Magnetic DNA affinity purification of ecdysteroid receptor.

A new method for rapid purification to near homogeneity of the ecdysteroid receptor (EcdR) from Drosophila melanogaster nuclear extract is presented. In the first step of the purification procedure the EcdR molecules were radiolabelled with [3H]ponasterone A and the [3H]ponasterone A-EcdR complexes were chromatographed under very mild conditions on Fractogel EMD TMAE(s) ion-exchanger. A 23-fold purified receptor was obtained which can be stored in liquid N2 without loss of activity. The second step involved the use of a magnetic DNA affinity technique where the double stranded hsp 27 oligonucleotide containing EcdR binding sequence was biotin 5'-end labelled and bound to monodisperse superparamagnetic particles coated with streptavidin (Dynabeads M-280 Streptavidin) giving magnetic DNA affinity beads. The chromatographed EcdR-ponasterone A complexes were bound to the magnetic DNA affinity beads and by magnetic separation, wash and elution, a 29,000-fold enriched EcdR preparation was obtained within 1.5 h. This procedure can be applied for other EcdR sources with minor modifications.     

Nectar thieves or invited pollinators? A case study of tansy flowers and common house mosquitoes

Mosquitoes are generally considered nectar thieves that do not contribute to the pollination of the flowers they visit. Here we tested the hypothesis that the common house mosquito, Culex pipiens, contributes to the pollination of tansies, Tanacetum vulgare, and possibly the pollination of other members of the Asteraceae (Achillea millefolium, Leucanthemum vulgare, Solidago canadensis). To test this hypothesis, we (1) field-collected mosquitoes probing inflorescences of T. vulgare, A. millefolium, and L. vulgare, and recorded the number and distribution of pollen grains on their bodies, (2) exposed laboratory-reared Cx. pipiens to inflorescences of T. vulgare, A. millefolium, and S. canadensis, and (3) ran pollination experiments with Cx. pipiens and T. vulgare in a greenhouse. We found (1) that 41 of 164 field-collected Cx. pipiens carried pollen, (2) that 48, 34, and 34 % of Cx. pipiens accumulated pollen from T. vulgare, A. millefolium, and S. canadensis, respectively, during floral visits of greenhouse-grown specimens, and (3) that cross-pollination by Cx. pipiens resulted in significant seed set of T. vulgare in pollination experiments. Based on our observations that Cx. pipiens are frequent floral visitors and are able to carry pollen and to induce seed set in T. vulgare, it is clear that Cx. pipiens plays a role in the pollination of T. vulgare, and possibly other members of the Asteraceae.     

Does nitrogen deposition affect soil microfungal diversity and soil N and P dynamics in a high Arctic ecosystem?

In a high Arctic polar semidesert ecosystem (ambient N deposition c. 0.1 g N m⁻² a⁻¹), the effects of N enrichment on the diversity of soil microfungi and on N content and availability in organic and mineral soils were determined. Three N (total: 0, 0.5, 5 g N m⁻² a⁻¹) and two P (total 0, 1 g m⁻² a⁻¹) treatments were applied, since P may limit response to N in this ecosystem. Organic and mineral soils were sampled in June and August in the second year of treatment for microfungi, pH, moisture content, and total N and P. In the third year, soils were resampled for extractable and total N and P. The fungi isolated were typical of high pH soils in the High Arctic and Antarctic. The species richness and diversity of soil microfungi were very low, with ranges as follows: Shannon diversity, 0.56–1.5; richness, 2–6; evenness, 0.79–0.9. There was no significant effect of treatment on the frequency of occurrence of different taxa of soil microfungi. Time of sampling also had no significant impact on fungal assemblages, although different, more diverse communities were isolated from organic, rather than mineral, soils. Nitrate-N in organic soil decreased significantly when P was added alone, but not when P and N were added together. Addition of 0.5 g N m⁻² a⁻¹, a rate deposition already occurring in Greenland and Iceland, appeared to exceed N demand even when P limitation was relieved. There was no apparent soil acidification as a result of the N treatments.     

