The Preservation and Spectral Detection of Historic Museum Specimen Microbial Mat Biosignatures Within Martian Dust: Lessons Learned for Mars Exploration and Sample Return.

The key building blocks for life on Mars could be preserved within potentially habitable paleo-depositional settings with their detection possible by utilizing mid-infrared spectroscopy; however, a definite identification and confirmation of organic or even biological origin will require the samples to be returned to Earth. In the present study, Fourier-transform infrared (FTIR) spectroscopic techniques were used to characterize both mineralogical and organic materials within Mars dust simulant JSC Mars-1 and ancient Antarctic cyanobacterial microbial mats from 1901 to 1904 Discovery Expedition. When FTIR spectroscopy is applied to cyanobacterial microbial mat communities, the resulting spectra will reflect the average biochemical composition of the mats rather than taxa-specific spectral patterns of the individual organisms and can thus be considered as a total chemical analysis of the mat colony. This study also highlights the potential difficulties in the detection of these communities on Mars and which spectral biosignatures will be most detectable within geological substrates. Through the creation and analysis of a suite of dried microbial mat material and Martian dust simulant mixtures, the spectral signatures and wavenumber positions of CHx aliphatic hydrocarbons and the C-O and O-H bands of polysaccharides remained detectable and may be detectable within sample mixtures obtained through Mars Sample Return activities.

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.

Microbial Ecology of an Arctic Travertine Geothermal Spring: Implications for Biosignature Preservation and Astrobiology.

Jotun springs in Svalbard, Norway, is a rare warm environment in the Arctic that actively forms travertine. In this study, we assessed the microbial ecology of Jotun's active (aquatic) spring and dry spring transects. We evaluated the microbial preservation potential and mode, as well as the astrobiological relevance of the travertines to marginal carbonates mapped at Jezero Crater on Mars (the Mars 2020 landing site). Our results revealed that microbial communities exhibited spatial dynamics controlled by temperature, fluid availability, and geochemistry. Amorphous carbonates and silica precipitated within biofilm and on the surface of filamentous microorganisms. The water discharged at the source is warm, with near neutral pH, and undersaturated in silica. Hence, silicification possibly occurred through cooling, dehydration, and partially by a microbial presence or activities that promote silica precipitation. CO2 degassing and possible microbial contributions induced calcite precipitation and travertine formation. Jotun revealed that warm systems that are not very productive in carbonate formation may still produce significant carbonate buildups and provide settings favorable for fossilization through silicification and calcification. Our findings suggest that the potential for amorphous silica precipitation may be essential for Jezero Crater's marginal carbonates because it significantly increases the preservation potential of putative martian organisms.

Unveiling Challenging Microbial Fossil Biosignatures from Rio Tinto with Micro-to-Nanoscale Chemical and Ultrastructural Imaging.

Understanding the nature and preservation of microbial traces in extreme environments is crucial for reconstructing Earth's early biosphere and for the search for life on other planets or moons. At Rio Tinto, southwestern Spain, ferric oxide and sulfate deposits similar to those discovered at Meridiani Planum, Mars, entomb a diversity of fossilized organisms, despite chemical conditions commonly thought to be challenging for life and fossil preservation. Investigating this unique fossil microbiota can elucidate ancient extremophile communities and the preservation of biosignatures in acidic environments on Earth and, potentially, Mars. In this study, we use an innovative multiscale approach that combines the state-of-the-art synchrotron X-ray nanoimaging methods of ptychographic X-ray computed laminography and nano-X-ray fluorescence to reveal Rio Tinto's microfossils at subcellular resolution. The unprecedented nanoscale views of several different specimens within their geological and geochemical contexts reveal novel intricacies of preserved microbial communities. Different morphotypes, ecological interactions, and possible taxonomic affinities were inferred based on qualitative and quantitative 3D ultrastructural information, whereas diagenetic processes and metabolic affinities were inferred from complementary chemical information. Our integrated nano-to-microscale analytical approach revealed previously invisible microbial and mineral interactions, which complemented and filled a gap of spatial resolution in conventional methods. Ultimately, this study contributes to the challenge of deciphering the faint chemical and morphological biosignatures that can indicate life's presence on the early Earth and on distant worlds.

Cosmic kidney disease: an integrated pan-omic, physiological and morphological study into spaceflight-induced renal dysfunction.

