Fluid-deposited graphite and its geobiological implications in early Archean gneiss from Akilia, Greenland

Graphite, interpreted as altered bioorganic matter in an early Archean, ca. 3.83‐Ga‐old quartz‐amphibole‐pyroxene gneiss on Akilia Island, Greenland, has previously been claimed to be the earliest trace of life on Earth. Our petrographic and Raman spectroscopy data from this gneiss reveal the occurrence of graphitic material with the structure of nano‐crystalline to crystalline graphite in trails and clusters of CO₂, CH₄ and H₂O bearing fluid inclusions. Irregular particles of graphitic material without a fluid phase, representing decrepitated fluid inclusions are common in such trails too, but occur also as dispersed individual or clustered particles. The occurrence of graphitic material associated with carbonic fluid inclusions is consistent with an abiologic, fluid deposited origin during a poly‐metamorphic history. The evidence for fluid‐deposited graphitic material greatly complicates any claim about remnants of early life in the Akilia rock.     

A new occurrence of ambient inclusion trails from the ~1900‐million‐year‐old Gunflint Formation,Ontario: nanocharacterization and testing of potential formation mechanisms

Ambient inclusion trails (AITs) are tubular microstructures thought to form when a microscopic mineral crystal is propelled through a fine‐grained rock matrix. Here, we report a new occurrence of AITs from a fossilized microbial mat within the 1878‐Ma Gunflint Formation, at Current River, Ontario. The AITs are 1–15 μm in diameter, have pyrite as the propelled crystal, are infilled with chlorite and have been propelled through a microquartz (chert) or chlorite matrix. AITs most commonly originate at the boundary between pyrite‐ and chlorite‐rich laminae and chert‐filled fenestrae, with pyrite crystals propelled into the fenestrae. A subset of AITs originate within the fenestrae, rooted either within the chert or within patches of chlorite. Sulphur isotope data (³⁴S/³²S) obtained in situ from AIT pyrite have a δ³⁴S of ?8.5 to +8.0 ‰, indicating a maximum of ~30 ‰ fractionation from Palaeoproterozoic seawater sulphate (δ³⁴S ≈ +20 ‰). Organic carbon is common both at the outer margins of the fenestrae and in patches of chlorite where most AITs originate, and can be found in smaller quantities further along some AITs towards the terminal pyrite grain. We infer that pyrite crystals now found within the AITs formed via the action of heterotrophic sulphate‐reducing bacteria during early diagenesis within the microbial mat, as pore waters were becoming depleted in seawater sulphate. Gases derived from this process such as CO₂ and H₂S were partially trapped within the microbial mat, helping produce birds‐eye fenestrae, while rapid microquartz precipitation closed porosity. We propose that propulsion of the pyrite crystals to form AITs was driven by two complementary mechanisms during burial and low‐grade metamorphism: firstly, thermal decomposition of residual organic material providing CO₂, and potentially CH₄, as propulsive gases, plus organic acids to locally dissolve the microquartz matrix; and secondly, reactions involving clay minerals that potentially led to enhanced quartz solubility, plus increases in fluid and/or gas pressure during chlorite formation, with chlorite then infilling the AITs. This latter mechanism is novel and represents a possible way to generate AITs in environments lacking organic material.     

Septate‐tubular textures in 2.0‐Ga pillow lavas from the Pechenga Greenstone Belt: a nano‐spectroscopic approach to investigate their biogenicity

Pillow lava rims and interpillow hyaloclastites from the upper part of the Pechenga Greenstone Belt, Kola Peninsula, N‐Russia contain rare tubular textures 15–20 μm in diameter and up to several hundred μm long in prehnite–pumpellyite to lower greenschist facies meta‐volcanic glass. The textures are septate with regular compartments 5–20 μm across and exhibit branching, stopping and no intersecting features. Synchrotron micro‐energy dispersive X‐ray was used to image elemental distributions; scanning transmission X‐ray microscopy, Fe L‐edge and C K‐edge were used to identify iron and carbon speciation at interfaces between the tubular textures and the host rock. In situ U–Pb radiometric dating by LA‐MC‐ICP‐MS (laser ablation multicollector inductively coupled plasma mass spectrometry) of titanite from pillow lavas yielded a metamorphic age of 1790 ± 89 Ma. Focused ion‐beam milling combined with transmission electron microscopy was used to analyze the textures in three dimensions. Electron diffraction showed that the textures are mineralized by orientated pumpellyite. On the margins of the tubes, an interface between mica or chlorite and the pumpellyite shows evidence of dissolution reactions where the pumpellyite is replaced by mica/chlorite. A thin poorly crystalline Fe‐phase, probably precipitated out of solution, occurs at the interface between pumpellyite and mica/chlorite. This sequence of phases leads to the hypothesis that the tubes were initially hollow, compartmentalized structures in volcanic glass that were mineralized by pumpellyite during low‐grade metamorphism. Later, a Fe‐bearing fluid mineralized the compartments between the pumpellyite and lastly the pumpellyite was partially dissolved and replaced by chlorite during greenschist metamorphism. The most plausible origin for a septate‐tubular texture is a progressive etching of the host matrix by several generations of microbes and subsequently these tubes were filled by authigenic mineral precipitates. This preserves the textures in the rock record over geological time. The micro textures reported here thus represent a pumpellyite‐mineralized trace fossil that records a Paleoproterozoic sub‐seafloor biosphere.     

