An appraisal of simple tree-mounted shelters for non-lethal monitoring of weta (Orthoptera: Anostostomatidae and Rhaphidophoridae) in New Zealand nature reserves

When monitoring rare insect species, or when surveying faunas within nature reserves, it is desirable not to use indiscriminate lethal sampling techniques. In this investigation we assessed the usefulness of simple tree-mounted wooden shelters to monitor endemic weta (Orthoptera) in nature reserves in Canterbury, New Zealand. Fifty shelters were placed out at six sites and examined at three-monthly intervals for a year. A wide variety of invertebrates were found utilizing the shelters, with Arachnida, Blattodea and Collembola being the most common occupants. After three months over 80% of the shelters exhibited signs of use by invertebrates, increasing to 96% after 12 months. Only seven tree weta (Anostostomatidae) and one (dead) ground weta (Hemiandrus sp.) were observed in the shelters over the full 12 month period. There were 52 observations of cave weta (Rhaphidophoridae) in the shelters, 36 of which occurred at one site, Orton Bradley Park. Occupation of the shelters by cave weta was not affected by soil conditions, light intensity or aspect of the shelter. However, cave weta exhibited a preference for shelters less than 50 cm above the ground and for shelters attached to kanuka and vines. Although weta were found in only a small proportion (9%) of the shelters, this method proved useful in confirming the presence of weta without risk of harming vulnerable populations. These shelters are inexpensive and easy to manufacture and have potential for long-term non-lethal monitoring of weta and as a collection/carriage device for live specimens used in conservation translocations.     

In vitro FRAP identifies the minimal requirements for Mad2 kinetochore dynamics

BACKGROUND: Mad1 and Mad2 are constituents of the spindle-assembly checkpoint, a device coupling the loss of sister-chromatid cohesion at anaphase to the completion of microtubule attachment of the sister chromatids at metaphase. Fluorescence recovery after photobleaching (FRAP) revealed that the interaction of cytosolic Mad2 with kinetochores is highly dynamic, suggesting a mechanism of catalytic activation of Mad2 at kinetochores followed by its release in a complex with Cdc20. The recruitment of cytosolic Mad2 to kinetochores has been attributed to a stable receptor composed of a distinct pool of Mad2 tightly bound to Mad1. Whether specifically this interaction accounts for the kinetochore dynamics of Mad2 is currently unknown. RESULTS: To gain a precise molecular understanding of the interaction of Mad2 with kinetochores, we reconstituted the putative Mad2 kinetochore receptor and developed a kinetochore recruitment assay with purified components. When analyzed by FRAP in vitro, this system faithfully reproduced the previously described in vivo dynamics of Mad2, providing an unequivocal molecular account of the interaction of Mad2 with kinetochores. Using the same approach, we dissected the mechanism of action of p31(comet), a spindle-assembly checkpoint inhibitor. CONCLUSIONS: In vitro FRAP is a widely applicable approach to dissecting the molecular bases of the interaction of a macromolecule with an insoluble cellular scaffold. The combination of in vitro fluorescence recovery after photobleaching with additional fluorescence-based assays in vitro can be used to unveil mechanism, stoichiometry, and kinetic parameters of a macromolecular interaction, all of which are important for modeling protein interaction networks.     

Changes and Tracking of Fat Mass in Pubertal Girls

Puberty is a critical period in body composition development. The influence of puberty on the development of fat mass asks for further investigation. We investigated the development of fat mass during puberty in a longitudinal prospective study in 152 healthy nonobese white girls, initial ages between 9 to 12 years. The influence of menarcheal age and the existing of tracking of fat mass have been analyzed. In 10 years time, participants were measured on eight time points. Various anthropometric data were collected, breast development was staged according to Tanner and body composition was determined with the dual‐energy X‐ray absorptiometry (DXA) scan. Calculations were made with the use of a linear mixed model. Fat mass increases from 7.9 kg (23.6%) at B1 to 18.5 kg (29.3%) at B5. Fat mass is higher in girls with an early menarche than in girls with a late menarche from B2. Girls in the quartile with initially the lowest fat mass have a chance of being in the same quartile after 10 years of 77% (P < 0.001). Girls in the quartile with initially the highest fat mass, have a risk of staying in the highest quartile of 55% (P < 0.001). Menarcheal age is of great influence on the development of fat mass. Girls with an early menarche, will have a bigger fat mass, especially at the end of puberty. Tracking of fat mass exists: a high amount of fat mass in early puberty will continue to exist at young adulthood.     

