The importance of microscopic examinations of eggshells: Discrimination of bioalteration and diagenetic overprints from biological features

Although fossilization usually favors the preservation of calcium carbonate biominerals, diagenetic alterations might still produce erratic patterns that overprint the original biological structures. This investigation tries to discriminate in the fossil record “pathological” eggshells from diagenetic induced features as well as determine their origin, and aims, when possible, to provide alternative parsimonious interpretations to the origin of some of these rare and erratic features. In the past, most dinosaur eggshell studies failed to combine cathodoluminescence, scanning electron microscopy, transmitted and polarized light microscopic observations and were limited to only one or several of these examinations, which might have contributed to misinterpretations by lack of thorough observations. Sauropod eggshells from Faidella (Spain) and Auca Mahuevo (Argentina) provide ideal proxies to perform this research, as they display aberrant crystallographic features that have been or could be considered pathological. Under cathodoluminescence, the specimens fluoresce tremendously, indicating a strong diagenetic component in their make up. Guide by this information, further transmitted and polarized light microscopic examinations reveal microscopic dissolution fronts, which otherwise would have been left unnoted. The proposed hypothesis for the Faidella specimens in that organic filaments, which represent up to 2% of the shell composition, were exposed on the internal wall surfaces of pore canals where the calcium carbonate had been dissolved during a first diagenetic event. As such, the exposed extremities of the organic filaments likely triggered the formation of pseudo cores that mimic those in the membrana testacea during oogenesis. Observations based on the Argentinean specimens indicate that an added extra external structural layer is also separated from the original biological eggshell by a dissolution and recrystallization front. In addition to this abiotic process, artifact formations induced by bacterial mediation, a topic treated in an earlier publication, was also common at Auca Mahuevo. Without combined microscopic and cathodoluminiscence observations, the Faidella and Auca Mahuevo megaloolithid eggshells could be easily considered pathological eggshells. This would bias ensuing phylogenetic, paleobiological, and paleoenvironmental interpretations.A pesar de que la fosilizacion favorece la conservacion de los biominerales de carbonato calcico, las alteraciones diageneticas pueden llegar a producir patrones erraticos que se sobrepongan a las estructuras biologicas originales. Esta investigacion intenta discriminar en el registro fosil cascaras “patologicas” con rasgos diageneticamente inducidos, determinando su origen y procura, en la medida de lo posible, dar una interpretacion parsimoniosa alternativa al origen de algunos de estos rasgos extranos y erraticos. En el pasado, la mayoria de los estudios en cascaras de dinosaurios se vieron limitados al uso de uno o dos de estos procedimientos (catodoluminiscencia, microscopia electronica, observaciones con microscopio de luz transmitida y polarizada), lo que puede haber contribuido a interpretaciones erroneas debido a observaciones incompletas. Las cascaras de huevos de sauropodos del yacimiento de Faidella (Espana) y de Auca Mahuevo (Argentina) muestran condiciones ideales para realizar esta investigacion, ya que denotan rasgos critalográficos aberrantes que habían sido o podrían ser considerados como patológicos. Mediante catodoluminiscencia, los ejemplares muestran una violenta fluorescencia, lo que indica un fuerte componente diagenético en su composición. Guiados por esta informacion, los analisis posteriores con microscopia de transmision y luz polarizada revelan frentes de disoluciones microscópicas, que de otro modo no habrían sido apreciadas. La hipótesis que se propone para los ejemplares de Faidella es que filamentos orgánicos, que representan hasta el 2% de la composición de las cáscaras, estaban expuestos en las superficies de las paredes internas de los canales de los poros donde el carbonato calcico se hubo disuelto durante el primer evento diagetico. Dada esta condicion, los extremos de los filamentos orgánicos expuestos, muy probablemente dispararon la formación de pseudo núcleos que imitan los de la membrana testacea durante la oogenesis. Las observaciones basadas en los ejemplares argentinos indican que una capa externa extra esta separada de la cascara biologica original por una disolucion y recristalizacion. Ademas de este proceso abiotico, en Auca Mahuevo son tambien frecuentes rasgos inducidos por bacterias, asunto tratado en una publicacion anterior. Sin la combinacion de la combinacion de estos metodos, las cascaras de huevo de Faidella y Auca Mahuevo podrian haber sido fácilmente consideradas como patológicas, lo que sesgaría otras interpretaciones como las filogenéticas, paleobiológicas y paleoambientales

