Role of Caribbean Islands in the diversification and biogeography of Neotropical Heraclides swallowtails

Numerous hypotheses on the evolution of Neotropical biodiversity have stimulated research to provide a better understanding of diversity dynamics and distribution patterns of the region. However, few studies integrate molecular and morphological data with complete sampling of a Neotropical group, and so there has been little synthesis of the multiple processes governing biodiversity through space and time. Here, a total‐evidence phylogenetic approach is used to reconstruct the evolutionary history of the butterfly subgenus Heraclides. We used DNA sequences for two mitochondrial genes and one nuclear gene and coded 133 morphological characters of larvae and adults. A robust and well‐resolved phylogeny was obtained using several analytical approaches, while molecular dating and biogeographical analyses indicated an early Miocene origin (22 Mya) in the Caribbean Islands. We inferred six independent dispersal events from the Caribbean to the mainland, and three from the mainland to the Caribbean, and we suggest that cooling climates with decreasing sea levels may have contributed to these events. The time‐calibrated tree is best explained by a museum model of diversity in which both speciation and extinction rates remained constant through time. By assessing both continental and fine‐scale biodiversity patterns, this study provides new findings, for instance that islands may act as source of diversity rather than as a sink, to explain spatio‐temporal macroevolutionary processes within the Neotropical region.     

Are extra‐pair males different from cuckolded males? A case study and a meta‐analytic examination

Traditional models for female extra‐pair matings assume that females benefit indirectly from extra‐pair mating behaviour. Under these so‐called adaptive models, extra‐pair males are hypothesized to have more compatible genotypes, larger body size, exaggerated ornaments or to be older than cuckolded males. Alternatively, (‘nonadaptive’) models that consider female extra‐pair matings to be a by‐product posit that female extra‐pair mating can be maintained even if there is no benefit to females. This could happen if, for example, males gained fitness benefits from extra‐pair mating, while female and male extra‐pair mating behaviours were genetically correlated. Extra‐pair males are also expected to be older and larger if this improves their ability to convince or coerce females to mate. We investigated whether a female's extra‐pair mates differed from her cuckolded mate in both genetic and phenotypic traits by analysing data from an insular house sparrow population. We found that extra‐pair males were older than cuckolded males, consistent with both models. However, in contrast to the expectations from from adaptive models, extra‐pair and cuckolded males were of similar genetic relatedness, and hence expected compatibility, with the female, and had comparable body size and secondary sexual traits. We also updated previous meta‐analyses examining differences between extra‐pair and cuckolded males. The meta‐analytic results matched results from our house sparrow case study. Although we cannot completely exclude indirect benefits for females, nonadaptive models may better explain female extra‐pair matings. These neglected alternative models deserve more research attention, and this should improve our understanding of the evolution of mating systems.     

Estrogen receptors in medaka (Oryzias latipes) and estrogenic environmental contaminants: an in vitro-in vivo correlation

In many vertebrates, estrogens are necessary to promote the growth and differentiation of the female reproductive system during development, and have important reproductive roles in both males and females. Medaka (Oryzias latipes) has three estrogen receptor (ER) subtypes, ERα, ERβ1 and ERβ2. To evaluate the three medaka ER (mER)-ligand interactions, we applied the ERE-luciferase reporter assay system to characterize each ER subtype. In this transient transfection assay system using mammalian cells, the mER proteins displayed estrogen-dependent activation. 17β-Estradiol (E(2)) and op'-DDT showed high activation irrespective of ERs. Endosulfan also exhibited activation; with less/no transactivity measured using other pesticides, i.e., heptachlor, carbendazim, deltamethrin, acephate, dimethoate and amitraz. It was generally observed that ERβ2 had higher activation potential than ERα and ERβ1. To understand the molecular mechanism of estrogen action via ER, we also conducted E(2) treatment where we observed a trigger in ERβ2 expression upon E(2) exposure. The present data suggest that ERβ2 is essential for female gonad maintenance. The data were supported by induction of vitellogenin (VTG) mRNA in the liver and reduced VTG receptor mRNA expression in the gonad of both sexes. The present work will provide a basic tool allowing future studies to examine the receptor-ligand interactions and endocrine disrupting mechanisms, and also expands our knowledge of estrogen action on reproductive development in medaka.     

