Raman imaging of microbial colonization in rock-some analytical aspects.

Raman imaging allows one to obtain spatially resolved chemical information in a nondestructive manner. Herein, we present analytical aspects of effective in situ and in vivo Raman imaging of algae and cyanobacteria from within their native rock habitats. Specifically, gypsum and halite inhabited by endolithic communities from the hyperarid Atacama Desert were analyzed. Raman imaging of these phototrophic colonization reveals a pigment composition within the aggregates that helps in understanding some of their adaptation strategies to survive in this harsh polyextreme environment. The study is focused on methodical aspects of Raman imaging acquisition and subsequent data processing. Point imaging is compared with line imaging in terms of their image quality, spatial resolution, spectral signal-to-noise ratio, time requirements, and risk of laser-induced sample alteration. The roles of excitation wavelength, exposure time, and step size of the imaging grid on successful Raman imaging results are also discussed. Graphical abstract.

Fundamental drivers for endolithic microbial community assemblies in the hyperarid Atacama Desert.

In hyperarid deserts, endolithic microbial communities colonize the rocks' interior as a survival strategy. Yet, the composition of these communities and the drivers promoting their assembly are still poorly understood. We analysed the diversity and community composition of endoliths from four different lithic substrates - calcite, gypsum, ignimbrite and granite - collected in the hyperarid zone of the Atacama Desert, Chile. By combining microscopy, mineralogy, spectroscopy and high throughput sequencing, we found these communities to be highly specific to their lithic substrate, although they were all dominated by the same four main phyla, Cyanobacteria, Actinobacteria, Chloroflexi and Proteobacteria. Our finding indicates a fine scale diversification of the microbial reservoir driven by substrate properties. The data suggest that the overall rock chemistry and the light transmission properties of the substrates are not essential drivers of community structure and composition. Instead, we propose that the architecture of the rock, i.e., the space available for colonization and its physical structure, linked to water retention capabilities, is ultimately the driver of community diversity and composition at the dry limit of life.

Functional interactions of archaea, bacteria and viruses in a hypersaline endolithic community.

Halite endoliths in the Atacama Desert represent one of the most extreme ecosystems on Earth. Cultivation-independent methods were used to examine the functional adaptations of the microbial consortia inhabiting halite nodules. The community was dominated by haloarchaea and functional analysis attributed most of the autotrophic CO2 fixation to one unique cyanobacterium. The assembled 1.1 Mbp genome of a novel nanohaloarchaeon, Candidatus Nanopetramus SG9, revealed a photoheterotrophic life style and a low median isoelectric point (pI) for all predicted proteins, suggesting a 'salt-in' strategy for osmotic balance. Predicted proteins of the algae identified in the community also had pI distributions similar to 'salt-in' strategists. The Nanopetramus genome contained a unique CRISPR/Cas system with a spacer that matched a partial viral genome from the metagenome. A combination of reference-independent methods identified over 30 complete or near complete viral or proviral genomes with diverse genome structure, genome size, gene content and hosts. Putative hosts included Halobacteriaceae, Nanohaloarchaea and Cyanobacteria. Despite the dependence of the halite community on deliquescence for liquid water availability, this study exposed an ecosystem spanning three phylogenetic domains, containing a large diversity of viruses and predominance of a 'salt-in' strategy to balance the high osmotic pressure of the environment.

Raman imaging in geomicrobiology: endolithic phototrophic microorganisms in gypsum from the extreme sun irradiation area in the Atacama Desert.

