Fossil preservation in the Neoproterozoic Doushantuo phosphorite Lagerstätte, South China

Phosphorites of the late Neoproterozoic Doushantuo Formation exposed in the vicinity of Weng'an, Guizhou Province, and Chadian, Shaanxi Province, South China, contain exceptionally well-preserved algal thalli, acritarchs, and globular microfossils interpreted as animal embryos. Combined optical microscopic and SEM observations provide insights into the taphonomy of phosphatized fossils. Algal cells and tissues are variably resistant to decay, and within preserved populations permineralization began at varying stages of degradation. In consequence, there is a spectrum of quality in cellular preservation. Algal cell walls, acritarch vesicles, and embryo envelopes are commonly encrusted by an isopachous rim of apatite, with cell interiors filled by collophane and later diagenetic dolomite. In contrast, blastomere surfaces of animal embryos are encrusted primarily by minute phosphatic spherules and filaments, possibly reflecting an immediately postmortem infestation of bacteria that provided nucleation sites for phosphate crystal growth. Thus, the same processes that gave rise to Phanerozoic phosphatized Lagerstätten - phosphatic encrustation, and impregnation, probably mediated by microbial activity - effected soft-tissue preservation in the Doushantuo Lagerstätte. It remains unclear how phosphatic ions and organic macro-molecules interact at the molecular level and to what extent specific microbial metabolisms or microenvironmental conditions control the phosphatization of soft tissues. New observations of phosphatized Doushantuo fossils include: a second locality (Chadian) for Wengania globosa , interpreted as an algal thallus and previously known only from Weng'an; microtunnels in Weng'an phosphorites interpreted as pyrite trails; and new taxa described from Weng'an: Meghystrichosphaeridium reticulatum (acritarch), Sarcinophycus radiatus (algal thallus), and one unnamed problematic form.     

Fossil preservation in the Neoproterozoic Doushantuo phosphorite Lagerstatte, South China.

Phosphorites of the late Neoproterozoic Doushantuo Formation exposed in the vicinity of Weng'an, Guizhou Province, and Chadian, Shaanxi Province, South China, contain exceptionally well-preserved algal thalli, acritarchs, and globular microfossils interpreted as animal embryos. Combined optical microscopic and SEM observations provide insights into the taphonomy of phosphatized fossils. Algal cells and tissues are variably resistant to decay, and within preserved populations permineralization began at varying stages of degradation. In consequence, there is a spectrum of quality in cellular preservation. Algal cell walls, acritarch vesicles, and embryo envelopes are commonly encrusted by an isopachous rim of apatite, with cell interiors filled by collophane and later diagenetic dolomite. In contrast, blastomere surfaces of animal embryos are encrusted primarily by minute phosphatic spherules and filaments, possibly reflecting an immediately postmortem infestation of bacteria that provided nucleation sites for phosphate crystal growth. Thus, the same processes that gave rise to Phanerozoic phosphatized Lagerstatten--phosphatic encrustation, and impregnation, probably mediated by microbial activity--effected soft-tissue preservation in the Doushantuo Lagerstatte. It remains unclear how phosphatic ions and organic macromolecules interact at the molecular level and to what extent specific microbial metabolisms or microenvironmental conditions control the phosphatization of soft tissues. New observations of phosphatized Doushantuo fossils include: a second locality (Chadian) for Wengania globosa, interpreted as an algal thallus and previously known only from Weng'an; microtunnels in Weng'an phosphorites interpreted as pyrite trails; and new taxa described from Weng'an: Meghystrichosphaeridium reticulatum (acritarch), Sarcinophycus radiatus (algal thallus), and one unnamed problematic form.     

