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After Viking proved that long-term, meaningful scientific data could be sent reliably from the martian surface, the field fundamentally shifted. Its success reshaped how scientists and engineers formulated their research and planned future missions. In a similar way, the Ingenuity Mars Helicopter's demonstration of flight changes the paradigm, enabling new ways to do science on Mars and other planetary bodies. Rotorcraft enable increased range and coverage, access to hazardous terrain, and access to the atmospheric boundary layer. These capabilities expand the scientific reach of future missions. In astrobiology, aerial explorers could revolutionize the study of subsurface cavities and potential refuges for life, the detection of trace gases, high-resolution geological mapping, sampling or sample retrieval, and planetary protection. The technology driving improvements in both standalone rotorcraft and coordinated fleets of aerial vehicles is progressing quickly. As these systems mature, they are poised to become essential tools in planetary exploration.

Following the Viking experiments in 1976, many of the original inferences of biological metabolism have been replicated via abiotic mechanisms we now know are plausible on Mars' surface. While in many cases subsequent experiments have cast doubt on whether Viking truly detected life, numerous other studies since Viking have greatly expanded our knowledge of life's limits and microbial metabolism. In particular, increased characterization of Earth's subsurface has revealed the astounding complexity and adaptability of life, highlighting chemically based metabolisms as potentially strong targets for future life detection missions. Over the same time frame, we have gained knowledge of putatively more habitable regions in Mars' subsurface, relative to the original Viking lander surface sites, that could host similar organisms. In this review, we discuss the wealth of knowledge concerning the habitability of zones across Mars' surface/subsurface, and we suggest specific microbial metabolisms that should be targeted in future life detection missions based on laboratory and field studies under analogous conditions on Earth and with consideration of recommendations from the larger Astrobiology community. The ability to leverage these advancements in subsurface research toward the incorporation of increased specificity in future life detection efforts is additionally discussed in the context of current Mars subsurface mission progress and planetary protection and defense concerns.

Questions about our earliest beginnings have filled thinking minds for millennia across cultures, faiths, and wide-ranging frontiers of research. Asking where we come from is as fundamental as astrobiology's driving query of "are we alone?" These days, we often link these questions as we explore life beyond our planet and solar system. Scientific steps toward answers have been big and frequent, but the pathways remain highly varied. Achieving something even close to a consensus has been elusive. The one thing we all can agree on, however, is that our understanding of Earth's earliest stages, and that of our solar system, has advanced by leaps and bounds over recent decades. No longer must we explore life's beginnings with little knowledge of how and when planetary habitability first developed and, more specifically, what the world was like roughly 4.4-4.2 billion years ago-a reasonable time estimate for the initial steps in the progression toward life. The simple distillation of this view is that models for life's earliest chapters, including experimental simulations of prebiotic chemistry, can and should be designed around an increasingly sophisticated understanding of Earth's initial boundary conditions, including the timing and controls on the emergence of oceans, the atmosphere, and tectonics-along with their coupled evolutions.

Desert cyanobacteria of the Chroococcidiopsis genus, due to their remarkable desiccation and radiation tolerance, are considered model candidates in the field of astrobiology. For this reason, three strains, namely Chroococcidiopsis sp. CCMEE 029, 057, and 064 have been exposed in the dried state to space- and Mars-like conditions throughout laboratory simulations and real space exposure using the EXPOSE facility installed outside the International Space Station. However, how they can recover upon rehydration and repair the damage accumulated under extreme conditions on Earth and in space remains to be fully elucidated through omics-based investigations. Hence, comparative genomics of these three Chroococcidiopsis strains offered a powerful lens to explore the genetic components of their capability to persist under extreme conditions. The analysis of newly obtained gapless genome assemblies of the laboratory-maintained reference strains of CCMEE 029, 057, and 064 allowed the identification of conserved and non-shared genes involved in reactive oxygen species detoxification, desiccation tolerance, DNA protection, and repair. Moreover, a biosynthetic gene cluster for scytonemin production was identified in every strain, while strain CCMEE 057 also harbored the genes for the biosynthesis of a mycosporine-like amino-acid. Such insights are crucial for understanding the adaptation strategies employed by microorganisms to survive in dry, radiation-intense environments, offering clues about the potential for life beyond Earth.

