New ecosystems in the deep subsurface follow the flow of water driven by geological activity.

Eukarya have been discovered in the deep subsurface at several locations in South Africa, but how organisms reach the subsurface remains unknown. We studied river-subsurface fissure water systems and identified Eukarya from a river that are genetically identical for 18S rDNA. To further confirm that these are identical species one metazoan species recovered from the overlying river interbred successfully with specimen recovered from an underlying mine at -1.4 km. In situ seismic simulation experiments were carried out and show seismic activity to be a major force increasing the hydraulic conductivity in faults allowing organisms to create ecosystems in the deep subsurface. As seismic activity is a non-selective force we recovered specimen of algae and Insecta that defy any obvious other explanation at a depth of -3.4 km. Our results show there is a steady flow of surface organisms to the deep subsurface where some survive and adapt and others perish. As seismic activity is also present on other planets and moons in our solar system the mechanism elucidated here may be relevant for future search and selection of landing sites in planetary exploration.

Fluctuations in populations of subsurface methane oxidizers in coordination with changes in electron acceptor availability.

The concentrations of electron donors and acceptors in the terrestrial subsurface biosphere fluctuate due to migration and mixing of subsurface fluids, but the mechanisms and rates at which microbial communities respond to these changes are largely unknown. Subsurface microbial communities exhibit long cellular turnover times and are often considered relatively static-generating just enough ATP for cellular maintenance. Here, we investigated how subsurface populations of CH4 oxidizers respond to changes in electron acceptor availability by monitoring the biological and geochemical composition in a 1339 m-below-land-surface (mbls) fluid-filled fracture over the course of both longer (2.5 year) and shorter (2-week) time scales. Using a combination of metagenomic, metatranscriptomic, and metaproteomic analyses, we observe that the CH4 oxidizers within the subsurface microbial community change in coordination with electron acceptor availability over time. We then validate these findings through a series of 13C-CH4 laboratory incubation experiments, highlighting a connection between composition of subsurface CH4 oxidizing communities and electron acceptor availability.

Eukaryotic opportunists dominate the deep-subsurface biosphere in South Africa.

Following the discovery of the first Eukarya in the deep subsurface, intense interest has developed to understand the diversity of eukaryotes living in these extreme environments. We identified that Platyhelminthes, Rotifera, Annelida and Arthropoda are thriving at 1.4 km depths in palaeometeoric fissure water up to 12,300 yr old in South African mines. Protozoa and Fungi have also been identified; however, they are present in low numbers. Characterization of the different species reveals that many are opportunistic organisms with an origin due to recharge from surface waters rather than soil leaching. This is the first known study to demonstrate the in situ distribution of biofilms on fissure rock faces using video documentation. Calculations suggest that food, not dissolved oxygen is the limiting factor for eukaryal population growth. The discovery of a group of Eukarya underground has important implications for the search for life on other planets in our solar system.

Nematoda from the terrestrial deep subsurface of South Africa.

Since its discovery over two decades ago, the deep subsurface biosphere has been considered to be the realm of single-cell organisms, extending over three kilometres into the Earth's crust and comprising a significant fraction of the global biosphere. The constraints of temperature, energy, dioxygen and space seemed to preclude the possibility of more-complex, multicellular organisms from surviving at these depths. Here we report species of the phylum Nematoda that have been detected in or recovered from 0.9-3.6-kilometre-deep fracture water in the deep mines of South Africa but have not been detected in the mining water. These subsurface nematodes, including a new species, Halicephalobus mephisto, tolerate high temperature, reproduce asexually and preferentially feed upon subsurface bacteria. Carbon-14 data indicate that the fracture water in which the nematodes reside is 3,000-12,000-year-old palaeometeoric water. Our data suggest that nematodes should be found in other deep hypoxic settings where temperature permits, and that they may control the microbial population density by grazing on fracture surface biofilm patches. Our results expand the known metazoan biosphere and demonstrate that deep ecosystems are more complex than previously accepted. The discovery of multicellular life in the deep subsurface of the Earth also has important implications for the search for subsurface life on other planets in our Solar System.