现代丰年虾卵的腐解实验——对早期化石胚胎研究的启示

早寒武世和埃迪卡拉纪中的球状化石,一些已被归入可能的后生动物胚胎化石,由于具较为完好的三维保存方式以及近乎完美的胚胎发育序列,为早期后生动物的起源、分类、谱系演化及发育生物学提供了难得的实证材料。然而随着研究的深入,多数寒武纪胚胎的生物学分类位置未定;而数量异常巨大、又有独自的保存方式的晚元古代陡山沱组胚胎的真伪和生物学归属,更是争议未消。通过对现生生物胚胎的实验埋藏研究,可以揭示出各类生物胚胎在腐解、埋藏各阶段的保存潜力,而现代胚胎在各实验埋藏阶段形态、结构的变化,也能为化石胚胎的研究提供重要的实证材料。本文就是通过对虾卵胚胎各发育阶段腐解保存潜力的实验模拟研究,试图为球状化石的形成机制和化石归属提供一些实验室依据。     

Organic compositions of the embryo-bearing lowermost Cambrian Kuanchuanpu formation on the northern Yangtze platform, China

We investigated the origin of organic matter and paleoceanographic conditions of the embryo-bearing lowermost Cambrian Kuanchuanpu Formation in Shaanxi Province, China, in terms of sedimentary facies, fossil morphometry, and organic geochemistry, including biomarker compositions. The results indicated the abundance of algae and eukaryotic phytoplankton that fueled a diverse ecosystem including cnidarians and small shelly fossils. The water column contained enough oxygen to provide habitat for benthic cnidarians such as Olivooides. On the other hand, low pristane/phytane ratios, detection of squalane, and hopane composition indicate reductive conditions within the sediments, and that the redox front was likely near the water-sediment interface. Common occurrences of pyrite and barite indicate that redox reactions of sulfur species were activated at the water-sediment interface. Such circumstances and the high phosphate concentration in ambient water may recall the presence of sulfur-oxidizing bacteria that promoted the phosphatization of animal embryos, as suggested for in the Neoproterozoic Doushantuo Formation. Nitrogen was the limiting nutrient for primary productivity, and the high phosphate concentration in the ocean promoted the phosphatization of animal embryos at the reductive water–sediment interface.     

A new look at saponite formation and its implications for early animal records in the Ediacaran of South China

Acanthomorphic acritarch fossils, including some interpreted to be the fossils of the earliest animal embryos, first appear in the lower Doushantuo Formation of the Yangtze Gorges area (YGA). Further, the complete paleontological and geochemical record for the YGA has played a central role in defining the global biological and geochemical backdrop that presaged and witnessed the dawn of diverse animal life. Despite the importance of the YGA in our understanding of Neoproterozoic Earth history, basic aspects about its depositional history remain debated. Foremost among the controversies, extensively studied sections in the YGA were recently tied to deposition in an alkaline lake, casting new but contentious light on the environments of early animal evolution and the broader significance of geochemical records from the YGA. Arguments for a lacustrine setting hinged on the presence of trioctahedral clays (saponite–corrensite). However, this clay type commonly forms in other environments, including the weathering profiles of mafic and ultramafic volcanics. Using a coupled geochemical and sedimentological approach, we argue that the trioctahedral clays in the lower Doushantuo of the YGA are better explained as weathering products from a regional mafic‐to‐ultramafic hinterland delivered by rivers to a shelf or lagoon in the Yangtze Gorges Basin. These novel provenance relationships for YGA sediments and associated clays are consistent with a marine setting for the early animal records and must factor in our current understanding of the broader geochemical fabric of the Doushantuo Formation.     

Precambrian animal life: Taphonomy of phosphatized metazoan embryos from southwest China

Phosphatized fossils from the Neoproterozoic Doushantuo Formation have provided valuable insight into the early evolution of metazoans, but the preservation of these spectacular fossils is not yet fully understood. This research begins to address this issue by performing a detailed specimen-based taphonomic analysis of the Doushantuo Formation phosphatized metazoan embryos. A total of 206 embryos in 65 thin sections from the Weng'an Phosphorite Member of the Doushantuo Formation were examined and their levels of pre-phosphatization decay estimated. The data produced from this examination reveal a strong taphonomic bias toward earlier (2-cell and 4-cell) cleavage stages, which tend to be well-preserved, and away from later (8-cell and 16-cell) cleavage stages, which tend to exhibit evidence for slight to intense levels of organic decay. In addition, the natural abundances of these embryos tend to decrease with advancement in cleavage stage, and no evidence of more advanced (beyond 16-cell) cleavage stages or eventual adult forms were found in this study. One possible explanation for this taphonomic bias toward early cleavage stages is that later cleavage stages and adult forms were more physically delicate, allowing them to be more easily damaged during burial and reworking, allowing for more rapid decay. The spectacular preservation of these embryos was probably aided by their likely internal enrichment in phosphate-rich yolk, which would have caused their internal dissolved phosphate levels to reach critical levels with only miniscule organic decay, thereby hastening phosphatization. If internal sources of phosphate did indeed play a role in the phosphatization of these embryos, it may explain their prolific abundance in these rocks compared to other phosphatized fossils as well as indicating that metazoans lacking such internal phosphate sources were likely much more difficult to preserve. The phosphatic fossils of the Doushantuo Formation, therefore, provide an indispensable, yet restricted, window into Neoproterozoic life and metazoan origins.     