Biosignature detection remains a key challenge in astrobiology, yet robust mineral biosignatures remain limited. Raman spectroscopy is increasingly applied in planetary exploration, but its high-dimensional spectral information has not yet been fully exploited for biosignature discrimination using data-driven approaches. Here, we integrate Raman spectroscopy with interpretable machine learning to distinguish biotic from abiotic apatite, a ubiquitous phosphate mineral in terrestrial and extraterrestrial environments. We compile 331 apatite Raman spectra from abiotic and biotic sources and extract 21 band-resolved spectral features. Principal component analysis reveals systematic separation between abiotic and biotic endmembers. A random forest classifier achieves 96.8% accuracy on an independent test set. Robustness is confirmed by multiple validation schemes, including leave-one-source-out cross-validation across 60 independent data sources, indicating that model performance generalizes beyond source- or instrument-specific artifacts. Feature importance identifies two dominant controls: phosphate-band broadening as a structural indicator of disorder and the carbonate-band intensity as a chemical signature of substitution. Density-functional calculations reproduce these features in simulated spectra and indicate that carbonate substitution doubles phosphate-tetrahedral distortion and increases formation energies by two orders of magnitude. Mechanically, higher carbonate contents during biomineral apatite formation reduce crystallinity and broaden Raman bands. We propose that the trained machine-learning model and a two-feature decision map enable the rapid probabilistic discrimination of unknown apatite samples. Our Raman-based machine-learning framework establishes a broadly applicable and mission-relevant strategy for deep-time archives and future planetary missions.

The anhydrobiotic (living without water) yeast Saccharomyces cerevisiae has gained considerable interest for its use in a variety of fields, from investigation of the biological effects of deep space radiation exposure to its utilization as a radiation dosimeter and its applicability in the study of preservation of microbial life in martian regolith. A complete radiobiological characterization of yeast during anhydrobiosis must be undertaken to ensure the informed interpretation of these works. This study investigated the radiobiological properties of a desiccated recombinational repair-deficient rad51 knockout strain of S. cerevisiae. We focused on radiation tolerance, oxygen enhancement ratio (OER), and relative biological effectiveness (RBE). Desiccation-induced radioresistance was significantly higher compared with hydrated yeast; it required a threefold higher dose for an equivalent biological response. Desiccated yeast exhibited no OER, which indicates the absence of oxygen-dependent radiosensitization due to the lack of indirect damage pathways. RBE measurements for a 74 MeV proton beam (1.10 ± 0.01) and a neutron beam (12.2 ± 0.7) align with prior studies and demonstrate consistency between desiccated and hydrated systems when appropriately scaled. These findings support the use of the desiccated yeast model as a robust and translatable system for addressing fundamental questions in astrobiology and radiobiology.

Biological evolution confronts situations in which the modification of a trait to improve the performance of one function may diminish the performance of another. Similar trade-offs occur in economics and engineering, where they are evaluated with the concept of Pareto optimality. "Pareto optimal" solutions are solutions such that performance cannot be improved for any task without sacrificing performance for another task. Solutions outside the Pareto optimal set are likely to be uncompetitive in that they could be improved without negative consequences. Biologists have argued that optimization for multiple biological functions restricts the variety of evolutionarily stable phenotypes to a Pareto set within traitspace, while promoting diversity within this set. Here, we consider whether evidence for such optimality could serve as evidence of life in astrobiology. We propose that objects whose properties lie demonstrably within a region constrained by trade-offs between biologically relevant functions are more likely to be biogenic; examples discussed here include bacterial morphology, mycelial networks, and the selection of molecules. Conversely, objects with comparable characteristics to known forms of life that exist outside the relevant Pareto set are less likely to be biological. We conclude that the detection of Pareto optimality may disclose functionality and, hence, biogenicity in unfamiliar materials.

The detection of amino acids in extraterrestrial environments has important implications for astrobiology and prebiotic chemistry, yet the pathways to their syntheses and their photostability under such conditions remain unclear. In this study, we employ femtosecond UV-pump visible-probe spectroscopy to investigate the ultrafast relaxation dynamics of non-aromatic amino acids in aqueous environments, representative of those found in Murchison-type meteorites and pristine Bennu samples. Using excitation wavelengths around 210 nm and probing in the visible range, two distinct decay lifetimes are identified, revealing the coexistence of internal conversion and fluorescence decay mechanisms. Internal conversion lifetimes range from 25 to 32 ps, showing efficient non-radiative relaxation processes, while fluorescence decays have a slower timescale of about 1 ns. These findings can help clarify the excited-state relaxation pathways that influence the photostability and survival of these amino acids.

Fleeting public interest in the Viking mission inspired Carl Sagan to devise a new mode of science communication. One result was the Cosmos television series. Carl's global success offended many in the scientific community, and he was punished despite his solid research credentials. Almost 50 years later, the importance of public outreach by scientists seems to have reached greater professional acceptance. Key Words: Science communication-Viking-Cosmos-Mars-Public outreach. Astrobiology 26, 35S-37S.