PKC-alpha and TAK-1 are intermediates in the activation of c-Jun NH2-terminal kinase by hypoxia-reoxygenation

c-Jun NH(2)-terminal kinase (JNK), a member of the MAPK family of protein kinases, is a stress-response kinase that is activated by proinflammatory cytokines and growth factors coupled to membrane receptors or through nonreceptor pathways by stimuli such as heat shock, UV irradiation, protein synthesis inhibitors, and conditions that elevate the levels of reactive oxygen intermediates (ROI). Ischemia followed by reperfusion or hypoxia with reoxygenation represents a condition of high oxidative stress where JNK activation is associated with elevated ROI. We recently demonstrated that the activation of JNK by this condition is initiated by ROI generated by mitochondrial electron transport and involves sequential activation of the proline-rich kinase 2 and the small GTP-binding factors Rac-1 and Cdc42. Here we present evidence that protein kinase C (PKC) and transforming growth factor-beta-activated kinase-1 (TAK-1) are also components of this pathway. Inhibition of PKC with the broad-range inhibitor calphostin C, the PKC-alpha/beta-selective inhibitor Go9367, or adenovirus-expressing dominant-negative PKC-alpha blocked the phosphorylation of proline-rich kinase 2 and JNK. Reoxygenation activated the mitogen-activated protein kinase kinase kinase, TAK-1, and promoted the formation of a complex containing Rac-1, TAK-1, and JNK but not apoptosis-stimulating kinase-1 or p21-activated kinase-1, which was detected within the first 10 min of reoxygenation. These results identify two new components, PKC and TAK-1, that have not been previously described in this signaling pathway.     

Contrasting abundance and contribution of clonal proliferation to the population structure of the corkscrew sea anemone Bartholomea annulata in the tropical Western Atlantic

Clonal propagation is an important life history trait for many sessile organisms, and often leads to the formation of monoclonal aggregations. In the marine environment, sea anemones have been model species for testing theory regarding the evolution of sex and understanding the contribution of sexual versus asexual reproduction to the population structure in facultatively clonal animals. However, little attention has been paid to tropical actiniarians. The corkscrew anemone Bartholomea annulata is common in tropical marine habitats in the western Atlantic and Caribbean; it forms small aggregations (2–4 anemones) on coral reefs and larger aggregations (>10 anemones) in mangrove habitats. We used field surveys and molecular analyses to investigate patterns of distribution, abundance, and genetic structure of aggregations formed by B. annulata on a reef in the US Virgin Islands and in a unique mangrove habitat in the Florida Keys. Abundance was greatest at the abandoned rock quarry mangrove habitat in the Florida Keys, where anemones formed continuously distributed aggregations carpeting the exposed limestone walls. Genetic diversity assessed via intersimple sequence repeats (ISSRs) and six microsatellite loci revealed that asexual reproduction plays only a minor role in the formation of both small and large anemone aggregations. Specifically, ISSR analyses showed that only ~10% of anemone aggregations were clonal in the US Virgin Islands, while microsatellite genotyping identified clonality in only 1 of 35 aggregations. In the Florida Keys, only four clonal genotypes were recovered within aggregations, but eight clones, representing 33% of the total surveyed population, had individuals in multiple aggregations. Thus, population structure of B. annulata appears to rely primarily on sexual reproduction, although asexual reproduction may play a nontrivial role in some environments. Mechanisms that drive the formation of genotypically diverse aggregations remain unknown, but may include attraction toward conspecifics, shared use of preferred habitats, or the local retention of larvae in partially enclosed habitats.     

Abundance and clonal replication in the tropical corallimorpharian Rhodactis rhodostoma

Abstract. The corallimorpharian Rhodactis rhodostoma appears to be an opportunistic species capable of rapidly monopolizing patches of unoccupied shallow substrate on tropical reefs. On a fringing coral reef at Eilat, Israel, northern Red Sea, we examined patterns of abundance and clonal replication in R. rhodostoma in order to understand the modes and rates of spread of polyps across the reef flat. Polyps were abundant on the inner reef flat (maximum 1510 polyps m⁻² and 69% cover), rare on the outer reef flat, and completely absent on the outer reef slope at >3 m depth. Individuals cloned throughout the year via 3 distinct modes: longitudinal fission, inverse budding, and marginal budding. Marginal budding is a replicative mode not previously described. Cloning mode varied significantly with polyp size. Approximately 9% of polyps cloned each month, leading to a clonal doubling time of about 1 year. The rate of cloning varied seasonally and depended on day length and seawater temperature, except for a brief reduction in cloning during midsummer when polyps spawned gametes. Polyps of R. rhodostoma appear to have replicated extensively following a catastrophic low-tide disturbance in 1970, and have become an alternate dominant to stony corals on parts of the reef flat.     

