Variable and Large Losses of Diagnostic Biomarkers After Simulated Cosmic Radiation Exposure in Clay- and Carbonate-Rich Mars Analog Samples.

Mars has been exposed to ionizing radiation for several billion years, and as part of the search for life on the Red Planet, it is crucial to understand the impact of radiation on biosignature preservation. Several NASA and ESA missions are looking for evidence of ancient life in samples collected at depths shallow enough that they have been impacted by galactic cosmic rays (GCRs). In this study, we exposed a diverse set of Mars analog samples to 0.9 Megagray (MGy) of gamma radiation to mimic 15 million years of exposure on the Martian surface. We measured no significant impact of GCRs on the total organic carbon (TOC) and bulk stable C isotopes in samples with initial TOC concentration > 0.1 wt. %; however, diagnostic molecular biosignatures presented a wide range of degradation that didn't correlate to factors like mineralogy, TOC, water content, and surface area. Exposure dating suggests that the surface of Gale crater has been irradiated at more than five times our dose, yet using this relatively low dose and "best-case scenario" geologically recalcitrant biomarkers, large and variable losses were nevertheless evident. Our results empasize the importance of selecting sampling sites at depth or recently exposed at the Martian surface.

Five state factors control progressive stages of freshwater salinization syndrome.

Factors driving freshwater salinization syndrome (FSS) influence the severity of impacts and chances for recovery. We hypothesize that spread of FSS across ecosystems is a function of interactions among five state factors: human activities, geology, flowpaths, climate, and time. (1) Human activities drive pulsed or chronic inputs of salt ions and mobilization of chemical contaminants. (2) Geology drives rates of erosion, weathering, ion exchange, and acidification-alkalinization. (3) Flowpaths drive salinization and contaminant mobilization along hydrologic cycles. (4) Climate drives rising water temperatures, salt stress, and evaporative concentration of ions and saltwater intrusion. (5) Time influences consequences, thresholds, and potentials for ecosystem recovery. We hypothesize that state factors advance FSS in distinct stages, which eventually contribute to failures in systems-level functions (supporting drinking water, crops, biodiversity, infrastructure, etc.). We present future research directions for protecting freshwaters at risk based on five state factors and stages from diagnosis to prognosis to cure.

Rapid Radiolytic Degradation of Amino Acids in the Martian Shallow Subsurface: Implications for the Search for Extinct Life.

Amino acids are fundamental to life as we know them as the monomers of proteins and enzymes. They are also readily synthesized under a variety of plausible prebiotic conditions and are common in carbon-rich meteorites. Thus, they represent a reasonable class of organics to target in the search for prebiotic chemistry or chemical evidence of life on Mars. However, regardless of their origin, amino acids and other organic molecules present in near-surface regolith and rocks on Mars can be degraded by exposure to cosmic rays that can penetrate to a depth of a few meters. We exposed several pure amino acids in dry and hydrated silicate mixtures and in mixtures of silicates with perchlorate salts to gamma radiation at various temperatures and radiation doses representative of the martian near-subsurface. We found that irradiation of amino acids mixed with dry silica powder increased the rate of amino acid radiolysis, with the radiolysis constants of amino acids in silicate mixtures at least a factor of 10 larger compared with the radiolysis constants of amino acids alone. The addition of perchlorate salts to the silicate samples or hydration of silicate samples further accelerated the rate of amino acid destruction during irradiation and increased the radiolysis constants by a factor of ∼1.5. Our results suggest that even low-molecular-weight amino acids could degrade in just ∼20 million years in the top 10 cm of the martian surface regolith and rock, and even faster if the material contains elevated abundances of hydrated silicate minerals or perchlorates. We did not detect evidence of amino acid racemization after gamma radiation exposure of the samples, which indicates that the chirality of some surviving amino acids may still be preserved. Our experimental results suggest serious challenges for the search of ancient amino acids and other potential organic biosignatures in the top 2 m of the martian surface.