How did bacterial ancestors reproduce? Lessons from L‐form cells and giant lipid vesicles

In possible scenarios on the origin of life, protocells represent the precursors of the first living cells. To study such hypothetical protocells, giant vesicles are being widely used as a simple model. Lipid vesicles can undergo complex morphological changes enabling self‐reproduction such as growth, fission, and extra‐ and intravesicular budding. These properties of vesicular systems may in some way reflect the mechanism of reproduction used by protocells. Moreover, remarkable similarities exist between the morphological changes observed in giant vesicles and bacterial L‐form cells, which represent bacteria that have lost their rigid cell wall, but retain the ability to reproduce. L‐forms feature a dismantled cellular structure and are unable to carry out classical binary fission. We propose that the striking similarities in morphological transitions of L‐forms and giant lipid vesicles may provide insights into primitive reproductive mechanisms and contribute to a better understanding of the origin and evolution of mechanisms of cell reproduction. Editor's suggested further reading in BioEssays Synthesizing artificial cells from giant unilamellar vesicles: State‐of‐the art in the development of microfluidic technology Abstract     

Phosphatized multicellular algae in the Neoproterozoic Doushantuo Formation, China, and the early evolution of florideophyte red algae

Phosphatic sediments of the Late Neoproterozoic (ca. 600 million years old [Myr]) Doushantuo Formation at Weng'an, South China, contain fossils of multicellular algae preserved in anatomical detail. As revealed by light microscopy and scanning electron microscopy, these fossils include both simple pseudoparenchymatous thalli with apical growth but no cortex-medulla differentiation and more complex thalli characterized by cortex-medulla differentiation and structures interpretable as carposporophytes, suggesting a multiphasic life cycle. Simple pseudoparenchymatous thalli, represented by Wengania, Gremiphyca, and Thallophycoides, are interpreted as stem group florideophytes. In contrast, complex pseudoparenchymatous thalli, such as Thallophyca and Paramecia, compare more closely to fossil and living corallinaleans than to other florideophyte orders, although they also differ in some important aspects (e.g., lack of biocalcification). These more complex thalli are interpreted as early stem group corallinaleans that diverged before Paleozoic stem groups such as Arenigiphyllum, Petrophyton, Graticula, and Archaeolithophyllum. This phylogenetic interpretation implies that (1) the phylogenetic divergence between the Florideophyceae and its sister group, the Bangiales, must have taken place before Doushantuo time-an inference supported by the occurrence of bangialean fossils in Mesoproterozoic rocks; (2) the initial diversification of the florideophytes occurred no later than the Doushantuo time; and (3) the corallinalean clade had a "soft" (uncalcified) evolutionary history in the Neoproterozoic before evolving biocalcification in the Paleozoic and undergoing crown group diversification in the Mesozoic.     

Porphyromonas gingivalis as a Model Organism for Assessing Interaction of Anaerobic Bacteria with Host Cells