Ambient Inclusion Trails (AITs) from the Neoproterozoic Gangolihat Formation,Lesser Himalaya,India

Discovery of well-preserved Ambient Inclusion Trails (AITs) is reported for the first time in India from stromatolitic dolomite unit of the Neoproterozoic Gangolihat Formation, Kumaun Lesser Himalaya. AITs are distinct microtubular structures formed by migration of a mineral in a rock substrate. They exhibit several noncrystallographic morphologies such as curved and helical types among others. Mode of field occurrence, association, and petrographic textures suggest that AITs are indigenous to the host rock. Based on the physico-chemical conditions necessary for the formation of AITs, involvement of a biological process is considered for the genesis of AITs in the Gangolihat Formation.     

Garnet-filled trails associated with carbonaceous matter mimicking microbial filaments in Archean basalt

The study of the earliest traces of life on Earth can be complicated by abiotically formed biomorphs. We report here the finding of clustered micrometer-sized filaments of iron- and calcium-rich garnets associated with carbonaceous matter in an agate amygdale from a 2.7-billion-year-old basalt of the Maddina Formation, Western Australia. The distribution of carbonaceous matter and the mineral phases composing the filaments were analyzed using a combination of confocal laser scanning microscopy, laser-Raman micro-spectroscopy, focused ion beam sectioning and transmission electron microscopy. The results allow consideration of possible biogenic and abiotic processes that produced the filamentous structures. The filaments have a range of sizes, morphologies and distributions similar to those of certain modern iron-mineralized filamentous bacteria and some ancient filamentous structures interpreted as microfossils. They also share a high morphological similarity with tubular structures produced by microbial boring activity. However, the microstructures and the distribution of carbonaceous matter are more suggestive of an abiotic origin for the filaments. They are characteristic features of trails produced by the displacement of inclusions associated with local dissolution of their silica matrix. Organic compounds found in kerogen or bitumen inclusions may have contributed significantly to the dissolution of the quartz (or silica gel) matrix driving filamentous growth. Discriminating the products of such abiotic organic-mediated processes from filamentous microfossils or microbial borings is important to the interpretation of the scarce Precambrian fossil record and requires investigation down to the nanoscale.     

Soil-borne wheat mosaic virus inclusion bodies: structural, compositional and staining properties

Anatomy and cytochemistry of inclusion bodies induced by Soil‐borne wheat mosaic virus infection were studied in roots and leaves to learn more about the nature of inclusions and their roles in pathogenesis. Acid Fuchsin, Giemsa stain, Toluidine Blue and Trypan Blue stains facilitated visualization of inclusion bodies. Combined, simultaneous staining with Acid Fuchsin and Toluidine Blue clearly differentiated inclusion bodies from host nuclei. The overall anatomy, composition and structure of virus inclusions in leaves and roots were generally similar, as shown by phase contrast, differential interference contrast, epifluorescence, laser scanning confocal and transmission electron microscopy. Both were often closely associated with host nuclei; both were comprised of intertwined masses of tubular material, presumably endoplasmic reticulum, and in which varied numbers and sizes of vacuolar cavities occurred. Leaf inclusions, however, were typically larger and more vacuolate than those in roots. Lipids were found to be significant constituents of both the tubular and vacuolar components of inclusions, indicated by positive staining with Nile Red and Sudan Black. Inclusion bodies in both leaves and roots lost their structural and compositional integrity, eventually becoming disorganized and devoid of clearly identifiable components as host tissue aged and symptom expression advanced. Significant results of this study include the first published examination of virus inclusion bodies in root tissue, the degree of structural detail of inclusion body anatomy revealed by laser scanning confocal microscopy and the presence of an extensive lipid component in virus inclusion bodies.     