Missions into Deep Space are planned this decade. Yet the health consequences of exposure to microgravity and galactic cosmic radiation (GCR) over years-long missions on indispensable visceral organs such as the kidney are largely unexplored. We performed biomolecular (epigenomic, transcriptomic, proteomic, epiproteomic, metabolomic, metagenomic), clinical chemistry (electrolytes, endocrinology, biochemistry) and morphometry (histology, 3D imaging, miRNA-ISH, tissue weights) analyses using samples and datasets available from 11 spaceflight-exposed mouse and 5 human, 1 simulated microgravity rat and 4 simulated GCR-exposed mouse missions. We found that spaceflight induces: 1) renal transporter dephosphorylation which may indicate astronauts' increased risk of nephrolithiasis is in part a primary renal phenomenon rather than solely a secondary consequence of bone loss; 2) remodelling of the nephron that results in expansion of distal convoluted tubule size but loss of overall tubule density; 3) renal damage and dysfunction when exposed to a Mars roundtrip dose-equivalent of simulated GCR.

Acclimation mechanism of microalgal photosynthetic apparatus under low atmospheric pressures - new astrobiological perspectives in a Mars-like atmosphere.

This study reveals a new acclimation mechanism of the eukaryotic unicellular green alga Chlorella vulgaris in terms of the effect of varying atmospheric pressures on the structure and function of its photosynthetic apparatus using fluorescence induction measurements (JIP-test). The results indicate that low (400mbar) and extreme low (2 atmosphere (simulating the Mars atmosphere), reveals that the impact of extremely low atmospheric pressure on PQ mobility within the photosynthetic membrane, coupled with the low density of an almost 100% CO2 Mars-like atmosphere, results to a similar photosynthetic efficiency to that on Earth. These findings pave the way for the identification of novel functional acclimation mechanisms of microalgae to extreme environments that are vastly distinct from those found on Earth.

Siderite and vivianite as energy sources for the extreme acidophilic bacterium Acidithiobacillus ferrooxidans in the context of mars habitability.

Past and present habitability of Mars have been intensely studied in the context of the search for signals of life. Despite the harsh conditions observed today on the planet, some ancient Mars environments could have harbored specific characteristics able to mitigate several challenges for the development of microbial life. In such environments, Fe2+ minerals like siderite (already identified on Mars), and vivianite (proposed, but not confirmed) could sustain a chemolithoautotrophic community. In this study, we investigate the ability of the acidophilic iron-oxidizing chemolithoautotrophic bacterium Acidithiobacillus ferrooxidans to use these minerals as its sole energy source. A. ferrooxidans was grown in media containing siderite or vivianite under different conditions and compared to abiotic controls. Our experiments demonstrated that this microorganism was able to grow, obtaining its energy from the oxidation of Fe2+ that came from the solubilization of these minerals under low pH. Additionally, in sealed flasks without CO2, A. ferrooxidans was able to fix carbon directly from the carbonate ion released from siderite for biomass production, indicating that it could be able to colonize subsurface environments with little or no contact with an atmosphere. These previously unexplored abilities broaden our knowledge on the variety of minerals able to sustain life. In the context of astrobiology, this expands the list of geomicrobiological processes that should be taken into account when considering the habitability of environments beyond Earth, and opens for investigation the possible biological traces left on these substrates as biosignatures.

Microbial preference for chlorate over perchlorate under simulated shallow subsurface Mars-like conditions.

The Martian surface and shallow subsurface lacks stable liquid water, yet hygroscopic salts in the regolith may enable the transient formation of liquid brines. This study investigated the combined impact of water scarcity, UV exposure, and regolith depth on microbial survival under Mars-like environmental conditions. Both vegetative cells of Debaryomyces hansenii and Planococcus halocryophilus, alongside with spores of Aspergillus niger, were exposed to an experimental chamber simulating Martian environmental conditions (constant temperatures of about - 11 °C, low pressure of approximately 6 mbar, a CO2 atmosphere, and 2 h of daily UV irradiation). We evaluated colony-forming units (CFU) and water content at three different regolith depths before and after exposure periods of 3 and 7 days, respectively. Each organism was tested under three conditions: one without the addition of salts to the regolith, one containing sodium chlorate, and one with sodium perchlorate. Our results reveal that the residual water content after the exposure experiments increased with regolith depth, along with the organism survival rates in chlorate-containing and salt-free samples. The survival rates of the three organisms in perchlorate-containing regolith were consistently lower for all organisms and depths compared to chlorate, with the most significant difference being observed at a depth of 10-12 cm, which corresponds to the depth with the highest residual water content. The postulated reason for this is an increase in the salt concentration at this depth due to the freezing of water, showing that for these organisms, perchlorate brines are more toxic than chlorate brines under the experimental conditions. This underscores the significance of chlorate salts when considering the habitability of Martian environments.