The Raman imaging method was successfully applied for mapping the distribution of biomolecules (e.g., pigments) associated with cryptoendolithic and hypoendolithic microorganisms, as well as the inorganic host mineral matrix that forms the habitat for the biota. To the best of our knowledge, this is the first comprehensive study in the field of geomicrobiology based on this technique. The studied microbial ecosystem was located nearly 3000 m above sea level within the driest desert on Earth, the Atacama in Chile. Enhancement of carotenoid Raman signal intensity close to the surface was registered at different areas of endolithic colonization dominated by algae, with cyanobacteria present as well. This is interpreted as an adaptation mechanism to the excessive solar irradiation. On the other hand, cyanobacteria synthesize scytonemin as a passive UV-screening pigment (found at both the hypoendolithic and cryptoendolithic positions). The distribution of the scytonemin Raman signal was mapped simultaneously with the surrounding mineral matrix. Thus, mapping was done of the phototrophic microorganisms in their original microhabitat together with the host rock environment. Important information which was resolved from the Raman imaging dataset of the host rock is about the hydration state of Ca-sulfate, demonstrated on the presence of gypsum (CaSO4·2H2O) and the absence of both anhydrite (CaSO4) and bassanite (CaSO4·1/2H2O). Obtaining combined "in situ" simultaneous information from the geological matrix (inorganic) together with the microbial biomolecules (organic) is discussed and concluded as an important advantage of this technique. We discuss how selection of the laser wavelength (785 and 514.5-nm) influences the Raman imaging results.

Microbial diversity and the presence of algae in halite endolithic communities are correlated to atmospheric moisture in the hyper-arid zone of the Atacama Desert.

The Atacama Desert is one of the oldest and driest deserts in the world, and its hyper-arid core is described as 'the most barren region imaginable'. We used a combination of high-throughput sequencing and microscopy methods to characterize the endolithic microbial assemblages of halite pinnacles (salt rocks) collected in several hyper-arid areas of the desert. We found communities dominated by archaea that relied on a single phylotype of Halothece cyanobacteria for primary production. A few other phylotypes of salt-adapted bacteria and archaea, including Salinibacter, Halorhabdus, and Halococcus were major components of the halite communities, indicating specific adaptations to the unique halite environments. Multivariate statistical analyses of diversity metrics clearly separated the halite communities from that of the surrounding soil in the Yungay area. These analyses also revealed distribution patterns of halite communities correlated with atmospheric moisture. Microbial endolithic communities from halites exposed to coastal fogs and high relative humidity were more diverse; their archaeal and bacterial assemblages were accompanied by a novel algae related to oceanic picoplankton of the Mamiellales. In contrast, we did not find any algae in the Yungay pinnacles, suggesting that the environmental conditions in this habitat might be too extreme for eukaryotic photosynthetic life.

Early evolution and ecology of camouflage in insects.

Taxa within diverse lineages select and transport exogenous materials for the purposes of camouflage. This adaptive behavior also occurs in insects, most famously in green lacewing larvae who nestle the trash among setigerous cuticular processes, known as trash-carrying, rendering them nearly undetectable to predators and prey, as well as forming a defensive shield. We report an exceptional discovery of a green lacewing larva in Early Cretaceous amber from Spain with specialized cuticular processes forming a dorsal basket that carry a dense trash packet. The trash packet is composed of trichomes of gleicheniacean ferns, which highlight the presence of wildfires in this early forest ecosystem. This discovery provides direct evidence of an early acquisition of a sophisticated behavioral suite in stasis for over 110 million years and an ancient plant-insect interaction.

Innovative approaches to accurately assess the effectiveness of biocide-based treatments to fight biodeterioration of Cultural Heritage monuments.

The development of diagnostic methods to accurately assess the effects of treatments on lithobiont colonization remains a challenge for the conservation of Cultural Heritage monuments. In this study, we tested the efficacy of biocide-based treatments on microbial colonization of a dolostone quarry, in the short and long-term, using a dual analytical strategy. We applied a metabarcoding approach to characterize fungal and bacterial communities over time, integrated with microscopy techniques to analyze the interactions of microorganisms with the substrate and evaluate the effectiveness. These communities were dominated by the bacterial phyla Actinobacteriota, Proteobacteria and Cyanobacteria, and the fungal order Verrucariales, which include taxa previously reported as biodeteriogenic agents and observed here associated with biodeterioration processes. Following the treatments, changes over time in the abundance profiles depend on taxa. While Cyanobacteriales, Cytophagales and Verrucariales decreased in abundance, other groups, such as Solirubrobacteriales, Thermomicrobiales and Pleosporales increased. These patterns could be related not only to the specific effects of the biocide on the different taxa, but also to different recolonization abilities of those organisms. The different susceptibility to treatments could be associated with the inherent cellular properties of different taxa, but differences in biocide penetration to endolithic microhabitats could be involved. Our results demonstrate the importance of both removing epilithic colonization and applying biocides to act against endolithic forms. Recolonization processes could also explain some of the taxon-dependent responses, especially in the long-term. Taxa showing resistance, and those benefiting from nutrient accumulation in the form of cellular debris following treatments, may have an advantage in colonizing treated areas, pointing to the need for long-term monitoring of a wide range of taxa. This study highlights the potential utility of combining metabarcoding and microscopy to analyze the effects of treatments and design appropriate strategies to combat biodeterioration and establish preventive conservation protocols.