Deconstructing Earth’s oldest ichnofossil record from the Pilbara Craton,West Australia: Implications for seeking life in the Archean subseafloor

Microtextures of titanite (CaTiSiO₅) in exceptionally preserved Archean pillow lavas have been proposed as the earliest examples of microbial ichnofossils. An origin from microbial tunneling of seafloor volcanic glass that is subsequently chloritized and the tunnels infilled by titanite has been argued to record the activities of subseafloor microbes. We investigate the evidence in pillow lavas of the 3.35 Ga Euro Basalt from the Pilbara Craton, Western Australia, to evaluate the biogenicity of the microtextures. We employ a combination of light microscopy and chlorite mineral chemical analysis by EPMA (electron probe micro‐analysis) to document the environment of formation and analyze their ultrastructure using FIB‐TEM (focussed ion beam combined with transmission electron microscopy) to investigate their mode of growth. Petrographic study of the original and re‐collected material identified an expanded range of titanite morphotypes along with early anatase growth forming chains and aggregates of coalesced crystallites in a sub‐greenschist facies assemblage. High‐sensitivity mapping of FIB lamellae cut across the microtextures confirm that they are discontinuous chains of coalesced crystallites that are highly variable in cross section and contain abundant chlorite inclusions, excluding an origin from the mineralization of previously hollow microtunnels. Comparison of chlorite mineral compositions to DSDP/IODP data reveals that the Euro Basalt chlorites are similar to recent seafloor chlorites. We advance an abiotic origin for the Euro Basalt microtextures formed by spontaneous nucleation and growth of titanite and/anatase during seafloor‐hydrothermal metamorphism. Our findings reveal that the Euro Basalt microtextures are not comparable to microbial ichnofossils from the recent oceanic crust, and we question the evidence for life in these Archean lavas. The metamorphic reactions that give rise to the growth of the Euro Basalt microtextures could be commonplace in Archean pillow lavas and need to be excluded when seeking traces of life in the subseafloor on the early Earth.     

Tracking and predation on earthworms by the invasive terrestrial planarian Bipalium adventitium (Tricladida, Platyhelminthes)

The potential ecological impact of exotic terrestrial planarians will be determined in part by their sensory abilities and predatory behavior. It has been suggested that these flatworms may only encounter their earthworm prey by chance, hence restricting the breadth of species they will feed upon and the number of microhabitats in which predator-prey interactions occur. We hypothesized that those flatworms that have already successfully invaded North America (genus Bipalium) actually detect and follow chemical trails of earthworms and possess the behavioral repertoire needed to feed on the prey in a range of microhabitats. We examined: (1) the tendency of Bipalium adventitium to follow chemical trails left by injured and un-injured earthworms; (2) the behavioral repertoire and predatory success of B. adventitium feeding on three earthworm species in subterranean tunnels; and (3) the response of flatworms to the reportedly defensive secretions of the earthworm Eisenia fetida in tunnels. B. adventitium detected and followed trails of earthworm mucus and secretions left by injured and un-injured earthworms. Flatworms followed trails on a range of substrates and pursued and captured three species of earthworms in subterranean tunnels, including individuals many times their mass. Although most behavior exhibited during underground attacks was similar to that reported for surface encounters, the flatworms also behaved in ways that blocked earthworm escape from tunnels. The flatworms were less successful at preying on E. fetida than on Lumbricus rubellus and Lumbricus terrestris in underground tunnels and showed some aversion to the secretions from E. fetida.     

Intranuclear Inclusions in the Ameboid Cells of Protostelium zonatum*

SYNOPSIS. Two morphologically distinct types of intranuclear inclusions are found in ameboid cells of the protostelid mycetozoan Protostelium zonatum. One type of inclusion is a coiled tubular structure which in cross section appears as cisternae and oval to elliptical vesicles 40–60 nm in diameter. These tubular and vesicular structures are formed by a unit membrane that is connected directly with the inner nuclear membrane. The other type of inclusion is a membrane-bound structure that contains amorphous and/or fibrous material. These inclusions usually are present at several locations in a nucleus. No similar structures occur in the cytoplasm.     