Volcanic fumaroles are extreme habitats with high temperatures, sulfur‑rich gases, and low pH, creating challenging niches for life. Soil microbiota in these environments remain underexplored, particularly at near‑vent sites with pH below 2. We characterized bacterial communities in hyperacid soils of Taiwan's Tatun Volcanic Group to examine how season and soil properties shape these communities. We sampled 39 soils and 8 hot spring waters from four fumaroles during dry and wet seasons. Soil pH, nutrients (nitrate, ammonium), organic matter, and altitude were recorded, and bacterial diversity was profiled via 16S rRNA gene sequencing. Pseudomonadota overwhelmingly dominated the communities, followed by Bacillota and Actinomycetota. Wet-season soils displayed more homogeneous assemblages, whereas dry-season soils exhibited greater heterogeneity. Soil taxonomy and texture also modulated patterns, with Andisols supporting more consistent communities than Inceptisols. Environmental variables such as nitrate, ammonium, organic matter, and altitude correlated with community composition. Heatmap and network analyses highlighted Delftia and Pseudomonas as key taxa. Extreme acidity and seasonal hydrology thus select for specialized bacterial assemblages in these fumarolic soils. These findings advance understanding of microbial adaptation to geochemical extremes and reveal how seasonal and edaphic factors structure communities, offering implications for biogeochemical cycling and astrobiology.

Experimentally modeling the habitability of extreme ocean world conditions, such as those on Jupiter's moon: Europa, would benefit from integrated microbe-virus model studies to provide both a microbial model and a virus capable of nutrient cycling. This work experimentally probes the microbe/virus combination Saci/STIV as a potential microbial model for habitability studies of Europa's ice-ocean interface in subsurface lakes with implications for habitats with extreme conditions and fluid mixing. This study experimentally modeled archaea/virus-integrated systems in one set of conditions that fall within the range of currently proposed for subsurface lakes in Europa's ice shell: 3.5% salinity, pH 3, rich in Na+/Fe2+/Mg2+/ SO 4 2 - /H+, fluctuating dissolved oxygen (D.O.), and 1-2 °C to explore habitability limits and candidate biosignatures. Planktonic Sulfolobus acidocaldarius (Saci) lysogens of Sulfolobus Turreted Icosahedral Virus (STIV) were included for the determination of potential habitability and biosignatures in both models. Extensive biofilm formation, biosulfur detection via Raman spectroscopy, viral mRNA/protein detection by RT-qPCR and western blotting, and successful viability assays under these conditions support the potential habitability under Europa-analog conditions. Cold-adapted Saci developed extensive biofilm rich in biosulfur globules, suggesting the possibility of sulfur-oxidizing metabolism. Finally, biosulfur and viral major capsid proteins (MCPs), with inter-domain conserved double jelly roll morphologies, were identified as candidate biosignatures for life on acidic icy ocean worlds. Such cryotolerant strains and the viral proteins proliferated from them may have applications in astrobiology, but also for extreme microbiology and beyond.

This article is an anecdotal recap of lessons learned from the ALH 84001 debate in light of the historical and recent debate over the Viking results. While it has been 50 years since Viking, it has also been 30 years since the announcement of possible indigenous martian life in the ALH 84001 meteorite. Much has been learned in the interim period, and many lessons are still being learned from the subsequent debate and the NASA Mars exploration and astrobiology programs it triggered.

James Lovelock was the first to lay out a physical and chemical basis for searching for life beyond Earth, based on the observation that the molecular features and patterns in classes of organics differ for compounds formed through biological versus nonbiological processes. This approach shaped the Viking molecular analysis experiment designed to search for organics, including potential signs of extinct or extant life, and it still remains fundamentally viable today. In the five decades since Viking, many missions have uncovered new information about the martian organic inventory, geological landscape, and changes to habitability over time, while advances in organic geochemistry on Earth enabled scientists to identify even more differences between biotic and abiotic organics, solidifying the utility of Lovelock's physical and chemical approach in the search for extraterrestrial life. Key Words: Search for Mars' Organics-Mars-Biosignatures-Life detection-Biomarkers. Astrobiology 26, 72S-78S.

Halophilic microorganisms are central to biotechnology, bioremediation, and astrobiology because they persist under extreme and polyextreme conditions analogous to extraterrestrial environments. Although metagenomics has transformed the study of halophilic biodiversity, available datasets remain fragmented and unevenly documented. To assess how halophilic metagenomic research reflects the global exploration of hypersaline environments, we analyzed PubMed-indexed studies and associated sequencing metadata deposited at the National Center for Biotechnology Information (NCBI) Sequence Read Archive (SRA) using a curation workflow. Our quantitative analysis reveals a severe geographic bias linked to uneven global research investment (Gini coefficient = 0.736), with a small number of countries contributing to many publicly available datasets. In contrast, the environmental distribution of these samples showed moderate ecological uniformity (Pielou's Evenness = 0.818), though we identified pervasive gaps in metadata completeness that hinder dataset interoperability. Our curated dataset highlights a strong research focus on polyextremophilic habitats, positioning these ecosystems as prime targets for biotechnological and astrobiological bioprospecting. Additionally, the geographical bias highlights the need for interoperable global data frameworks and more equitable investment in data generation and analysis, especially in underrepresented regions of the Global South.