Localization of synthesis of beta1,6-glucan in Saccharomyces cerevisiae

Beta1,6-Glucan is a key component of the yeast cell wall, interconnecting cell wall proteins, beta1,3-glucan, and chitin. It has been postulated that the synthesis of beta1,6-glucan begins in the endoplasmic reticulum with the formation of protein-bound primer structures and that these primer structures are extended in the Golgi complex by two putative glucosyltransferases that are functionally redundant, Kre6 and Skn1. This is followed by maturation steps at the cell surface and by coupling to other cell wall macromolecules. We have reinvestigated the role of Kre6 and Skn1 in the biogenesis of beta1,6-glucan. Using hydrophobic cluster analysis, we found that Kre6 and Skn1 show significant similarities to family 16 glycoside hydrolases but not to nucleotide diphospho-sugar glycosyltransferases, indicating that they are glucosyl hydrolases or transglucosylases instead of genuine glucosyltransferases. Next, using immunogold labeling, we tried to visualize intracellular beta1,6-glucan in cryofixed sec1-1 cells which had accumulated secretory vesicles at the restrictive temperature. No intracellular labeling was observed, but the cell surface was heavily labeled. Consistent with this, we could detect substantial amounts of beta1,6-glucan in isolated plasma membrane-derived microsomes but not in post-Golgi secretory vesicles. Taken together, our data indicate that the synthesis of beta1, 6-glucan takes place largely at the cell surface. An alternative function for Kre6 and Skn1 is discussed.     

A novel role for gag as a cis-acting element regulating RNA structure,dimerization and packaging in HIV-1 lentiviral vectors

Clinical usage of lentiviral vectors is now established and increasing but remains constrained by vector titer with RNA packaging being a limiting factor. Lentiviral vector RNA is packaged through specific recognition of the packaging signal on the RNA by the viral structural protein Gag. We investigated structurally informed modifications of the 5′ leader and gag RNA sequences in which the extended packaging signal lies, to attempt to enhance the packaging process by facilitating vector RNA dimerization, a process closely linked to packaging. We used in-gel SHAPE to study the structures of these mutants in an attempt to derive structure-function correlations that could inform optimized vector RNA design. In-gel SHAPE of both dimeric and monomeric species of RNA revealed a previously unreported direct interaction between the U5 region of the HIV-1 leader and the downstream gag sequences. Our data suggest a structural equilibrium exists in the dimeric viral RNA between a metastable structure that includes a U5–gag interaction and a more stable structure with a U5–AUG duplex. Our data provide clarification for the previously unexplained requirement for the 5′ region of gag in enhancing genomic RNA packaging and provide a basis for design of optimized HIV-1 based vectors.     

Olfactory sensitivity in tsetse flies: a daily rhythm

The diurnal tsetse Glossina morsitans morsitans bites especially in early morning and late afternoon; around midday feeding is at a low. In laboratory apparatus that measures the amount of locomotion under constant conditions over the photophase, the flies display a similar patterning of activity levels. The profile of daily rhythms for G. morsitans reported in the literature includes a number of motor and sensory motor systems that fluctuate cophasically. Lacking is a study on the patterning of the senses' response levels. In this paper we present the first instance of a daily modulation in the sense of smell. We stimulated the antennae with concentration series of host-derived odours and measured the spiking rate of cells at different times during the photophase. The concentration-response curves suggest that the sensitivity of antennal olfactory cells flows in parallel with the other daily rhythms. This was also reflected in electroantennograms (EAGs). The electroantennography was extended to G. fuscipes fuscipes, whose level of spontaneous locomotor activity--instead of following a U- shaped pattern--rises gradually over the photophase. Again, the EAGs appeared to parallel the species' locomotor activity. What we believe happens is that the organism tones down the sensitivity of its odour receptors during periods of anticipated inactivity for reasons of economy.      

Contrasting nutrient exports from a forested and an agricultural catchment in south-eastern Australia

Dissolved organic carbon (DOC) and total and inorganic nitrogen and phosphorus concentrations were determined over 3 years in headwater streams draining two adjacent catchments. The catchments are currently under different land use; pasture/grazing vs plantation forestry. The objectives of the work were to quantify C and nutrient export from these landuses and elucidate the factors regulating export. In both catchments, stream water dissolved inorganic nutrient concentrations exhibited strong seasonal variations. Concentrations were highest during runoff events in late summer and autumn and rapidly declined as discharge increased during winter and spring. The annual variation of stream water N and P concentrations indicated that these nutrients accumulated in the catchments during dry summer periods and were flushed to the streams during autumn storm events. By contrast, stream water DOC concentrations did not exhibit seasonal variation. Higher DOC and NO₃ ⁻ concentrations were observed in the stream of the forest catchment, reflecting greater input and subsequent breakdown of leaf-litter in the forest catchment. Annual export of DOC was lower from the forested catchment due to the reduced discharge from this catchment. In contrast however, annual export of nitrate was higher from the forest catchment suggesting that there was an additional NO₃ ⁻ source or reduction of a NO₃ ⁻ sink. We hypothesize that the denitrification capacity of the forested catchment has been significantly reduced as a consequence of increased evapotranspiration and subsequent decrease in streamflow and associated reduction in the near stream saturated area.