Trade-offs between reducing complex terminology and producing accurate interpretations from environmental DNA: Comment on "Environmental DNA: What's behind the term?" by Pawlowski et al., (2020).

In a recent paper, "Environmental DNA: What's behind the term? Clarifying the terminology and recommendations for its future use in biomonitoring," Pawlowski et al. argue that the term eDNA should be used to refer to the pool of DNA isolated from environmental samples, as opposed to only extra-organismal DNA from macro-organisms. We agree with this view. However, we are concerned that their proposed two-level terminology specifying sampling environment and targeted taxa is overly simplistic and might hinder rather than improve clear communication about environmental DNA and its use in biomonitoring. This terminology is based on categories that are often difficult to assign and uninformative, and it overlooks a fundamental distinction within eDNA: the type of DNA (organismal or extra-organismal) from which ecological interpretations are derived.

Biomarker stratigraphy in the Athel Trough of the South Oman Salt Basin at the Ediacaran-Cambrian Boundary.

The South Oman Salt Basin (SOSB) has been studied extensively for knowledge concerning the habitat of the enigmatic Ediacaran-Cambrian oils that are produced from that region. Geological, geochemical, geophysical, and geochronological investigations have all contributed to improved understanding of the range of late Neoproterozoic depositional environments recorded there. Of particular interest has been the deep Athel depocenter within the SOSB that features a silica-rich interval known as the Al Shomou Member or Athel Silicilyte and the co-eval A4 carbonate-evaporite sequence that straddles the Ediacaran-Cambrian boundary. The deep basin has been suggested to be anoxic and euxinic based on studies of sulfur isotopes, trace metal distributions and other proxies. Organic geochemistry has provided some clues concerning aspects of the depositional environments and microbial communities prevailing during this interval. However, ambiguities remain including a paucity of convincing molecular evidence for euxinia in the photic zone of the basin. Here, we present a comprehensive study of biomarker hydrocarbons, including steroids, triterpenoids, and carotenoids. Among the compounds detected is a distinctive array of aromatic carotenoids. Relatively low abundances of monoaromatic carotenoids, such as chlorobactane, okenane, and ß-isorenieratane, suggest the possibility of transient photic zone euxinia with a shallow chemocline or, perhaps, exogenous inputs from microbial mats. However, it is the dominance of renieratane and renierapurpurane over isorenieratane in diaromatic carotenoids and their association with abundant C38 and C39 carotenoids that identifies cyanobacteria as major contributors to the inventory of carotenoids. Our results, based on multiple lines of molecular evidence and statistical analysis, also suggest that the Athel Silicilyte was biogeochemically distinct from the other units of the Ara Group. Overall, our study has important implications for understanding other late Neoproterozoic depositional environments.

Author Correction: Effects of sampling effort on biodiversity patterns estimated from environmental DNA metabarcoding surveys.

A correction to this article has been published and is linked from the HTML and PDF versions of this paper. The error has been fixed in the paper.

eDNA metabarcoding as a new surveillance approach for coastal Arctic biodiversity.

Because significant global changes are currently underway in the Arctic, creating a large-scale standardized database for Arctic marine biodiversity is particularly pressing. This study evaluates the potential of aquatic environmental DNA (eDNA) metabarcoding to detect Arctic coastal biodiversity changes and characterizes the local spatio-temporal distribution of eDNA in two locations. We extracted and amplified eDNA using two COI primer pairs from ~80 water samples that were collected across two Canadian Arctic ports, Churchill and Iqaluit, based on optimized sampling and preservation methods for remote regions surveys. Results demonstrate that aquatic eDNA surveys have the potential to document large-scale Arctic biodiversity change by providing a rapid overview of coastal metazoan biodiversity, detecting nonindigenous species, and allowing sampling in both open water and under the ice cover by local northern-based communities. We show that DNA sequences of ~50% of known Canadian Arctic species and potential invaders are currently present in public databases. A similar proportion of operational taxonomic units was identified at the species level with eDNA metabarcoding, for a total of 181 species identified at both sites. Despite the cold and well-mixed coastal environment, species composition was vertically heterogeneous, in part due to river inflow in the estuarine ecosystem, and differed between the water column and tide pools. Thus, COI-based eDNA metabarcoding may quickly improve large-scale Arctic biomonitoring using eDNA, but we caution that aquatic eDNA sampling needs to be standardized over space and time to accurately evaluate community structure changes.