Emergent ribozyme behaviors in oxychlorine brines indicate a unique niche for molecular evolution on Mars.

Mars is a particularly attractive candidate among known astronomical objects to potentially host life. Results from space exploration missions have provided insights into Martian geochemistry that indicate oxychlorine species, particularly perchlorate, are ubiquitous features of the Martian geochemical landscape. Perchlorate presents potential obstacles for known forms of life due to its toxicity. However, it can also provide potential benefits, such as producing brines by deliquescence, like those thought to exist on present-day Mars. Here we show perchlorate brines support folding and catalysis of functional RNAs, while inactivating representative protein enzymes. Additionally, we show perchlorate and other oxychlorine species enable ribozyme functions, including homeostasis-like regulatory behavior and ribozyme-catalyzed chlorination of organic molecules. We suggest nucleic acids are uniquely well-suited to hypersaline Martian environments. Furthermore, Martian near- or subsurface oxychlorine brines, and brines found in potential lifeforms, could provide a unique niche for biomolecular evolution.

Quantifying the Potential for Nitrate-Dependent Iron Oxidation on Early Mars: Implications for the Interpretation of Gale Crater Organics.

Geological evidence and atmospheric and climate models suggest habitable conditions occurred on early Mars, including in a lake in Gale crater. Instruments aboard the Curiosity rover measured organic compounds of unknown provenance in sedimentary mudstones at Gale crater. Additionally, Curiosity measured nitrates in Gale crater sediments, which suggests that nitrate-dependent Fe2+ oxidation (NDFO) may have been a viable metabolism for putative martian life. Here, we perform the first quantitative assessment of an NDFO community that could have existed in an ancient Gale crater lake and quantify the long-term preservation of biological necromass in lakebed mudstones. We find that an NDFO community would have the capacity to produce cell concentrations of up to 106 cells mL-1, which is comparable to microbes in Earth's oceans. However, only a concentration of <104 cells mL-1, due to organisms that inefficiently consume less than 10% of precipitating nitrate, would be consistent with the abundance of organics found at Gale. We also find that meteoritic sources of organics would likely be insufficient as a sole source for the Gale crater organics, which would require a separate source, such as abiotic hydrothermal or atmospheric production or possibly biological production from a slowly turning over chemotrophic community.

Effect of vermicompost on rhizobiome and the growth of wheat on Martian regolith simulant.

As humanity embarks on the journey to establish permanent colonies on Mars, ensuring a reliable source of sustenance will be crucial. Therefore, detailed studies regarding crop cultivation using Martian simulants are of great importance. This study aimed to grow wheat on substrates based on soil and Martian simulants, with the addition of vermicompost, to investigate the differences in wheat development. Basic physical and chemical properties of substrates were examined, including determination of macro- and microelements as well as their microbiological properties. Plant growth parameters were also determined. The addition of vermicompost positively affected wheat grown on soil, but the effect on plants grown on substrate with Martian simulants was negligible. Comparing the microbiological and chemical components, it was observed that plants can defend themselves against the negative effects of growth on the Martian simulants, but their success depends on having the PGPR (Plant growth-promoting rhizobacteria) present, which can provide the plant with additional nitrogen. The presence of beneficial symbiotic microbiota will allow the wheat to wait out the negative growth time rather than adapt to the regolith environment.

Hydrochemical and isotopic characteristics of water sources for biological activity across a massive evaporite basin on the Tibetan Plateau: Implications for aquatic environments on early Mars.