Anaerobic bacteria far outnumber aerobes in many human niches such as the gut, mouth, and vagina. Furthermore, anaerobic infections are common and frequently of indigenous origin. The ability of some anaerobic pathogens to invade human cells gives them adaptive measures to escape innate immunity as well as to modulate host cell behavior. However, ensuring that the anaerobic bacteria are live during experimental investigation of the events may pose challenges. Porphyromonas gingivalis, a Gram-negative anaerobe, is capable of invading a variety of eukaryotic non-phagocytic cells. This article outlines how to successfully culture and assess the ability of P. gingivalis to invade human umbilical vein endothelial cells (HUVECs). Two protocols were developed: one to measure bacteria that can successfully invade and survive within the host, and the other to visualize bacteria interacting with host cells. These techniques necessitate the use of an anaerobic chamber to supply P. gingivalis with an anaerobic environment for optimal growth.The first protocol is based on the antibiotic protection assay, which is largely used to study the invasion of host cells by bacteria. However, the antibiotic protection assay is limited; only intracellular bacteria that are culturable following antibiotic treatment and host cell lysis are measured. To assess all bacteria interacting with host cells, both live and dead, we developed a protocol that uses fluorescent microscopy to examine host-pathogen interaction. Bacteria are fluorescently labeled with 2'',7''-Bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein acetoxymethyl ester (BCECF-AM) and used to infect eukaryotic cells under anaerobic conditions. Following fixing with paraformaldehyde and permeabilization with 0.2% Triton X-100, host cells are labeled with TRITC phalloidin and DAPI to label the cell cytoskeleton and nucleus, respectively. Multiple images taken at different focal points (Z-stack) are obtained for temporal-spatial visualization of bacteria. Methods used in this study can be applied to any cultivable anaerobe and any eukaryotic cell type.     

Are the new Ediacaran Doushantuo Megasphaera-like acritarchs early metazoans?

A recent report by Chen et al. (2014) describes the new Ediacaran Doushantuo acritarchs with fascinating features. Nested within Megaclonophycus-like fossils that exhibit palintomic cell division are multicellular spheroid structures termed matryoshkas, which are interpreted to be germ cells. Such distinctive features of cellular differentiation and germ-soma separation suggest affinities of these with early stem group metazoans. In this commentary, I suggest some alternative interpretations to those elaborated by Chen et al. (2014).     

Dynamics of the Nuclear Complement of Giant Cells Induced by Meloidogyne incognita

The total numbers of nuclei in giant cells induced by Meloidogyne incognita in pea, lettuce, tomato, and broad bean were determined. Mature giant cells from pea had the most nuclei per giant cell with a mean of 59 ± 23, lettuce had the fewest with 26 ± 16, and tomato and broad bean were intermediate. The rate of increase in numbers of nuclei for all plant species was greatest during the first 7 days after inoculation. No mitotic activity was observed in giant cells associated with adult nematodes. Number of nuclei per giant cell doubled each day during the period of greatest mitotic activity, but number of total chromosomes per giant cell increased 20-fold per day at the same time. The hypothesis is presented that factor(s) responsible for the polyploid, mulfinucleate condition characteristic of giant cells may be different from factor(s) responsible for aneuploid numbers of chromosome per nucleus or for nuclear aberrations such as the presence of linked nuclei.     

Eukaryotic Algae Associated with Mid-Proterozoic Black Chert in Northern China

During the past two decades, Precambrian research, especially in the respect of the occurfence of microbial fossils in silicified rocks has been achieved. It is still in argument, however, at what time the origin of the eukaryote, one of the major events in biological evolution appeared according to the different criteria of the low, er eukaryotic organism fossils identified. The difference between prokaryotic and eukaryotic algal fossils in the cell size of structure and morphological colony and the model of their reproduction in biologic evolution is interpreted based on the knowledge about living lower organisms in this paper. Eight genera and eight species of eukaryotic algal fossils, including three genera and three species (Proto- cosmarium panduratum, Closteriopsis taihangensis and Phyllophycoma sinensis) newly descover ed in black stromatolitic chert from Gaoyuzhuang Formation, Lingqiu, Shanxi Province, China, which is about 1,400–1,600 My in age are described and named. All of these are characterized by the big cell size and complex structure of colony in which the cells have been divided into different function in physiology, and some of them produce endospore and autospore which are comparable with their mother cell or colony in morphology. According to the morphological characters of complex and diverse microfossils, it is assumed that the evolution of eukaryotic organisms had been achieved and even evolved up to a higher level at least before 1,600 million years.     

Dimorphism in methane seep-dwelling ecotypes of the largest known bacteria

We present evidence for a dimorphic life cycle in the vacuolate sulfide-oxidizing bacteria that appears to involve the attachment of a spherical Thiomargarita-like cell to the exteriors of invertebrate integuments and other benthic substrates at methane seeps. The attached cell elongates to produce a stalk-like form before budding off spherical daughter cells resembling free-living Thiomargarita that are abundant in surrounding sulfidic seep sediments. The relationship between the attached parent cell and free-living daughter cell is reminiscent of the dimorphic life modes of the prosthecate Alphaproteobacteria, but on a grand scale, with individual elongate cells reaching nearly a millimeter in length. Abundant growth of attached Thiomargarita-like bacteria on the integuments of gastropods and other seep fauna provides not only a novel ecological niche for these giant bacteria, but also for animals that may benefit from epibiont colonization.     