Effects of sampling effort on biodiversity patterns estimated from environmental DNA metabarcoding surveys.

Environmental DNA (eDNA) metabarcoding can greatly enhance our understanding of global biodiversity and our ability to detect rare or cryptic species. However, sampling effort must be considered when interpreting results from these surveys. We explored how sampling effort influenced biodiversity patterns and nonindigenous species (NIS) detection in an eDNA metabarcoding survey of four commercial ports. Overall, we captured sequences from 18 metazoan phyla with minimal differences in taxonomic coverage between 18 S and COI primer sets. While community dissimilarity patterns were consistent across primers and sampling effort, richness patterns were not, suggesting that richness estimates are extremely sensitive to primer choice and sampling effort. The survey detected 64 potential NIS, with COI identifying more known NIS from port checklists but 18 S identifying more operational taxonomic units shared between three or more ports that represent un-recorded potential NIS. Overall, we conclude that eDNA metabarcoding surveys can reveal global similarity patterns among ports across a broad array of taxa and can also detect potential NIS in these key habitats. However, richness estimates and species assignments require caution. Based on results of this study, we make several recommendations for port eDNA sampling design and suggest several areas for future research.

Environmental DNA metabarcoding: Transforming how we survey animal and plant communities.

The genomic revolution has fundamentally changed how we survey biodiversity on earth. High-throughput sequencing ("HTS") platforms now enable the rapid sequencing of DNA from diverse kinds of environmental samples (termed "environmental DNA" or "eDNA"). Coupling HTS with our ability to associate sequences from eDNA with a taxonomic name is called "eDNA metabarcoding" and offers a powerful molecular tool capable of noninvasively surveying species richness from many ecosystems. Here, we review the use of eDNA metabarcoding for surveying animal and plant richness, and the challenges in using eDNA approaches to estimate relative abundance. We highlight eDNA applications in freshwater, marine and terrestrial environments, and in this broad context, we distill what is known about the ability of different eDNA sample types to approximate richness in space and across time. We provide guiding questions for study design and discuss the eDNA metabarcoding workflow with a focus on primers and library preparation methods. We additionally discuss important criteria for consideration of bioinformatic filtering of data sets, with recommendations for increasing transparency. Finally, looking to the future, we discuss emerging applications of eDNA metabarcoding in ecology, conservation, invasion biology, biomonitoring, and how eDNA metabarcoding can empower citizen science and biodiversity education.

Improving herpetological surveys in eastern North America using the environmental DNA method.

Among vertebrates, herpetofauna has the highest proportion of declining species. Detection of environmental DNA (eDNA) is a promising method towards significantly increasing large-scale herpetological conservation efforts. However, the integration of eDNA results within a management framework requires an evaluation of the efficiency of the method in large natural environments and the calibration of eDNA surveys with the quantitative monitoring tools currently used by conservation biologists. Towards this end, we first developed species-specific primers to detect the wood turtle (Glyptemys insculpta) a species at risk in Canada, by quantitative PCR (qPCR). The rate of eDNA detection obtained by qPCR was also compared to the relative abundance of this species in nine rivers obtained by standardized visual surveys in the Province of Québec (Canada). Second, we developed multi-species primers to detect North American amphibian and reptile species using eDNA metabarcoding analysis. An occurrence index based on the distribution range and habitat type was compared with the eDNA metabarcoding dataset from samples collected in seven lakes and five rivers. Our results empirically support the effectiveness of eDNA metabarcoding to characterize herpetological species distributions. Moreover, detection rates provided similar results to standardized visual surveys currently used to develop conservation strategies for the wood turtle. We conclude that eDNA detection rates may provide an effective semiquantitative survey tool, provided that assay calibration and standardization is performed.