First Evidence of Reproductive Adaptation to “Island Effect” of a Dwarf Cretaceous Romanian Titanosaur, with Embryonic Integument In Ovo

<div><h3>Background</h3><p>The Cretaceous vertebrate assemblages of Romania are famous for geographically endemic dwarfed dinosaur taxa. We report the first complete egg clutches of a dwarf lithostrotian titanosaur, from Toteşti, Romania, and its reproductive adaptation to the “island effect”.</p> <h3>Methodology/Findings</h3><p>The egg clutches were discovered in sequential sedimentary layers of the Maastrichtian Sânpetru Formation, Toteşti. The occurrence of 11 homogenous clutches in successive strata suggests philopatry by the same dinosaur species, which laid clutches averaging four ∼12 cm diameters eggs. The eggs and eggshells display numerous characters shared with the positively identified material from egg-bearing level 4 of the Auca Mahuevo (Patagonia, Argentina) nemegtosaurid lithostrotian nesting site. Microscopic embryonic integument with bacterial evidences was recovered in one egg. The millimeter-size embryonic integument displays micron size dermal papillae implying an early embryological stage at the time of death, likely corresponding to early organogenesis before the skeleton formation.</p> <h3>Conclusions/Significance</h3><p>The shared oological characters between the Haţeg specimens and their mainland relatives suggest a highly conservative reproductive template, while the nest decrease in egg numbers per clutch may reflect an adaptive trait to a smaller body size due to the “island effect”. The combined presence of the lithostrotian egg and its embryo in the Early Cretaceous Gobi coupled with the oological similarities between the Haţeg and Auca Mahuevo oological material evidence that several titanosaur species migrated from Gondwana through the Haţeg Island before or during the Aptian/Albian. It also suggests that this island might have had episodic land bridges with the rest of the European archipelago and Asia deep into the Cretaceous.</p> </div

Is the “Genyornis” egg of a mihirung or another extinct bird from the Australian dreamtime?

© 2016. This manuscript version is made available under the CC-BY-NC-ND 4.0 license http://creativecommons.org/licenses/by-nc-nd/4.0/. Following 24 month embargo from date of publication (Feb 1 2016)The iconic Australian Genyornis newtoni (Dromornithidae, Aves) is the sole Pleistocene member of an avian clade now hypothesized to be alternatively in Anseriformes or the sister group of crown Galloanseres. A distinctive type of fossil eggshell commonly found in eroding sand dunes, has been referred to Genyornis newtoni since the 1980s. The 126 by 97 mm Spooner Egg, dated at 54.7 ± 3.1 ka by optical dating of its enclosing sediments, is a complete specimen of this eggshell type that was reconstructed from fragments of a broken egg. We show that the size of the eggs from which this ‘Genyornis’ eggshell derives, either as predicted from measurements of fragments, or as indicated by the Spooner Egg, is unexpectedly small given the size of G. newtoni, which has an estimated mass of 275 kg, or about seven times the mass of the emu that has a similar sized egg. We compared the microstructure of the putative Genyornis eggshell to that of other dromornithids and a range of galloanseriform taxa using several microcharacterisation techniques. The ‘Genyornis’ eggshell displays a mosaic of oological characters that do not unambiguously support referral to any known modern bird. Its shell structure, coupled with chemical compounds in the accessory layer, makes it unlikely to have been laid by a dromornithid, whereas several characters support a megapode origin. A potential candidate for the bird that laid the putative ‘Genyornis’ eggs in the Pleistocene fossil avifaunal record has been ignored: Progura, a genus of extinct giant megapodes, whose species were widespread in Australia. Regression of egg size of megapodes and body mass shows that the Spooner Egg approximates the expected size for eggs laid by species of Progura. We advance the suggestion that the fossil eggshell hitherto referred to Genyornis newtoni, is more likely to have been laid by species of the giant extinct Progura. As megapodes, the species of Progura were obligate ectothermic incubators, which we suggest laid their eggs into a hole dug in sand like the modern megapode Macrocephalon maleo, thus explaining the abundant ‘Genyornis’ eggshell in sand dunes. Referral of this eggshell to Progura means that the fossil record of Genyornis newtoni is limited to bones and the timing of the extinction of this last dromornithid is unknown. In addition, structural similarities of eggshell in megapodes, the putative Genyornis eggshell and dromornithids, raise the possibility that these taxa are phylogenetically more closely related to each other than any is to anseriforms. Specifically, this means that dromornithids might be a sister group to galliforms rather than to or within anseriforms