Gypsum endolithic phototrophs under moderate climate (Southern Sicily): their diversity and pigment composition.

In this study, we used microscopic, spectroscopic, and molecular analysis to characterize endolithic colonization in gypsum (selenites and white crystalline gypsum) from several sites in Sicily. Our results showed that the dominant microorganisms in these environments are cyanobacteria, including: Chroococcidiopsis sp., Gloeocapsopsis pleurocapsoides, Gloeocapsa compacta, and Nostoc sp., as well as orange pigmented green microalgae from the Stephanospherinia clade. Single cell and filament sequencing coupled with 16S rRNA amplicon metagenomic profiling provided new insights into the phylogenetic and taxonomic diversity of the endolithic cyanobacteria. These organisms form differently pigmented zones within the gypsum. Our metagenomic profiling also showed differences in the taxonomic composition of endoliths in different gypsum varieties. Raman spectroscopy revealed that carotenoids were the most common pigments present in the samples. Other pigments such as gloeocapsin and scytonemin were also detected in the near-surface areas, suggesting that they play a significant role in the biology of endoliths in this environment. These pigments can be used as biomarkers for basic taxonomic identification, especially in case of cyanobacteria. The findings of this study provide new insights into the diversity and distribution of phototrophic microorganisms and their pigments in gypsum in Southern Sicily. Furthemore, this study highlights the complex nature of endolithic ecosystems and the effects of gypsum varieties on these communities, providing additional information on the general bioreceptivity of these environments.

Dark microbiome and extremely low organics in Atacama fossil delta unveil Mars life detection limits.

Identifying unequivocal signs of life on Mars is one of the most important objectives for sending missions to the red planet. Here we report Red Stone, a 163-100 My alluvial fan-fan delta that formed under arid conditions in the Atacama Desert, rich in hematite and mudstones containing clays such as vermiculite and smectites, and therefore geologically analogous to Mars. We show that Red Stone samples display an important number of microorganisms with an unusual high rate of phylogenetic indeterminacy, what we refer to as "dark microbiome", and a mix of biosignatures from extant and ancient microorganisms that can be barely detected with state-of-the-art laboratory equipment. Our analyses by testbed instruments that are on or will be sent to Mars unveil that although the mineralogy of Red Stone matches that detected by ground-based instruments on the red planet, similarly low levels of organics will be hard, if not impossible to detect in Martian rocks depending on the instrument and technique used. Our results stress the importance in returning samples to Earth for conclusively addressing whether life ever existed on Mars.

Comparative analysis of the microbial communities inhabiting halite evaporites of the Atacama Desert.

SUMMARY: Molecular biology and microscopy techniques were used to characterize the microbial communities inside halite evaporites from different parts of the Atacama Desert. Denaturing gradient gel electrophoresis (DGGE) analysis revealed that the evaporite rocks harbor communities predominantly made up of cyanobacteria, along with heterotrophic bacteria and archaea. Different DGGE profiles were obtained for the different sites, with the exception of the cyanobacterial profile, in which only one phylotype was detected across the three sites examined. Chroococcidiopsis-like cells were the only cyanobacterial components of the rock samples, although the phylogenetic study revealed their closer genetic affinity to Halothece genera. Gene sequences of the heterotrophic bacteria and archaea indicated their proximity to microorganisms found in other hypersaline environments. Microorganisms colonizing these halites formed microbial aggregates in the pore spaces between halite crystals, where microbial interactions occur. In this exceptional, salty, porous halite rock habitat, microbial consortia with a community structure probably conditioned by the environmental conditions occupy special microhabitats with physical and chemical properties that promote their survival.