Covered by vast eolian landforms, gravel deposits, and playas, the worldwide typical evaporite deposit land, Qaidam Basin, in northwestern China is analogous to early Mars when the aridification process had lasted for millions of years since the end of a wetter climate. This study aims to investigate the chemical and isotopic characteristics of waters in an evaporite-rich environment, as well as the habitable conditions therein, that have undergone a transformation similar to early Mars. In May 2023, a total of 26 water samples were collected across the representative central axis of a longitudinal aridity gradient in the Qaidam Basin, including categories of meteoric water, freshwater, standing water accumulated after precipitation, salty lacustrine water, and hypersaline brines to inspect compounds made up of carbon, nitrogen, phosphorus, sulfur, halogen, and metallic elements. As evaporation intensified, the salt types transformed from HCO3-Ca·Na to Cl·SO4-Na or ClMg. The dominance of carbonate will gradually be replaced by sulfate and chloride, leaving much more dilute and less detectable contents. The presence of trace ClO4-, ClO3-, ClO2-, and BrO3- was confirmed in a few of the sampled Qaidam waters, indicating the preservation of oxyhalides in waters within an arid region and possibly the presence of relevant microbial enzymes. The isotopes of water, carbonaceous, and nitrogenous compounds provide valuable references for either abiogenic or biogenic signatures. With undetectable amount, phosphorus was found to be the limiting nutrient in evaporative aquatic environments but not necessarily antibiosignatures. Overall, these results suggest that the paleo-lacustrine environments on Mars are more likely to preserve biosignatures if they feature the dominance of carbonate minerals, bioavailable nitrate, phosphorus, and organic carbon, the presence of thermodynamically unstable oxyhalides, and isotope ratios that point to the involvement of biological activity.

Martian microbes research and lessons learnt for forensic science.

A new method for looking for life outside the Earth is used as an example to demonstrate how ways of presenting complex scientific concepts to the general public, used in planetary science, could be used in forensic science. The work led to a pared down, practical definition of detectable Life for planetary exploration, An organised system capable of processing energy sources to its advantage. For nearly three quarters of Earth's history the only lifeforms were microbes, which are the target for looking for extraterrestrial life. Microbes are microscopic and may be sparsely distributed, but their metabolic products can form large, durable rocks, much easier to find and which may contain the organisms or their remains. There are similar challenges in presenting astrobiological and forensic science. Both may have to deal with very large or very small numbers which are not immediately comprehensible but can be understood by analogy. To increase the impact on the listener or reader, dramatic analogues are valuable, for example, referring to the mineralised microbial metabolic products as, "fossilised breath of bacteria" demands the audience's attention and engages them before more detailed explanations are given. The power of practical experiments or demonstrations is most important to reinforce what might otherwise be a fairly abstract concept. Surprisingly, most of these approaches can be made to work equally well in both spoken and written forms as well as in both sciences.

Intercropping on Mars: A promising system to optimise fresh food production in future martian colonies.

Future colonists on Mars will need to produce fresh food locally to acquire key nutrients lost in food dehydration, the primary technique for sending food to space. In this study we aimed to test the viability and prospect of applying an intercropping system as a method for soil-based food production in Martian colonies. This novel approach to Martian agriculture adds valuable insight into how we can optimise resource use and enhance colony self-sustainability, since Martian colonies will operate under very limited space, energy, and Earth supplies. A likely early Martian agricultural setting was simulated using small pots, a controlled greenhouse environment, and species compliant with space mission requirements. Pea (Pisum sativum), carrot (Daucus carota) and tomato (Solanum lycopersicum) were grown in three soil types ("MMS-1" Mars regolith simulant, potting soil and sand), planted either mixed (intercropping) or separate (monocropping). Rhizobia bacteria (Rhizobium leguminosarum) were added as the pea symbiont for Nitrogen-fixing. Plant performance was measured as above-ground biomass (g), yield (g), harvest index (%), and Nitrogen/Phosphorus/Potassium content in yield (g/kg). The overall intercropping system performance was calculated as total relative yield (RYT). Intercropping had clear effects on plant performance in Mars regolith, being beneficial for tomato but mostly detrimental for pea and carrot, ultimately giving an overall yield disadvantage compared to monocropping (RYT = 0.93). This effect likely resulted from the observed absence of Rhizobia nodulation in Mars regolith, negating Nitrogen-fixation and preventing intercropped plants from leveraging their complementarity. Adverse regolith conditions-high pH, elevated compactness and nutrient deficiencies-presumably restricted Rhizobia survival/nodulation. In sand, where more favourable soil conditions promoted effective nodulation, intercropping significantly outperformed monocropping (RYT = 1.32). Given this, we suggest that with simple regolith improvements, enhancing conditions for nodulation, intercropping shows promise as a method for optimising food production in Martian colonies. Specific regolith ameliorations are proposed for future research.