Ultrastructural and cytochemical investigations on the functional role of proteinaceous nuclear inclusions (PNIs) in chlorenchyma plant cells

Summary— Detailed investigations on the fine ultrastructural organization of different forms of proteinaceous nuclear inclusions (PNls) in chlorenchyma plant cells suggest they consist of the same elementary subunits. Previous high magnification of TEM micro-graphs had shown that the amorphous type of inclusion (A) was mainly composed of elementary fibrils measuring 3.0–3.5 nm in diameter, with no orderly spatial arrangement. New computer image treatments of electron micrographs allowed us to establish that the 8.0–13.0 nm thick filaments — forming the fibrillar (F), crystalline (C) and lamellar (L) inclusions — consist of two elementary fibrils which are coiled in a helix with variable pitch, depending on the type of inclusion. A further secondary coiling of two filaments, about 8.0–9.0 nm in diameter, gives the 20.0–25.0 nm thick tubules which form the characteristic tubular inclusion (T). Correlating the distributive data of PNIs with observations on their ultrastructural morphology and with micrographs of partial aggregation or disgregation patterns of the inclusions, led to the hypothesis that the different forms are not different classes of proteins, but simply different stages of structural complexity of the same protein. To determine whether the intranuclear inclusion protein is nucleolar or nucleolus-associated, cytochemical and immunocytochemical tests were performed on ultrathin sections or leaf lamina tissue in block. These techniques proved that PNIs do not belong to the class of argyrophilic proteins (AgNOR-proteins), and particularly not nucleolin and fibrillarin, two of the major nucleolar proteins. Structural similarities to other plant inclusions, especially P-proteins, and to animal and plant intermediate cytoskeletal filaments (IFs) are discussed with regard to the functional role of PNIs.     

Involvement of macroautophagy in the dissolution of neuronal inclusions

Ubiquitinated inclusions are a common feature of many neurodegenerative conditions. We have proposed that, at least in part, such inclusions may be formed due to dysfunction of the proteasome. We have modeled such proteasomal dysfunction by applying pharmacological inhibitors to cultured embryonic rat cortical neurons. This treatment leads to neuronal death and formation of ubiquitin/-synuclein-positive cytoplasmic inclusions. At late time points following proteasomal inhibition such inclusions are no longer discerned. Instead, many neurons accumulate small ubiquitinated aggregates, which may represent remnants of the inclusions. In this work we have examined a potential mechanism for inclusion dissolution. Electron microscopy images showed activation of macroautophagy at late time points after proteasomal inhibition. Labeling with LysoTracker Red, a dye that accumulates in acidic compartments, or immunostaining for the lysosomal enzyme Cathepsin D, showed an increase in globular staining. Cathepsin D co-localized partially with small ubiquitinated aggregates, but not inclusions. Application of an inhibitor of macroautophagy or of the vacuolar ATPase led to an increase in the number of inclusions and a decrease in small aggregates, whereas an activator of autophagy had the opposite effects. There was no significant change in apoptotic death following these manipulations. We conclude that, following proteasomal inhibition of cultured cortical neurons, there is activation of macroautophagy and of the lysosomal pathway. This activation results in dissolution of ubiquitinated inclusions into small aggregates, without directly impacting neuronal cell death. These data further support the idea that in this model inclusions and neuronal cell death are independent processes.     

Light and electron microscopy of virus inclusions inAmaranthus lividus cells

Summary Amaranthus plants infected with a virus of rod-shaped particles showed under the light microscope intracytoplasmic amorphous and crystalline inclusions.The submicroscopic organization of mesophyll cells from infectedAmaranthus leaves by electron microscopy is described. Besides big crystalline inclusions, long dark inclusions correspondent to needle-like inclusions observed by light microscopy are definable in the cytoplasm. The amorphous inclusion bodies were formed by an overgrown protrusion of vacuolate cytoplasm containing virus particles, long very dark stained inclusions forming dense bands and rings, normal elements of the cytoplasm such as mitochondria, endoplasmic reticulum and ribosomes, and some spherosomes. Inclusions and virus particles were not found in chloroplasts, mitochondria or nuclei of infected cells.     