Distinguishing geology from biology in the Ediacaran Doushantuo biota relaxes constraints on the timing of the origin of bilaterians

The Ediacaran Doushantuo biota has yielded fossils that include the oldest widely accepted record of the animal evolutionary lineage, as well as specimens with alleged bilaterian affinity. However, these systematic interpretations are contingent on the presence of key biological structures that have been reinterpreted by some workers as artefacts of diagenetic mineralization. On the basis of chemistry and crystallographic fabric, we characterize and discriminate phases of mineralization that reflect: (i) replication of original biological structure, and (ii) void-filling diagenetic mineralization. The results indicate that all fossils from the Doushantuo assemblage preserve a complex mélange of mineral phases, even where subcellular anatomy appears to be preserved. The findings allow these phases to be distinguished in more controversial fossils, facilitating a critical re-evaluation of the Doushantuo fossil assemblage and its implications as an archive of Ediacaran animal diversity. We find that putative subcellular structures exhibit fabrics consistent with preservation of original morphology. Cells in later developmental stages are not in original configuration and are therefore uninformative concerning gastrulation. Key structures used to identify Doushantuo bilaterians can be dismissed as late diagenetic artefacts. Therefore, when diagenetic mineralization is considered, there is no convincing evidence for bilaterians in the Doushantuo assemblage.     

Giant membrane vesicles as a model to study cellular substrate uptake dissected from metabolism

In order to use giant vesicles for substrate uptake studies in metabolically important tissues, we characterized giant vesicles isolated from heart, liver, skeletal muscle and adipose tissue. We investigated which cell types and which plasma membrane regions are involved in giant vesicle formation and we examined the presence of transporters for metabolic substrates. Analysis of giant vesicles with markers specific for distinct cell types and distinct domains of the plasma membrane reveals that the plasma membrane of parenchymal cells, but not endothelial cells, are the source of the vesicle membranes. In addition, plasma membrane regions enriched in caveolae and involved in docking of recycling vesicles from the endosomal compartment are retained in giant vesicles, indicating that KCl-induced alterations in recycling processes are involved in giant vesicle formation. Giant vesicles contain vesicular lumen consisting of the soluble constituents of the cytoplasm including, fatty-acid binding proteins. Furthermore, giant vesicles isolated from heart, liver, skeletal muscle and adipose tissue are similar in size (10–15 m) and shape and do not contain subcellular organelles, providing the advantage that substrate fluxes in the different organs can be studied independently of the surface/volume ratio but most importantly in the absence of intracellular metabolism.     

Enhanced levels of plant cell cycle inhibitors hamper root-knot nematode-induced feeding site development

Root-knot nematodes (RKN) are highly specialized, obligatory plant parasites. These animals reprogram root cells to form large, multinucleate, and metabolically active feeding cells (giant cells) that provide a continuous nutrient supply during 3–6 weeks of the nematode’s life. The establishment and maintenance of physiologically fully functional giant cells are necessary for the survival of these nematodes. As such, giant cells may be useful targets for applying strategies to reduce damage caused by these nematodes, aiming the reduction of their reproduction. We have recently reported the involvement of cell cycle inhibitors of Arabidopsis, named Kip-Related Proteins (KRPs), on nematode feeding site ontogeny. Our results have demonstrated that this family of cell cycle inhibitors can be envisaged to efficiently disrupt giant cell development, based on previous reports which showed that alterations in KRP concentration levels can induce cell cycle transitions. Herein, we demonstrated that by overexpressing KRP genes, giant cells development is severely compromised as well as nematode reproduction. Thus, control of root-knot nematodes by modulating cell cycle-directed pathways through the enhancement of KRP protein levels may serve as an attractive strategy to limit damage caused by these plant parasites.     

