virus (ODV). Next, we investigated the effect of KNK437, a known inhibitor of inducible HSPs, in BmNPV-infected BmN cells and found that both reduced BV production and delayed viral DNA replication were observed in virus-infected cells treated with KNK437. Furthermore, the formation of occlusion bodies (OBs) was not observed in KNK437-treated cells because this compound reduced the promoter activity of the polyhedrin gene severely. Collectively, the present results suggest that BmHSC70-4 is a novel structural protein of BmNPV and may have important roles in BmNPV propagation.

Incongruence among mitochondrial, chloroplast and nuclear gene trees in Pinus subgenus Strobus (Pinaceae).

Introgression has been considered to be one of main factors leading to phylogenetic incongruence among different datasets at lower taxonomic levels. In the plants of Pinaceae, the mtDNA, cpDNA, and nuclear DNA (nrDNA) may have different evolutionary histories through introgression because they are inherited maternally, paternally and biparentally, respectively. We compared mtDNA, cpDNA, and two low-copy nrDNA phylogenetic trees in the genus Pinus subgenus Strobus, in order to detect unknown past introgression events in this group. nrDNA trees were mostly congruent with the cpDNA tree, and supported the recent sectional and subsectional classification system. In contrast, mtDNA trees split the members of sect. Quinquefoliae into two groups that were not observed in the other gene trees. The factors constituting incongruence may be divided into the following two categories: the different splits within subsect. Strobus, and the non-monophyly of subsect. Gerardianae. The former was hypothesized to have been caused by the past introgression of cpDNA, mtDNA or both between Eurasian and North American species through Beringia. The latter was likely caused by the chimeric structure of the mtDNA sequence of P. bungeana, which might have originated through past hybridization, or through a horizontal transfer event and subsequent recombination.

Airborne-pollen pool and mating pattern in a hybrid zone between Pinus pumila and P. parviflora var. pentaphylla.

The reproductive isolation barriers and the mating patterns among Pinus pumila, P. parviflora var. pentaphylla and their hybrids were examined by flowering phenology and genetic assays of three life stages: airborne-pollen grains, adults and seeds, in a hybrid zone on Mount Apoi, Hokkaido, Japan. Chloroplast DNA composition of the airborne-pollen was determined by single-pollen polymerase chain reaction. Mating patterns were analysed by estimating the molecular hybrid index of the seed parent, their seed embryos and pollen parents. The observation of flowering phenology showed that the flowering of P. pumila precedes that of P. parviflora var. pentaphylla by about 6 to 10 days within the same altitudinal ranges. Although this prezygotic isolation barrier is effective, the genetic assay of airborne-pollen showed that the two pine species, particularly P. pumila, still have chances to form F(1) hybrid seeds. Both parental species showed a strong assortative mating pattern; F(1) seeds were found in only 1.4% of seeds from P. pumila mother trees and not at all in P. parviflora var. pentaphylla. The assortative mating was concluded as the combined result of flowering time differentiation and cross-incompatibility. In contrast to the parental species, hybrids were fertilized evenly by the two parental species and themselves. The breakdown of prezygotic barriers (intermediate flowering phenology) and cross-incompatibility may account for the unselective mating. It is suggested that introgression is ongoing on Mount Apoi through backcrossing between hybrids and parental species, despite strong isolation barriers between the parental species.