Estimating fish abundance and biomass from eDNA concentrations: variability among capture methods and environmental conditions.

Environmental DNA (eDNA) promises to ease noninvasive quantification of fish biomass or abundance, but its integration within conservation and fisheries management is currently limited by a lack of understanding of the influence of eDNA collection method and environmental conditions on eDNA concentrations in water samples. Water temperature is known to influence the metabolism of fish and consequently could strongly affect eDNA release rate. As water temperature varies in temperate regions (both seasonally and geographically), the unknown effect of water temperature on eDNA concentrations poses practical limitations on quantifying fish populations using eDNA from water samples. This study aimed to clarify how water temperature and the eDNA capture method alter the relationships between eDNA concentration and fish abundance/biomass. Water samples (1 L) were collected from 30 aquaria including triplicate of 0, 5, 10, 15 and 20 Brook Charr specimens at two different temperatures (7 °C and 14 °C). Water samples were filtered with five different types of filters. The eDNA concentration obtained by quantitative PCR (qPCR) varied significantly with fish abundance and biomass and types of filters (mixed-design ANOVA, P < 0.001). Results also show that fish released more eDNA in warm water than in cold water and that eDNA concentration better reflects fish abundance/biomass at high temperature. From a technical standpoint, higher levels of eDNA were captured with glass fibre (GF) filters than with mixed cellulose ester (MCE) filters and support the importance of adequate filters to quantify fish abundance based on the eDNA method. This study supports the importance of including water temperature in fish abundance/biomass prediction models based on eDNA.

Complex genetic patterns in closely related colonizing invasive species.

Anthropogenic activities frequently result in both rapidly changing environments and translocation of species from their native ranges (i.e., biological invasions). Empirical studies suggest that many factors associated with these changes can lead to complex genetic patterns, particularly among invasive populations. However, genetic complexities and factors responsible for them remain uncharacterized in many cases. Here, we explore these issues in the vase tunicate Ciona intestinalis (Ascidiacea: Enterogona: Cionidae), a model species complex, of which spA and spB are rapidly spreading worldwide. We intensively sampled 26 sites (N = 873) from both coasts of North America, and performed phylogenetic and population genetics analyses based on one mitochondrial fragment (cytochrome c oxidase subunit 3-NADH dehydrogenase subunit I, COX3-ND1) and eight nuclear microsatellites. Our analyses revealed extremely complex genetic patterns in both species on both coasts. We detected a contrasting pattern based on the mitochondrial marker: two major genetic groups in C. intestinalis spA on the west coast versus no significant geographic structure in C. intestinalis spB on the east coast. For both species, geo-graphically distant populations often showed high microsatellite-based genetic affinities whereas neighboring ones often did not. In addition, mitochondrial and nuclear markers provided largely inconsistent genetic patterns. Multiple factors, including random genetic drift associated with demographic changes, rapid selection due to strong local adaptation, and varying propensity for human-mediated propagule dispersal could be responsible for the observed genetic complexities.

Disentangling invasion processes in a dynamic shipping-boating network.