Purification and Characterization of AsES Protein A Subtilisin Secreted by Acremonium Strictum is a Novel Plant Defense Elicitor.

In this work, the purification and characterization of an extracellular elicitor protein, designated AsES, produced by an avirulent isolate of the strawberry pathogen Acremonium strictum, are reported. The defense eliciting activity present in culture filtrates was recovered and purified by ultrafiltration (cutoff, 30 kDa), anionic exchange (Q-Sepharose, pH 7.5), and hydrophobic interaction (phenyl-Sepharose) chromatographies. Two-dimensional SDS-PAGE of the purified active fraction revealed a single spot of 34 kDa and pI 8.8. HPLC (C2/C18) and MS/MS analysis confirmed purification to homogeneity. Foliar spray with AsES provided a total systemic protection against anthracnose disease in strawberry, accompanied by the expression of defense-related genes (i.e. PR1 and Chi2-1). Accumulation of reactive oxygen species (e.g. H2O2 and O2̇̄) and callose was also observed in Arabidopsis. By using degenerate primers designed from the partial amino acid sequences and rapid amplification reactions of cDNA ends, the complete AsES-coding cDNA of 1167 nucleotides was obtained. The deduced amino acid sequence showed significant identity with fungal serine proteinases of the subtilisin family, indicating that AsES is synthesized as a larger precursor containing a 15-residue secretory signal peptide and a 90-residue peptidase inhibitor I9 domain in addition to the 283-residue mature protein. AsES exhibited proteolytic activity in vitro, and its resistance eliciting activity was eliminated when inhibited with PMSF, suggesting that its proteolytic activity is required to induce the defense response. This is, to our knowledge, the first report of a fungal subtilisin that shows eliciting activity in plants. This finding could contribute to develop disease biocontrol strategies in plants by activating its innate immunity

A comparison of the radiosensitisation ability of 22 different element metal oxide nanoparticles using clinical megavoltage X-rays

