Barcoding rotifer biodiversity in Mediterranean ponds using diapausing egg banks

The biodiversity of Mediterranean freshwater bodies is among the most threatened worldwide; therefore, its accurate estimation is an urgent issue. However, traditional methods are likely to underestimate freshwater zooplankton biodiversity due to its high species seasonality and cryptic diversity. We test the value of applying DNA barcoding to diapausing egg banks, in combination with the creation of a reference collection of DNA barcodes using adult individual samples, to characterize rotifer communities. We use monogonont rotifers from two lakes in Doñana National Park and one from Ruidera Natural Park in Spain as models to create a reference collection of DNA barcodes for taxonomically diagnosed adult individuals sampled from the water column, to compare with the sequences obtained from individual eggs from the diapausing egg banks. We apply two different approaches to carry out DNA taxonomy analyses, the generalized mixed Yule coalescent method (GMYC) and the Automatic Barcode Gap Discovery (ABGD), to the obtained sequences and to publicly available rotifer sequences. We obtained a total of 210 new rotifer COI sequences from all three locations (151 diapausing eggs and 59 adults). Both GMYC and ABGD generated the same 35 operational taxonomic units (OTUs), revealing four potential cryptic species. Most sequences obtained from diapausing eggs (85%) clustered with sequences obtained from morphologically diagnosed adults. Our approach, based on a single sediment sample, retrieved estimates of rotifer biodiversity higher than or similar to those of previous studies based on a number of seasonal samples. This study shows that DNA barcoding of diapausing egg banks is an effective aid to characterize rotifer diversity in Mediterranean freshwater bodies

Neurone decapping characterization by atomic force microscopy: A topological systematic analysis

WE tested a new approach to cell decapping on rat cerebellar neurones, and observed its effects on cell topography by atomic force microscopy (AFM). The results clearly demonstrate the effectiveness of our decapping approach, and also the ability of AFM to reveal fine details of the decapped cells. Specifically, varying the conditions and duration of the decapping process modifies the extent of the decapping. Such a method can be used to investigate the cytoplasm with surface sensitive techniques

X-Ray Secondary-Emission Microscopy (Xsem) of Neurons

We present the first X-ray secondary (photoelectron) emission microscopy (XSEM) pictures and video microimages of an uncoated and unstained neuron specimen. This novel kind of synchrotron radiation microscopy is suitable for local chemical analysis with a lateral resolution in the micron range. We explored the details of the neuron system, demonstrated chemical contrast by scanning the photon energy, studied in real time the photoelectron emitting properties of the specimen's components, and made preliminary tests of the radiation damage. These results significantly enhance the potential role of photoemission techniques in the life sciences and specifically in neurobiology

Native and modified uncoated neurons observed by atomic force microscopy

Dried, fixed uncoated neuron granule cells and their neurites have been imaged in air by an atomic force microscope (AFM) in the repulsive regime of contact mode. Neurons have also been observed after swelling with glutamate or decapping. Modifications induced by glutamate resulted in a drastic reduction in the cell body height (from 1.5 to 2.0 mu m for the untreated ones down to 0.7-1.0 mu m) and in an increased corrugation of the granule cell membrane. In the decapped neurons AFM revealed decreasing cell body height for an increasing amount of removed material during the decapping procedure. These results demonstrate that AFM is the ideal technique to observe cell corrugation and height modifications during cell treatments of neurobiological interest. (C) 1996 American Vacuum Society

Photoemission analysis of chemical differences between the membrane and cytoplasm of neuronal cells

We demonstrate the possibility of using x-ray photoemission spectroscopy (XPS) to distinguish between the chemical properties of the neuron membrane and of the cytoplasm in brain cells. This was possible by analysing cells de-capped by using a newly developed technique. The XPS spectra clearly show chemical differences between the cell cytoplasm and the membrane, for example between nitrogen in -NH2 and -NH3, which are characteristic of the proteins and membrane phospholipids, respectively. A preliminary (and not conclusive) interpretation of all the observed spectroscopic features is given

Scanning Photoemission Microscopy on Maximum Reaches 0.1 Micron Resolution

We present the first results from the upgraded version of the scanning photoemission spectromicroscope MAXIMUM, bared on synchrotron undulator fight and on a multilayer-coated Schwarzschild objective. The upgrade involved nearly all parts of the instrument, notably the beamline and the electron analysis system. Micro-images of Fresnel zone plates and of metal test patterns on semiconductor substrates reached a new record in lateral resolution, well beyond 0.1 micron. The first spectromicroscopy tests were also successfully performed on the new instrument, with analysis of f and d core levels in different systems