Investigating Microbial Biosignatures in Aeolian Environments Using Micro-X-Ray: Simulation of PIXL Instrument Analyses at Jezero Crater Onboard the Perseverance Mars 2020 Rover.

Assessing the past habitability of Mars and searching for evidence of ancient life at Jezero crater via the Perseverance rover are the key objectives of NASA's Mars 2020 mission. Onboard the rover, PIXL (Planetary Instrument for X-ray Lithochemistry) is one of the best suited instruments to search for microbial biosignatures due to its ability to characterize chemical composition of fine scale textures in geological targets using a nondestructive technique. PIXL is also the first micro-X-ray fluorescence (XRF) spectrometer onboard a Mars rover. Here, we present guidelines for identifying and investigating a microbial biosignature in an aeolian environment using PIXL-analogous micro-XRF (µXRF) analyses. We collected samples from a modern wet aeolian environment at Padre Island, Texas, that contain buried microbial mats, and we analyzed them using µXRF techniques analogous to how PIXL is being operated on Mars. We show via µXRF technique and microscope images the geochemical and textural variations from the surface to ∼40 cm depth. Microbial mats are associated with heavy-mineral lags and show specific textural and geochemical characteristics that make them a distinct biosignature for this environment. Upon burial, they acquire a diffuse texture due to the expansion and contraction of gas-filled voids, and they present a geochemical signature rich in iron and titanium, which is due to the trapping of heavy minerals. We show that these intrinsic characteristics can be detected via µXRF analyses, and that they are distinct from buried abiotic facies such as cross-stratification and adhesion ripple laminations. We also designed and conducted an interactive survey using the Padre Island µXRF data to explore how different users chose to investigate a biosignature-bearing dataset via PIXL-like sampling strategies. We show that investigating biosignatures via PIXL-like analyses is heavily influenced by technical constraints (e.g., the XRF measurement characteristics) and by the variety of approaches chosen by different scientists. Lessons learned for accurately identifying and characterizing this biosignature in the context of rover-mission constraints include defining relative priorities among measurements, favoring a multidisciplinary approach to the decision-making process of XRF measurements selection, and considering abiotic results to support or discard a biosignature interpretation. Our results provide guidelines for PIXL analyses of potential biosignature on Mars.

Genomic and morphological characterization of Knufia obscura isolated from the Mars 2020 spacecraft assembly facility.

Members of the family Trichomeriaceae, belonging to the Chaetothyriales order and the Ascomycota phylum, are known for their capability to inhabit hostile environments characterized by extreme temperatures, oligotrophic conditions, drought, or presence of toxic compounds. The genus Knufia encompasses many polyextremophilic species. In this report, the genomic and morphological features of the strain FJI-L2-BK-P2 presented, which was isolated from the Mars 2020 mission spacecraft assembly facility located at the Jet Propulsion Laboratory in Pasadena, California. The identification is based on sequence alignment for marker genes, multi-locus sequence analysis, and whole genome sequence phylogeny. The morphological features were studied using a diverse range of microscopic techniques (bright field, phase contrast, differential interference contrast and scanning electron microscopy). The phylogenetic marker genes of the strain FJI-L2-BK-P2 exhibited highest similarities with type strain of Knufia obscura (CBS 148926T) that was isolated from the gas tank of a car in Italy. To validate the species identity, whole genomes of both strains (FJI-L2-BK-P2 and CBS 148926T) were sequenced, annotated, and strain FJI-L2-BK-P2 was confirmed as K. obscura. The morphological analysis and description of the genomic characteristics of K. obscura FJI-L2-BK-P2 may contribute to refining the taxonomy of Knufia species. Key morphological features are reported in this K. obscura strain, resembling microsclerotia and chlamydospore-like propagules. These features known to be characteristic features in black fungi which could potentially facilitate their adaptation to harsh environments.

Report of the 6th (Virtual) Meeting on the Planetary Protection Knowledge Gaps for Human Missions to Mars on June 1-2, 2022.