Background: A wide range of nanoparticles (NPs), composed of different elements and their compounds, are being developed by several groups as possible radiosensitisers, with some already in clinical trials. However, no systematic experimental survey of the clinical X-ray radiosensitising potential of different element nanoparticles has been made. Here, we directly compare the irradiation-induced (10 Gy of 6-MV X-ray photon) production of hydroxyl radicals, superoxide anion radicals and singlet oxygen in aqueous solutions of the following metal oxide nanoparticles: Al2O3, SiO2, Sc2O3, TiO2, V2O5, Cr2O3, MnO2, Fe3O4, CoO, NiO, CuO, ZnO, ZrO2, MoO3, Nd2O3, Sm2O3, Eu2O3, Gd2O3, Tb4O7, Dy2O3, Er2O3 and HfO2. We also examine DNA damage due to these NPs in unirradiated and irradiated conditions. Results: Without any X-rays, several NPs produced more radicals than water alone. Thus, V2O5 NPs produced around 5-times more hydroxyl radicals and superoxide radicals. MnO2 NPs produced around 10-times more superoxide anions and Tb4O7 produced around 3-times more singlet oxygen. Lanthanides produce fewer hydroxyl radicals than water. Following irradiation, V2O5 NPs produced nearly 10-times more hydroxyl radicals than water. Changes in radical concentrations were determined by subtracting unirradiated values from irradiated values. These were then compared with irradiation-induced changes in water only. Irradiation-specific increases in hydroxyl radical were seen with most NPs, but these were only significantly above the values of water for V2O5, while the Lanthanides showed irradiation-specific decreases in hydroxyl radical, compared to water. Only TiO2 showed a trend of irradiation-specific increase in superoxides, while V2O5, MnO2, CoO, CuO, MoO3 and Tb4O7 all demonstrated significant irradiation-specific decreases in superoxide, compared to water. No irradiation-specific increases in singlet oxygen were seen, but V2O5, NiO, CuO, MoO3 and the lanthanides demonstrated irradiation-specific decreases in singlet oxygen, compared to water. MoO3 and CuO produced DNA damage in the absence of radiation, while the highest irradiation-specific DNA damage was observed with CuO. In contrast, MnO2, Fe3O4 and CoO were slightly protective against irradiation-induced DNA damage. Conclusions: Beyond identifying promising metal oxide NP radiosensitisers and radioprotectors, our broad comparisons reveal unexpected differences that suggest the surface chemistry of NP radiosensitisers is an important criterion for their success

Paleobiology of titanosaurs: reproduction, development, histology, pneumaticity, locomotion and neuroanatomy from the South American fossil record

Fil: García, Rodolfo A.. Instituto de Investigación en Paleobiología y Geología. Museo Provincial Carlos Ameghino. Cipolletti; ArgentinaFil: Salgado, Leonardo. Instituto de Investigación en Paleobiología y Geología. General Roca. Río Negro; ArgentinaFil: Fernández, Mariela. Inibioma-Centro Regional Universitario Bariloche. Bariloche. Río Negro; ArgentinaFil: Cerda, Ignacio A.. Instituto de Investigación en Paleobiología y Geología. Museo Provincial Carlos Ameghino. Cipolletti; ArgentinaFil: Carabajal, Ariana Paulina. Museo Carmen Funes. Plaza Huincul. Neuquén; ArgentinaFil: Otero, Alejandro. Museo de La Plata. Universidad Nacional de La Plata; ArgentinaFil: Coria, Rodolfo A.. Instituto de Paleobiología y Geología. Universidad Nacional de Río Negro. Neuquén; ArgentinaFil: Fiorelli, Lucas E.. Centro Regional de Investigaciones Científicas y Transferencia Tecnológica. Anillaco. La Rioja; Argentin

New Specimens of Nemegtomaia from the Baruungoyot and Nemegt Formations (Late Cretaceous) of Mongolia

Two new specimens of the oviraptorid theropod Nemegtomaia barsboldi from the Nemegt Basin of southern Mongolia are described. Specimen MPC-D 107/15 was collected from the upper beds of the Baruungoyot Formation (Campanian-Maastrichtian), and is a nest of eggs with the skeleton of the assumed parent of Nemegtomaia on top in brooding position. Much of the skeleton was damaged by colonies of dermestid coleopterans prior to its complete burial. However, diagnostic characters are recovered from the parts preserved, including the skull, partial forelimbs (including the left hand), legs, and distal portions of both feet. Nemegtomaia represents the fourth known genus of oviraptorid for which individuals have been found on nests of eggs. The second new specimen, MPC-D 107/16, was collected a few kilometers to the east in basal deposits of the Nemegt Formation, and includes both hands and femora of a smaller Nemegtomaia individual. The two formations and their diverse fossil assemblages have been considered to represent sequential time periods and different environments, but data presented here indicate partial overlap across the Baruungoyot-Nemegt transition. All other known oviraptorids from Mongolia and China are known exclusively from xeric or semi-arid environments. However, this study documents that Nemegtomaia is found in both arid/aeolian (Baruungoyot Formation) and more humid/fluvial (Nemegt Formation) facies