Improved composting of Undaria pinnatifida seaweed by inoculation with Halomonas and Gracilibacillus sp. isolated from marine environments

Composting of the Undaria pinnatifida (wakame) seaweed was conducted after inoculation with 6×10(8) CFU g(-1)Halomonas sp. AW4 and the alginate-degrading bacterium Gracilibacillus sp. A7. Inoculation with strains A7 and AW4 resulted in 27.8% and 24.7% degradation of U. pinnatifida dry mass after 168 h, whereas only 17.5% degradation occurred in the uninoculated control. The C/N ratio decreased in the A7 and AW4 inoculated compost by 7.0% and 9.2% after 72 h, but increased by 11.5% in the control. Inoculation with A7 resulted in 2.8 times faster degradation of alginate and 1.2 and 1.6 times higher levels of reducing sugars and unsaturated sugars than inoculation with AW4. The compost produced from the inoculation with A7 had low plant toxicity as measured by germination experiment. The results suggest that inoculation of wakame with alginate-degrading bacteria not only shortened the length of composting but also created seaweed compost with good fertilizer qualities.     

Enhanced cytokine secretion from primary macrophages due to Dectin-1 mediated uptake of CpG DNA/β-1,3-glucan complex

Unmethylated CpG sequences (CpG DNA) can induce Th1 response and thus become a potential immunotherapeutic agent. A key step in the treatment is to transport CpG DNA to its receptor TLR9 located in the endocytosis pathway of target immune cells. For the effective transport, we prepared a novel complex from a β-1,3-glucan schizophyllan (SPG) and CpG DNA, and administered the complex to murine peritoneal macrophages that had been previously activated by thioglycollate and expressed a major β-1,3-glucan receptor Dectin-1 on the cellular surface. Flow cytometric analysis and microscopic observation showed that the complex was taken up by the macrophage through Dectin-1 mediated pathway. Indeed, ELISA demonstrated that IL-12 production was increased sigmoidally with increasing SPG/CpG DNA ratio in the complexation, and reached the maximum at the SPG-rich composition. In the present work, we describe a new approach to deliver CpG DNA to immune cells by use of a β-1,3-glucan/DNA complex.     

A trial of production of the plant-derived high-value protein in a plant factory: Photosynthetic photon fluxes affect the accumulation of recombinant miraculin in transgenic tomato fruits

One of the ultimate goals of plant science is to test a hypothesis obtained by basic science and to apply it to agriculture and industry. A plant factory is one of the ideal systems for this trial. Environmental factors affect both plant yield and the accumulation of recombinant proteins for industrial applications within transgenic plants. However, there have been few reports studying plant productivity for recombinant protein in closed cultivation systems called plant factories.To investigate the effects of photosynthetic photon flux (PPF) on tomato fruit yield and the accumulation of recombinant miraculin, a taste-modifying glycoprotein, in transgenic tomato fruits, plants were cultivated at various PPFs from 100 to 400 (µmol m⁻² s⁻¹) in a plant factory. Miraculin production per unit of energy used was highest at PPF100, although miraculin production per unit area was highest at PPF300. The commercial productivity of recombinant miraculin in transgenic tomato fruits largely depended on light conditions in the plant factory. Our trial will be useful to consider the trade-offs between the profits from production of high-value materials in plants and the costs of electricity.Key words: light, molecular farming, recombinant miraculin, taste-modifying protein, transgenic tomato     

Development of a quantification method for digoxin, a typical P-glycoprotein probe in clinical and non-clinical studies, using high performance liquid chromatography-tandem mass spectrometry: the usefulness of negative ionization mode to avoid competitive adduct-ion formation