This paper reports the sixth in a series of meetings held under the auspices of COSPAR (with space agencies support) to identify, refine and prioritize the knowledge gaps that need to be addressed for planetary protection for crewed missions to Mars, as well as to describe where and how needed data can be obtained. This approach is consistent with current scientific understanding and COSPAR policy, that the presence of a biological hazard in Martian material cannot be ruled out, and appropriate mitigations need to be in place. The workshops in the series were intentionally organized to obtain a diverse set of inputs from subject matter experts across a range of expertise on conduct of a potential future crewed Mars exploration mission, identifying and leveraging precursor ground, cis-lunar crewed and Mars robotic activities that can be used to close knowledge gaps. The knowledge gaps addressed by this meeting series fall into three major themes: 1. Microbial and human health monitoring; 2. Technology and operations for biological contamination control, and; 3. Natural transport of biological contamination on Mars. This report describes the findings of the 2022 meeting, which focused on measures needed to protect the crew and the returning Mars samples during the mission, both on the Martian surface and during the return to Earth. Much of this approach to crewed exploration is well aligned with the Principles and Guidelines for Human Missions to Mars described in section 9.3 of the current (2021) COSPAR policy, in terms of goals and intent. There were three specific recommendations.

Breathing life into Mars: Terraforming and the pivotal role of algae in atmospheric genesis.

The Martian environment, characterized by extreme aridity, frigid temperatures, and a lack of atmospheric oxygen, presents a formidable challenge for potential terraforming endeavors. This review article synthesizes current research on utilizing algae as biocatalysts in the proposed terraforming of Mars, assessing their capacity to facilitate Martian atmospheric conditions through photosynthetic bioengineering. We analyze the physiological and genetic traits of extremophile algae that equip them for survival in extreme habitats on Earth, which serve as analogs for Martian surface conditions. The potential for these organisms to mediate atmospheric change on Mars is evaluated, specifically their role in biogenic oxygen production and carbon dioxide sequestration. We discuss strategies for enhancing algal strains' resilience and metabolic efficiency, including genetic modification and the development of bioreactors for controlled growth in extraterrestrial environments. The integration of algal systems with existing mechanical and chemical terraforming proposals is also examined, proposing a synergistic approach for establishing a nascent Martian biosphere. Ethical and ecological considerations concerning introducing terrestrial life to extra-planetary bodies are critically appraised. This appraisal includes an examination of potential ecological feedback loops and inherent risks associated with biological terraforming. Biological terraforming is the theoretical process of deliberately altering a planet's atmosphere, temperature, and ecosystem to render it suitable for Earth-like life. The feasibility of a phased introduction of life, starting with microbial taxa and progressing to multicellular organisms, fosters a supportive atmosphere on Mars. By extending the frontier of biotechnological innovation into space, this work contributes to the foundational understanding necessary for one of humanity's most audacious goals-the terraforming of another planet.

Tissue-specific dose equivalents of secondary mesons and leptons during galactic cosmic ray exposures for mars exploration.

During a human mission to Mars, astronauts would be continuously exposed to galactic cosmic rays (GCR) consisting of high energy protons and heavier ions coming from outside our solar system. Due to their high energy, GCR ions can penetrate spacecraft and space habitat structures, directly reaching human organs. Additionally, they generate secondary particles when interacting with shielding materials and human tissues. Baryon secondaries have been the focus of many previous studies, while meson and lepton secondaries have been considered to a much lesser extent. In this work, we focus on assessing the tissue-specific dose equivalents and the effective dose for males of secondary mesons and leptons for the interplanetary cruise phase and the surface phase on Mars. We also provide the energy distribution of the secondary pions in each human organ since they are dominant compared to other mesons and leptons. For this calculation, the PHITS3.27 Monte Carlo simulation toolkit is used to compute the energy spectra of particles in organs in a realistic human phantom. Based on the simulation data, the dose equivalent has been estimated with radiation quality factors in ICRP Publication 60 and in the latest NASA Space Cancer Risk model (NSCR-2022). The effective dose is then assessed with the tissue weighting factors in ICRP Publication 103 and in the NSCR model, separately. The results indicate that the contribution of secondary mesons and leptons to the total effective dose is 6.1 %, 9.1 %, and 11.3 % with the NSCR model in interplanetary space behind 5, 20, and 50 g/cm2 aluminum shielding, respectively, with similar values using the ICRP model. The outcomes of this work lead to an improved understanding of the potential health risks induced by secondary particles for exploration missions to Mars and other destinations.