Defining linkages between the GSC and NSF's LTER program: how the Ecological Metadata Language (EML) relates to GCDML and other outcomes

The Genomic Standards Consortium (GSC) invited a representative of the Long-Term Ecological Research (LTER) to its fifth workshop to present the Ecological Metadata Language (EML) metadata standard and its relationship to the Minimum Information about a Genome/Metagenome Sequence (MIGS/MIMS) and its implementation, the Genomic Contextual Data Markup Language (GCDML). The LTER is one of the top National Science Foundation (NSF) programs in biology since 1980, representing diverse ecosystems and creating long-term, interdisciplinary research, synthesis of information, and theory. The adoption of EML as the LTER network standard has been key to build network synthesis architectures based on high-quality standardized metadata. EML is the NSF-recognized metadata standard for LTER, and EML is a criteria used to review the LTER program progress. At the workshop, a potential crosswalk between the GCDML and EML was explored. Also, collaboration between the LTER and GSC developers was proposed to join efforts toward a common metadata cataloging designer's tool. The community adoption success of a metadata standard depends, among other factors, on the tools and trainings developed to use the standard. LTER's experience in embracing EML may help GSC to achieve similar success. A possible collaboration between LTER and GSC to provide training opportunities for GCDML and the associated tools is being explored. Finally, LTER is investigating EML enhancements to better accommodate genomics data, possibly integrating the GCDML schema into EML. All these action items have been accepted by the LTER contingent, and further collaboration between the GSC and LTER is expected.     

Quantitative traits associated with adaptation of three barley (Hordeum vulgare L) cultivars to suboptimal iron supply

A laboratory method was developed that allows determination of in situ net nitrification with high sensitivity and at high temporal resolution. Nitrate in soils is quantitatively converted into nitrous oxide under strictly anaerobic conditions in the presence of 10 kPa acetylene by the soil endogenous denitrifier population, with the N₂O detected by a gas chromatograph equipped with a ⁶³Ni electron capture detector. Thus, even low net nitrification rates, i.e. small net increases in soil nitrate concentrations can easily be detected. Comparison of results using this method with results obtained using the classical in situ incubation method (buried bag soil incubation) revealed excellent agreement. Application of the new method allowed both determination of the seasonal pattern of net nitrification as well as correlation analysis between in situ NO and N₂O flux rates and in situ net nitrification rates of the forest soils studied. Regardless of the forest site studied (spruce, spruce limed, beech), and during each year of a 3 years period (1995–1997), net nitrification varied strongly with season and was least during winter and greatest during summer. The long-term annual, mean rate of net nitrification for the untreated spruce site, the limed spruce site and the beech site were 1.54 ± 0.27 mg N kg^–1 sdw d^–1, 1.92 ± 0.23 mg N kg^–1 sdw d^–1 and 1.31 ± 0.23 mg N kg^–1 sdw d^–1, respectively. In situ rates of nitrification and NO and N₂O emission were strongly correlated for all sites suggesting that nitrification was the dominate source of NO as well as N₂O.     

Dispersal by bagworm larvae, Metisa plana: effects of population density, larval sex, and host plant attributes

Abstract 1. The work reported here investigated the incidence of dispersal by bagworm larvae Metisa plana (Walker) (Lepidoptera: Psychidae), a species with apterous females and winged males.
2. A mark–recapture study conducted in a plantation of oil palm revealed that a significant proportion of larvae undertakes multiple episodes of ballooning, and suggested that ballooning represents a strategy for foraging both within and between hosts.
3. Experiments conducted in a controlled cage environment indicated that increasing population density and defoliation of oil palm both promote dispersal by larvae.
4. Neonatal larvae typically remained on the host where they emerged, indicating that pupation site of late-instar females influences the distribution of their progeny, and suggesting a high level of genetic relatedness in localised bagworm populations.
5. Density-dependent dispersal may generate relatively uniform distributions of M. plana in plantations of oil palm, by simultaneously stabilising populations on heavily infested palms and redistributing larvae on lightly infested palms.
6. The rate of ballooning was greater for female than for male larvae, possibly resulting from the sex-specific impact of population density on reproductive success or from late-instar females seeking suitable oviposition sites for their future progeny.