The P2 of Wheat yellow mosaic virus rearranges the endoplasmic reticulum and recruits other viral proteins into replication‐associated inclusion bodies

Viruses commonly modify host endomembranes to facilitate biological processes in the viral life cycle. Infection by viruses belonging to the genus Bymovirus (family Potyviridae) has long been known to induce the formation of large membranous inclusion bodies in host cells, but their assembly and biological roles are still unclear. Immunoelectron microscopy of cells infected with the bymovirus Wheat yellow mosaic virus (WYMV) showed that P1, P2 and P3 are the major viral protein constituents of the membranous inclusions, whereas NIa‐Pro (nuclear inclusion‐a protease) and VPg (viral protein genome‐linked) are probable minor components. P1, P2 and P3 associated with the endoplasmic reticulum (ER), but only P2 was able to rearrange ER and form large aggregate structures. Bioinformatic analyses and chemical experiments showed that P2 is an integral membrane protein and depends on the active secretory pathway to form aggregates of ER membranes. In planta and in vitro assays demonstrated that P2 interacts with P1, P3, NIa‐Pro or VPg and recruits these proteins into the aggregates. In vivo RNA labelling using WYMV‐infected wheat protoplasts showed that the synthesis of viral RNAs occurs in the P2‐associated inclusions. Our results suggest that P2 plays a major role in the formation of membranous compartments that house the genomic replication of WYMV.     

ELECTRON MICROSCOPIC OBSERVATIONS ON TUBULAR STRUCTURES INVOLVED IN CRYSTAL FORMATION IN THE RED ALGA PLEONOSPORIUM SQUARRULOSUM (HARV.) ABBOTT1

Hexagonal or angular crystalline inclusions in Pleonosporium (Naeg.) Hauck vegetative cells were examined using electron microscopy. Ultrastructural analysis reveals that the inclusions initially contain tubular elements resembling microtubules but, with continued differentiation, are transformed into rod containing crystals. The tubular structures initially measure 25 nm in diameter. Scattered tubules become arranged in a parallel and alternate pattern and undergo subsequent enlargement to approximately 29 nm. Following enlargement, each tubule apparently disaggregates into rods that form a crystal having hexagonally arranged rod-like subunits. It is suggested that these tubules may represent microtubules and the resultant crystals are composed of tubulin.     

Effect of phorid fly density on the foraging of Atta vollenweideri leafcutter ants in the field

Leafcutter ants in the genus Atta (Hymenoptera: Formicidae: Attini) are considered major pests of agriculture and forestry in the Neotropics. Phorid flies (Diptera: Phoridae) have been proposed as viable candidates for biological control of ants because of the importance of their trait‐mediated effects on their hosts. However, the impact of different densities of phorid flies has never been assessed in the field. Experiments were conducted by isolating 3‐m sections of Atta vollenweideri Forel foraging trails with tunnels, and sampling ants in trails with 0, 1, or 4 Eibesfeldtphora trilobata Disney female parasitoid flies. Samples were collected every 30 min from these trails. We also collected a sample before introducing the parasitoids and another one 30 min after removing them from the trail. We measured traffic of ants on the trails, weight and type of plant material transported, and the proportion and size of the workers collected. The presence of phorids on the trails reduced the ant traffic and amount of plant material transported into the nests and decreased the proportion of workers on the trails in the size range preferred as hosts by the flies. The effect on worker size, as well as the lag effect recorded after phorids were removed from the tunnels, was more pronounced with four phorids. The presence of phorids also affected the weight of monocotyledon and dicotyledon material transported. Even at the minimum density possible, phorids significantly influenced a key aspect of the colony life, the food intake through foraging. From an applied point of view, our results show that releases of these phorids into the field should not necessarily involve many individuals to reduce foraging by A. vollenweideri, making them potentially useful candidates for biological control of these ants.     

Intranuclear crystalline inclusions in spermatozoa ofLumbricus terrestris

Summary In the nuclei of atypical spermatids ofLumbricus terrestris granular or filamentous inclusions are surrounded by dense chromatin. Aggregation and condensation of chromatin in nuclei during spermatid differentiation coincide with increase in density, granularity, and the subsequent crystallization of the intranuclear inclusion. In mature spermatozoa, the crystalline inclusion displaying an irregular shape is composed of parallel repeating units measuring 50–80 Å. The subunits sometimes possess a clear central cavity.Atypical spermatozoa, possessing inclusions that distort their normally cylindrical shape, possess typical acrosomes, middle pieces, and flagella. Spermatozoa bearing intranuclear crystals are rarely observed in the seminal receptacles ofLumbricus.These intranuclear inclusions probably represent proteinaceous material that is not eliminated during nuclear differentiation. Their sole existence in the nuclei of spermatozoa, their transformation into crystalline structures during spermiogenesis, and their similarity to crystals in virus infected plant and animal cells suggest a viral origin.Supported by a training grant (GM-00582-07) from the Public Health Service.     