Double fossilization in eukaryotic microorganisms from Lower Cretaceous amber.

BACKGROUND: Microfossils are not only useful for elucidating biological macro- and microevolution but also the biogeochemical history of our planet. Pyritization is the most important and extensive mode of preservation of animals and especially of plants. Entrapping in amber, a fossilized resin, is considered an alternative mode of biological preservation. For the first time, the internal organization of 114-million-year-old microfossils entrapped in Lower Cretaceous amber is described and analyzed, using adapted scanning electron microscopy in backscattered electron mode in association with energy dispersive X-ray spectroscopy microanalysis. Double fossilization of several protists included in diverse taxonomical groups and some vegetal debris is described and analyzed. RESULTS: In protists without an exoskeleton or shell (ciliates, naked amoebae, flagellates), determinate structures, including the nuclei, surface envelopes (cortex or cytoplasmic membrane) and hyaloplasm are the main sites of pyritization. In protists with a biomineralized skeleton (diatoms), silicon was replaced by pyrite. Permineralization was the main mode of pyritization. Framboidal, subhedral and microcrystalline are the predominant pyrite textures detected in the cells. Abundant pyritized vegetal debris have also been found inside the amber nuggets and the surrounding sediments. This vegetal debris usually contained numerous pyrite framboids and very densely packed polycrystalline pyrite formations infilled with different elements of the secondary xylem. CONCLUSION: Embedding in amber and pyritization are not always alternative modes of biological preservation during geological times, but double fossilization is possible under certain environmental conditions. Pyritization in protists shows a quite different pattern with regard to plants, due to the different composition and cellular architecture in these microorganisms and organisms. Anaerobic sulphate-reducing bacteria could play a crucial role in this microbial fossilization.

Response of Endolithic Chroococcidiopsis Strains From the Polyextreme Atacama Desert to Light Radiation.

Cyanobacteria exposed to high solar radiation make use of a series of defense mechanisms, including avoidance, antioxidant systems, and the production of photoprotective compounds such as scytonemin. Two cyanobacterial strains of the genus Chroococcidiopsis from the Atacama Desert - which has one of the highest solar radiation levels on Earth- were examined to determine their capacity to protect themselves from direct photosynthetically active (PAR) and ultraviolet radiation (UVR): the UAM813 strain, originally isolated from a cryptoendolithic microhabitat within halite (NaCl), and UAM816 strain originally isolated from a chasmoendolithic microhabitat within calcite (CaCO3). The oxidative stress induced by exposure to PAR or UVR + PAR was determined to observe their short-term response, as were the long-term scytonemin production, changes in metabolic activity and ultrastructural damage induced. Both strains showed oxidative stress to both types of light radiation. The UAM813 strain showed a lower acclimation capacity than the UAM816 strain, showing an ever-increasing accumulation of reactive oxygen species (ROS) and a smaller accumulation of scytonemin. This would appear to reflect differences in the adaptation strategies followed to meet the demands of their different microhabitats.

Inhabited subsurface wet smectites in the hyperarid core of the Atacama Desert as an analog for the search for life on Mars.

The modern Martian surface is unlikely to be habitable due to its extreme aridity among other environmental factors. This is the reason why the hyperarid core of the Atacama Desert has been studied as an analog for the habitability of Mars for more than 50 years. Here we report a layer enriched in smectites located just 30 cm below the surface of the hyperarid core of the Atacama. We discovered the clay-rich layer to be wet (a phenomenon never observed before in this region), keeping a high and constant relative humidity of 78% (aw 0.780), and completely isolated from the changing and extremely dry subaerial conditions characteristic of the Atacama. The smectite-rich layer is inhabited by at least 30 halophilic species of metabolically active bacteria and archaea, unveiling a previously unreported habitat for microbial life under the surface of the driest place on Earth. The discovery of a diverse microbial community in smectite-rich subsurface layers in the hyperarid core of the Atacama, and the collection of biosignatures we have identified within the clays, suggest that similar shallow clay deposits on Mars may contain biosignatures easily reachable by current rovers and landers.