Highly sensitive and accurate liquid chromatography-tandem mass spectrometry (LC/MS/MS) methods have been developed and validated for measuring digoxin (DGX), a typical P-glycoprotein probe, in human plasma, rat plasma, and rat brain. We extracted DGX and deuterium-labeled DGX (as internal standard) from sample fluids under basic conditions using acetonitrile and sodium chloride-saturated 0.1 mol/L sodium hydroxide. The upper organic layer was diluted with distilled water, and the resulting solution was injected into an LC/MS/MS system in negative ionization mode. Chromatographic separation was achieved on a C(18)-ODS column in the gradient mobile phase, which comprised 0.05% (w/v) ammonium carbonate (pH 9.0) and methanol at a flow rate of 0.7 mL/min. Regardless of the type of biological matrix, intra-day and inter-day validation tests demonstrated good linearity of calibration curves within ranges of 0.1-10 ng/mL for plasma and 0.5-50 ng/g for rat brain and gave excellent accuracy and precision of quality control samples at 4 concentration levels. Unlike existing methods, our approach uses negative ionization to avoid competitive adduct formation of DGX. Our method showed higher sensitivity and wider applicability to various types of biological matrices than existing methods. Our method will support clinical and preclinical investigation of in vivo P-glycoprotein functionality using DGX.     

Overview of the transcriptome profiles identified in hagfish, shark, and bichir: current issues arising from some nonmodel vertebrate taxa

Because of their crucial phylogenetic positions, hagfishes, sharks, and bichirs are recognized as key taxa in our understanding of vertebrate evolution. The expression patterns of the regulatory genes involved in developmental patterning have been analyzed in the context of evolutionary developmental studies. However, in a survey of public sequence databases, we found that the large-scale sequence data for these taxa are still limited. To address this deficit, we used conventional Sanger DNA sequencing and a next-generation sequencing technology based on 454 GS FLX sequencing to obtain expressed sequence tags (ESTs) of the Japanese inshore hagfish (Eptatretus burgeri; 161,482 ESTs), cloudy catshark (Scyliorhinus torazame; 165,819 ESTs), and gray bichir (Polypterus senegalus; 34,336 ESTs). We deposited the ESTs in a newly constructed database, designated the "Vertebrate TimeCapsule." The ESTs include sequences from genes that can be effectively used in evolutionary developmental studies; for instance, several encode cartilaginous extracellular matrix proteins, which are central to an understanding of the ways in which evolutionary processes affected the skeletal elements, whereas others encode regulatory genes involved in craniofacial development and early embryogenesis. Here, we discuss how hagfishes, sharks, and bichirs contribute to our understanding of vertebrate evolution, we review the current status of the publicly available sequence data for these three taxa, and we introduce our EST projects and newly developed database.     

Production of ectoine through a combined process that uses both growing and resting cells of <Emphasis Type="Italic">Halomonas salina</Emphasis> DSM 5928<Superscript>T</Superscript>

Using ectoine-excreting strain Halomonas salina DSM 5928^T, we developed a new process for high-efficiency production of ectoine, which involved a combined process of batch fermentation by growing cells and production by resting cells. In the first stage, batch fermentation was carried out using growing cells under optimal fermentation conditions. The second stage was the production phase, in which ectoine was synthesized and excreted by phosphate-limited resting cells. Optimal conditions for synthesis and excretion of ectoine during batch fermentation in a 10 l fermentor were 0.5 mol l⁻¹ NaCl and an initial monosodium glutamate concentration of 80 g l⁻¹ respectively. The pH was adjusted to 7.0 and the temperature was maintained at 33°C. In phosphate-limited resting cells medium, monosodium glutamate and NaCl concentration was 200 g l⁻¹ and 0.5 mol l⁻¹, respectively, as well as pH was 7.0. The total concentration of ectoine produced was 14.86 g l⁻¹, the productivity and yield of ectoine was 7.75 g l⁻¹ day⁻¹ and 0.14 g g⁻¹, respectively, and the percentage of ectoine excreted was 79%. These levels of ectoine production and excretion are the highest reported to date.