The relative importance of multiple vectors to the initial establishment, spread and population dynamics of invasive species remains poorly understood. This study used molecular methods to clarify the roles of commercial shipping and recreational boating in the invasion by the cosmopolitan tunicate, Botryllus schlosseri. We evaluated (i) single vs. multiple introduction scenarios, (ii) the relative importance of shipping and boating to primary introductions, (iii) the interaction between these vectors for spread (i.e. the presence of a shipping-boating network) and (iv) the role of boating in determining population similarity. Tunicates were sampled from 26 populations along the Nova Scotia, Canada, coast that were exposed to either shipping (i.e. ports) or boating (i.e. marinas) activities. A total of 874 individuals (c. 30 per population) from five ports and 21 marinas was collected and analysed using both mitochondrial cytochrome c oxidase subunit I gene (COI) and 10 nuclear microsatellite markers. The geographical location of multiple hotspot populations indicates that multiple invasions have occurred in Nova Scotia. A loss of genetic diversity from port to marina populations suggests a stronger influence of ships than recreational boats on primary coastal introductions. Population genetic similarity analysis reveals a dependence of marina populations on those that had been previously established in ports. Empirical data on marina connectivity because of boating better explains patterns in population similarities than does natural spread. We conclude that frequent primary introductions arise by ships and that secondary spread occurs gradually thereafter around individual ports, facilitated by recreational boating.

Cytochrome P450-catalyzed degradation of nicotine: fundamental parameters determining hydroxylation by cytochrome P450 2A6 at the 5'-carbon or the n-methyl carbon.

The oxidation of (2'S)-nicotine in the active site of human cytochrome P450 2A6 has been subjected to a detailed analysis by theoretical quantum mechanical/molecular mechanical (QM/MM) calculations linked with a theoretical and experimental study of the associated isotope effects. The study has focused on seeking an explanation as to why oxidation at the 5'-carbon position (A) is favored over oxidation at the methyl carbon (CMe) position (B). It is deduced that the choice of hydrogen for abstraction is not determined by geometric features of the active site, but by the lower energy barrier associated with 5' oxidation. N-Demethylation leading to N-hydroxymethylnornicotine requires ca. 6.5 kcal/mol more energy to transfer a hydrogen atom than is required for oxidation on the carbon 5'. Neither protonation of the pyrrolidine nitrogen (N1') nor inclusion of a water molecule in the reaction process influences the balance between the two oxidation pathways. In both cases, the hydrogen transfer step is rate limiting. An analysis of the calculated kinetic isotope effects indicates that the presence of a (2)H in either the C5' or the CMepositions has a significant effect on the reaction kinetics. However, the experimental values of around 2.2-2.6 are considerably lower than those predicted by theoretical calculations (9.3 and 6.9 for C5' or the CMe positions, respectively, in the LS state of Cpd I), typical of the masking commonly found for CYP450 reactions. The fact that similar values are found for cotinine formation from both substrates, however, may indicate that the measured value is not that for H-abstraction but, rather, is a combined value for (2)H influence on electronic redistribution between iminium states of the pyrrolidine ring. This is the first time that oxidation at the C5' or the CMe positions has been directly compared and that isotope effects have been obtained for this reaction in a human cytochrome P450 reaction.

Jewelry boxes contaminated by Aspergillus oryzae: an occupational health risk?

In 2009, 100,000 jewelry boxes, manufactured in China, were delivered to a jewelry manufacturer in Besançon, France. All the boxes were contaminated by mold. Because the workers refused to handle these jewelry boxes, the company contacted our laboratory to determine how to deal with the problem. Three choices were available: (1) decontaminate the boxes, (2) return the boxes to the Chinese manufacturer, or (3) destroy the entire shipment. Based on microscopic identification, the culture analysis was positive for A. oryzae. This could not be confirmed by molecular techniques because of the genetic proximity of A. oryzae and A. flavus. Because A. flavus can produce aflatoxins, we tested for them using mass spectrometry. Aflatoxins B1, B2, G1, G2, and M1 were not detected; however, given the specifics of this situation, we could not discard the possibility of the presence of other aflatoxins, such as P1, B3, GM2, and ethoxyaflatoxin B2. We concluded that the contamination by A. oryzae was probably due to food products. However, because of the possible presence of aflatoxins, occupational health risks could not be entirely ruled out. The decision was therefore taken to destroy all the jewelry boxes by incineration. To avoid a similar situation we propose: (1) to maintain conditions limiting mold contamination during production (not eating on the work site, efficient ventilation systems); (2) to desiccate the products before sending them; and (3) to closely control the levels of dampness during storage and transport.