Haloterrigena sp. Strain SGH1, a Bacterioruberin-Rich, Perchlorate-Tolerant Halophilic Archaeon Isolated From Halite Microbial Communities, Atacama Desert, Chile.

An extreme halophilic archaeon, strain SGH1, is a novel microorganism isolated from endolithic microbial communities colonizing halites at Salar Grande, Atacama Desert, in northern Chile. Our study provides structural, biochemical, genomic, and physiological information on this new isolate living at the edge of the physical and chemical extremes at the Atacama Desert. SGH1 is a Gram-negative, red-pigmented, non-motile unicellular coccoid organism. Under the transmission electron microscope, strain SGH1 showed an abundant electro-dense material surrounding electron-lucent globular structures resembling gas vacuoles. Strain SGH1 showed a 16S rRNA gene sequence with a close phylogenetic relationship to the extreme halophilic archaea Haloterrigena turkmenica and Haloterrigena salina and has been denominated Haloterrigena sp. strain SGH1. Strain SGH1 grew at 20-40°C (optimum 37°C), at salinities between 15 and 30% (w/v) NaCl (optimum 25%) and growth was improved by addition of 50 mM KCl and 0.5% w/v casamino acids. Growth was severely restricted at salinities below 15% NaCl and cell lysis is avoided at a minimal 10% NaCl. Maximal concentrations of magnesium chloride and sodium or magnesium perchlorates that supported SGH1 growth were 0.5 and 0.15M, respectively. Haloterrigena sp. strain SGH1 accumulates bacterioruberin (BR), a C50 xanthophyll, as the major carotenoid. Total carotenoids in strain SGH1 amounted to nearly 400 µg BR per gram of dry biomass. Nearly 80% of total carotenoids accumulated as geometric isomers of BR: all-trans-BR (50%), 5-cis-BR (15%), 9-cis-BR (10%), 13-cis-BR (4%); other carotenoids were dehydrated derivatives of BR. Carotenogenesis in SGH1 was a reversible and salt-dependent process; transferring BR-rich cells grown in 25% (w/v) NaCl to 15% (w/v) NaCl medium resulted in depigmentation, and BR content was recovered after transference and growth of unpigmented cells to high salinity medium. Methanol extracts and purified BR isomers showed an 8-9-fold higher antioxidant activity than Trolox or ß-carotene. Both, plasma membrane integrity and mitochondrial membrane potential measurements under acute 18-h assays showed that purified BR isomers were non-toxic to cultured human THP-1 cells.

Microbial Cretaceous park: biodiversity of microbial fossils entrapped in amber.

Microorganisms are the most ancient cells on this planet and they include key phyla for understanding cell evolution and Earth history, but, unfortunately, their microbial records are scarce. Here, we present a critical review of fossilized prokaryotic and eukaryotic microorganisms entrapped in Cretaceous ambers (but not exclusively from this geological period) obtained from deposits worldwide. Microbiota in ambers are rather diverse and include bacteria, fungi, and protists. We comment on the most important microbial records from the last 25 years, although it is not an exhaustive bibliographic compilation. The most frequently reported eukaryotic microfossils are shells of amoebae and protists with a cell wall or a complex cortex. Likewise, diverse dormant stages (palmeloid forms, resting cysts, spores, etc.) are abundant in ambers. Besides, viral and protist pathogens have been identified inside insects entrapped in amber. The situation regarding filamentous bacteria and fungi is quite confusing because in some cases, the same record was identified consecutively as a member of these phylogenetically distant groups. To avoid these identification errors in the future, we propose to apply a more resolute microscopic and analytical method in amber studies. Also, we discuss the most recent findings about ancient DNA repair and bacterial survival in remote substrates, which support the real possibility of ancient DNA amplification and bacterial resuscitation from Cretaceous resins.