Resilience and alternative equilibria in a mire plant community after experimental disturbance by volcanic ash

Following disturbance events vegetation can either be resilient and return to its original state, or there can be shifts in vegetation composition and abundance patterns that may indicate alternative equilibiria. We conducted a long‐term field experiment that simulated impact by aerially transported volcanic ejecta (tephra) in a Sphagnum‐dominated plant community in order to test the effects of this type of large‐scale disturbance. Sixty plots of 1.4 × 1.4 m were established at Sarobetsu mire in northern Hokkaido, Japan and subjected to seven treatments (including the control) with natural tephra or ground glass shards differing in grain size, layer thickness and season of application. Water chemistry and vegetation were surveyed before tephra application and during five and eight years after the perturbation, respectively. Leaching of ions from fine‐grained glass shards caused a sustained increase of soil water pH and electric conductivity. Under coarser materials water quality differed little from the control, but a short‐lived peak of potassium suggested that mechanisms like nutrient release from decomposing plant material may influence water chemistry after volcanic disturbance. The perturbation initially reduced the cover of the dominant functional group (Sphagnum mosses) in all treatments; vascular plants were less affected. All species were able to recover by growing through the tephra, and open tephra surfaces were colonized by ubiquitous cryptogams, but not by spermatophytes. In contrast to the overall resilient behaviour of the vegetation, in some plots that received natural tephra an alternative state with high cover of the dwarf shrub Myrica gale developed. The patterns indicated that physical and chemical properties of the tephra determined the initial effects on plants, but that stochastic processes contributed to subsequent succession. These are likely to have effects on ecosystem functioning, e.g. hydrological processes and carbon sequestration.     

Critically testing olivine‐hosted putative martian biosignatures in the Yamato 000593 meteorite—Geobiological implications

On rocky planets such as Earth and Mars the serpentinization of olivine in ultramafic crust produces hydrogen that can act as a potential energy source for life. Direct evidence of fluid–rock interaction on Mars comes from iddingsite alteration veins found in martian meteorites. In the Yamato 000593 meteorite, putative biosignatures have been reported from altered olivines in the form of microtextures and associated organic material that have been compared to tubular bioalteration textures found in terrestrial sub‐seafloor volcanic rocks. Here, we use a suite of correlative, high‐sensitivity, in situ chemical, and morphological analyses to characterize and re‐evaluate these microalteration textures in Yamato 000593, a clinopyroxenite from the shallow subsurface of Mars. We show that the altered olivine crystals have angular and micro‐brecciated margins and are also highly strained due to impact‐induced fracturing. The shape of the olivine microalteration textures is in no way comparable to microtunnels of inferred biological origin found in terrestrial volcanic glasses and dunites, and rather we argue that the Yamato 000593 microtextures are abiotic in origin. Vein filling iddingsite extends into the olivine microalteration textures and contains amorphous organic carbon occurring as bands and sub‐spherical concentrations <300 nm across. We propose that a martian impact event produced the micro‐brecciated olivine crystal margins that reacted with subsurface hydrothermal fluids to form iddingsite containing organic carbon derived from abiotic sources. These new data have implications for how we might seek potential biosignatures in ultramafic rocks and impact craters on both Mars and Earth.     

Nature chimique des inclusions cristallines nucléaires des cellules parenchymateuses voisines de l'épithème chezStellaria media L.

Summary The chemical composition of crystalline inclusions, which have been detected in the double nuclear membrane with the electron microscope (Perrin 1969) was studied by enzymatic digestion. The crystals were digested in thin sections by pepsin and after 1 hour of incubation, nearly all the crystalline inclusions had disappeared entirely leaving empty spaces.These results demonstrate that the nuclear crystals are composed primarily of protein.     

Further studies on ultrastructure of plants infected withPetunia ringspot virus

Summary New methods of fixation and embedding have revealed in plants infected withPetunia ringspot some structural features not described before. These include the X-bodies, 80 per cent of which are formed by tubular elements which are responsible for the positive staining specific for Golgi apparatus. The tubular elements are morphologically similar to agranular endoplasmic reticulum as described in some animal cells. The rest of the inclusion is formed by normal cytoplasmic elements in which are embedded long rod-shaped tubules 600 Å wide and more than 7,000 Å long, cross sections of which are formed by 10 subunits. These subunits are arranged in a helical way to form the large tubules. These subunits are probably the actual virus particles, which would be icosahedral and would form tubular crystals. Icosahedral virus particles would also form true crystalline inclusions.It is not known what the role of the agranular endoplasmic reticulum and of some dense osmiophilic bodies found in it may be in the multiplication of the virus. However, these components induced by the virus infection probably result in the manufacture of some proteins or other substances necessary for virus multiplication.