A new preparation method to study fresh plant structures with X-ray computed tomography.

Since the development of X-ray computed tomography as a medical diagnostic tool, it was adapted and extended for many scientific applications, including plant structure research. As for many biological studies, sample preparation is of major importance to obtain good-quality images. Therefore, we present a new preparation method for fresh material which includes critical point drying and heavy metal staining. This technique enhances the contrast of fresh tissues, prevents artefacts such as tissue compression, and requires no embedding.

The comparative cellular architecture of the female gonoduct among tylenchoidea (nematoda: tylenchina).

The cellular architecture of the female gonoduct of 68 nematode populations representing 42 species belonging to Tylenchidae, Belonolaimidae, Hoplolaimidae and Meloinema is shown to have an overall similarity in cellular gonoduct structure. The oviduct consists of two rows of four cells; the spermatheca is comprised of 10 to 20 cells, and the uterus cells, except in the case of Psilenchus, are arranged in four (Tylenchidae) or three (Belonolaimidae, Hoplolaimidae and Meloinema) regular rows. Although the genus Meloinema is classified within Meloidogynidae, its spermatheca is clearly hoplolaimid-like and lacks the spherical shape with lobe-like protruding cells typical of Meloidogyne. Detailed morphology of expelled gonoducts may provide a valuable character set in phylogenetic analysis, and the cellular morphology of the spermatheca appears to be a distinguishing feature at species level, especially in the genera Tylenchus and Geocenamus. Ultrastructural data on the oviduct-spermatheca region of Meloidogyne incognita complement light-microscopic (LM) results. The combination of LM of expelled organs and transmission electron microscopy (TEM) on selected sections is put forward as a powerful tool to combine three-dimensional knowledge with ultrastructural detail.

Evaluation of fixation methods for ultrastructural study of Caenorhabditis elegans embryos.

Various fixation methods for transmission electron microscopy (TEM) were tested on Caenorhabditis elegans embryos. By combining various techniques, using 3.4% chitinase in combination with 1% alpha-chymotripsin, we were able to establish a new fixation procedure that for the first time preserves both membranes and internal cellular ultrastructure of C. elegans embryos in different stages of development. This unique procedure will enable a hitherto unattainable standard for TEM research of C. elegans embryos. Sectioning of specific developmental stages fixed with this method allows a detailed study of ultrastructural aspects of embryogenesis.

Isolation, characterization and immunolocalization of phosphorylcholine-substituted glycolipids in developmental stages of Caenorhabditis elegans.

Caenorhabditis elegans displays three neutral glycosphingolipids with structural homology to glycosphingolipids from the porcine nematode parasite, Ascaris suum. The present findings extend the degree of structural conservation between the two nematode species to glycosphingolipids with a phosphodiester substitution. Using a combination of hydrofluoric acid pretreatment, immunochemical characterization and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry, three zwitterionic, phosphorylcholine-substituted glycosphingolipids could be identified in the neutral glycolipid fraction of C. elegans. The components were isolated as their zwitterionic, phosphorylcholine-substituted, pyridylaminated oligosaccharides by HPLC. Structural analysis was performed using hydrofluoric acid treatment, partial acid hydrolysis, methylation analysis, gas chromatography-mass spectrometry, cleavage with exoglycosidases and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. Their chemical structures are proposed as: component Nz1, GalNAc(beta1-4)[phosphorylcholine]GlcNAc(beta1-3)Man(beta1-4)Glc-cera mide; component Nz2, Gal(alpha1-3)GalNAc(beta1-4)[phosphorylcholine]-GlcNAc(beta1-3)Man(be ta1-4)Glc-ceramide; and component Nz3, Gal(beta1-3)- Gal(alpha1-3)GalNAc(beta1-4)[phosphorylcholine]GlcNAc(beta1-3)Man(bet a1-4)Glc-ceramide. The oligosaccharide core is characteristic of the biosynthetic arthro-carbohydrate series of protostomial glycosphingolipids. The ceramide moiety was specified by a d17 : 1 sphingoid-base with iso-branching and anteiso-branching, and 2-hydroxy, saturated fatty acids as represented by docosanoic and tetracosanoic acids. Analysis of the spatial and temporal expression of the phosphorylcholine epitope, during embryonic and postembryonic development, showed it to be localized predominantly in seam cells and basement membranes, respectively. In early embryonic ontogenesis the phosphorylcholine epitope was only lipid bound, while in late embryonic and postembryonic development this epitope was both lipid bound and protein bound.