Morphological stasis of protists in lower cretaceous amber.

Paleomicrobiological studies of terrestrial and freshwater protists are extremely rare in comparison with studies of eukaryotic microfossils from marine ecosystems. Using optical and electron microscopy (SEM-BSE) for hard substrates, we have examined protists trapped in Lower Cretaceous amber from Peñacerrada (Alava, Spain). We present the earliest reasonably confident microfossils of three taxa: Excavata (Euglenozoa), that are similar to the extant genera Euglena and Phacus; Chlorophyceae identified as members of the genus Chlamydomonas, and finally, in the taxon Ciliophora (Chromalveolata), two ciliated protozoa identified as Colpoda (Class Colpodea) and Prorodon (Class Prostomatea). Morphological stasis is evident, and identification based on phenotypic traits indicates the existence of conservative phenotypes persisting over geological time scales.

Endolithic microbial habitats as refuges for life in polyextreme environment of the Atacama Desert.

The extremely harsh conditions of hyperarid deserts are a true challenge for microbial life. Microorganisms thriving in such polyextreme environments are fascinating as they can tell us more about life, its strategies and its boundaries than other groups of organisms. The Atacama Desert (North Chile) holds two world records of extreme environmental characteristics: the lowest rainfall and greatest surface ultraviolet radiation and total solar irradiance ever measured on Earth. Despite these limiting conditions for life, we recently identified several remarkable examples of endolithic habitats colonized by phototrophic and heterotrophic microorganisms in the hyperarid core of the Atacama Desert.

Discovery of carotenoid red-shift in endolithic cyanobacteria from the Atacama Desert.

The biochemical responses of rock-inhabiting cyanobacteria towards native environmental stresses were observed in vivo in one of the Earth's most challenging extreme climatic environments. The cryptoendolithic cyanobacterial colonization, dominated by Chroococcidiopsis sp., was studied in an ignimbrite at a high altitude volcanic area in the Atacama Desert, Chile. Change in the carotenoid composition (red-shift) within a transect through the cyanobacteria dominant microbial community (average thickness ~1 mm) was unambiguously revealed in their natural endolithic microhabitat. The amount of red shifted carotenoid, observed for the first time in a natural microbial ecosystem, is depth dependent, and increased with increasing proximity to the rock surface, as proven by resonance Raman imaging and point resonance Raman profiling. It is attributed to a light-dependent change in carotenoid conjugation, associated with the light-adaptation strategy of cyanobacteria. A hypothesis is proposed for the possible role of an orange carotenoid protein (OCP) mediated non-photochemical quenching (NPQ) mechanism that influences the observed spectral behavior. Simultaneously, information about the distribution of scytonemin and phycobiliproteins was obtained. Scytonemin was detected in the uppermost cyanobacteria aggregates. A reverse signal intensity gradient of phycobiliproteins was registered, increasing with deeper positions as a response of the cyanobacterial light harvesting complex to low-light conditions.

Endolithic cyanobacteria in halite rocks from the hyperarid core of the Atacama Desert.

In the driest parts of the Atacama Desert there are no visible life forms on soil or rock surfaces. The soil in this region contains only minute traces of bacteria distributed in patches, and conditions are too dry for cyanobacteria that live under translucent stones. Here we show that halite evaporite rocks from the driest part of the Atacama Desert are colonized by cyanobacteria. This colonization takes place just a few millimeters beneath the rock surface, occupying spaces among salt crystals. Our work reveals that these communities are composed of extremely resistant Chroococcidiopsis morphospecies of cyanobacteria and associated heterotrophic bacteria. This newly discovered endolithic environment is an extremely dry and, at the same time, saline microbial habitat. Photosynthetic microorganisms within dry evaporite rocks could be an important and previously unrecognized target for the search for life within our Solar System.