Cell-free synthesis of membrane proteins: Tailored cell models out of microsomes

Incorporation of proteins in biomimetic giant unilamellar vesicles (GUVs) is one of the hallmarks towards cell models in which we strive to obtain a better mechanistic understanding of the manifold cellular processes. The reconstruction of transmembrane proteins, like receptors or channels, into GUVs is a special challenge. This procedure is essential to make these proteins accessible to further functional investigation. Here we describe a strategy combining two approaches: cell-free eukaryotic protein expression for protein integration and GUV formation to prepare biomimetic cell models. The cell-free protein expression system in this study is based on insect lysates, which provide endoplasmic reticulum derived vesicles named microsomes. It enables signal-induced translocation and posttranslational modification of de novo synthesized membrane proteins. Combining these microsomes with synthetic lipids within the electroswelling process allowed for the rapid generation of giant proteo-liposomes of up to 50 μm in diameter. We incorporated various fluorescent protein-labeled membrane proteins into GUVs (the prenylated membrane anchor CAAX, the heparin-binding epithelial growth factor like factor Hb-EGF, the endothelin receptor ETB, the chemokine receptor CXCR4) and thus presented insect microsomes as functional modules for proteo-GUV formation. Single-molecule fluorescence microscopy was applied to detect and further characterize the proteins in the GUV membrane. To extend the options in the tailoring cell models toolbox, we synthesized two different membrane proteins sequentially in the same microsome. Additionally, we introduced biotinylated lipids to specifically immobilize proteo-GUVs on streptavidin-coated surfaces. We envision this achievement as an important first step toward systematic protein studies on technical surfaces.     

Signal diffusion and the mitigation of social exploitation in pneumococcal competence signalling

Quorum sensing (QS) in bacteria is thought to enable populations of cells to coordinately and cooperatively regulate gene expression for traits that confer group benefits. While this view has strong empirical and theoretical support, it is increasingly appreciated that QS under natural conditions may be incapable of monitoring bacterial numbers and, furthermore, that QS is evolutionarily unstable owing to conflicts of interest among competing cells. An alternative hypothesis, termed diffusion sensing (DS), proposes that autoinducer secretion monitors the diffusive properties of the local environment, with benefits that are directly realized by individual cells rather than populations. Here, we test central predictions of this hypothesis using the competence signalling system of Streptococcus pneumoniae as our model, which regulates the induction of natural transformation by the secretion and detection of a small diffusible peptide, CSP (competence-stimulating peptide). By experimentally manipulating the diffusive properties of the growth medium, we found that there is no fixed quorum for competence induction. Instead, induction cell density scales with diffusivity. In agreement with QS and DS expectations, we show that the benefit of signal exploitation by mutant cells that can use but not secrete CSP is strongly frequency-dependent. However, we also find that the magnitude of this benefit declines significantly as diffusion is reduced, a result more consistent with the predictions of DS. Together, these data provide strong support for the DS hypothesis for autoinducer response systems. More specifically, our results imply that autonomous rather than group benefits should be sought in order to more completely understand the role and evolution of CSP signalling in pneumococci.     

Membrane processes and biophysical characterization of living cells decorated with chromatic polydiacetylene vesicles

The structural complexity of the cell membrane makes analysis of membrane processes in living cells, as compared to model membrane systems, highly challenging. Living cells decorated with surface-attached colorimetric/fluorescent polydiacetylene patches might constitute an effective platform for analysis and visualization of membrane processes in situ. This work examines the biological and chemical consequences of plasma membrane labeling of promyelocytic leukemia cells with polydiacetylene. We show that the extent of fusion between incubated lipid/diacetylene vesicles and the plasma membrane is closely dependent upon the lipid composition of both vesicles and cell membrane. In particular, we find that cholesterol presence increased bilayer fusion between the chromatic vesicles and the plasma membrane, suggesting that membrane organization plays a significant role in the fusion process. Spectroscopic data and physiological assays show that decorating the cell membrane with the lipid/diacetylene patches reduces the overall lateral diffusion within the membrane bilayer, however polydiacetylene labeling does not adversely affect important cellular metabolic pathways. Overall, the experimental data indicate that the viability and physiological integrity of the surface-engineered cells are retained, making possible utilization of the platform for studying membrane processes in living cells. We demonstrate the use of the polydiacetylene-labeled cells for visualizing and discriminating among different membrane interaction mechanisms of pharmaceutical compounds.