Buccal capsule development as a consideration for phylogenetic analysis of Rhabditida (Nemata).

Bacterial feeding nematodes in the order Rhabditida including Zeldia punctata (Cephalobidae) and Caenorhabditis elegans (Rhabditidae) differ profoundly in the buccal capsule parts and associated cells. We carried out a range of tests to determine which buccal capsule parts and cells are evolutionarily homologous between the representative species of the two families. Tests included reconstruction of the buccal capsule and procorpus with transmission electron microscopy (TEM), nuclei position and morphology using 4, 6-diamidino-2-phenylindole (DAPI) staining, and cell lineage using four dimensional (4D) microscopy. The lining of the buccal capsule of Z. punctata and additional Cephalobidae includes four sets of muscular radial cells, ma, mb, mc and md, in contrast to C. elegans and additional Rhabditidae, which has two sets of epithelial cells (e1, e3) and two sets of muscle cells (m1, m2). Cell lineage of a nematode closely related to Z. punctata, Cephalobus cubaensis, supports the hypothesis that in cephalobids the e1 and e3 cells become hypodermal cells or are programmed to die. Our findings contradict all previous hypotheses of buccal capsule homology, and suggest instead that ma and mb in Z. punctata are homologous to m1 and m2 in C. elegans respectively. We also hypothesize that ma and mb could be homologous to primary and secondary sets of stylet-protractor muscle cells in the plant parasitic Tylenchida.

Germination of Bacillus thuringiensis spores in bacteriophagous nematodes (Nematoda: Rhabditida).

During screening of Bacillus thuringiensis isolates for nematicidal activity it was observed that spores of B. thuringiensis germinated in the intestine of bacteriophagous nematodes in the presence of antibiotics. This phenomenon was studied more closely by scanning electron microscopy. The nematodes were fed with bacterial spore-crystal mixtures in axenic culture medium supplemented with tetracyclin and chloramphenicol. Germination of spores was rare but was more frequently observed in Panagrellus redivivus than in other nematode species investigated. Germination of spores in the nematode intestine resulted in the colonization of the entire nematode within 24 hr. Crude nematode tissue preparations supported germination and subsequent growth of B. thuringiensis spores and vegetative cells. The mechanism for the loss of antibiotic activity in the nematode intestine is unknown. Since B. thuringiensis requires a nutrient-rich environment for reproduction, e.g., a cadaver, bacteriophagous nematodes may serve as suitable hosts for B. thuringiensis.

Tissue treatment for whole mount internal lectin staining in the nematodes Caenorhabditis elegans, Panagrolaimus superbus and Acrobeloides maximus.

Four different fixation schemes, using ten fluorescent-labelled lectins, were investigated for whole mount internal staining of three rhabditid nematodes: Caenorhabditis elegans, Panagrolaimus superbus and Acrobeloides maximus. Acetone-only fixation was found to give strong and reproducible staining, which could be prevented either by periodate treatment of the organisms or by specific inhibitory sugars of the lectins under investigation. Whereas the use of either phosphate or TRIS buffers had no effect on the staining pattern or the fluorescence intensity, the incubation time as well as the incubation temperature affected the staining reaction. The best results were obtained upon overnight incubation at 4 degrees C: the lectin staining could be inhibited in all cases, except for the intestinal brush border of C. elegans by the lectin of Lens culinaris.