Abstract EANA2026-61

Lava tubes and volcanic caves are among the most compelling terrestrial analogues for astrobiology because they combine energy limitation, mineral reactivity, and long-term physical protection from surface stressors. Within these environments, microscale reticulated filaments, which are mineralized filaments consisting of open square-shaped or diamond-shaped meshed reticles, are among the most enigmatic microbial features often found in different cave environments worldwide [1]. Their intricate morphology, association with extracellular polymeric substances (EPS), and the presence of microbial cells both within and surrounding the filaments have led researchers to infer a biogenic origin [2]. Despite their broad distribution and strong association with biomineralization, their precise microbial origins, formation mechanisms, and functional roles remain a mystery. Although previous studies have elucidated the microbial community composition of reticulated filaments [3,4], specific microbes/microbial communities responsible for the biomineralization of filaments have not been found thus far. We aim to analyze individual reticulated filaments from bulk speleothem samples obtained from lava tubes of the Canary Islands (Spain). Field emission scanning electron microscopy combined with energy dispersive spectroscopy (FESEM-EDS) was used to conduct a preliminary morphological and mineralogical analysis of the bulk samples. In addition, ultrathin sections of the filaments were analyzed to determine the presence of microbial cells using transmission electron microscopy (TEM). Individual filaments will be isolated from complex mineral deposits using focused ion beam scanning electron microscopy (FIB-SEM) with micromanipulation, avoiding the limitations of bulk-sample analyses. FIB-SEM will also be used to access DNA inside the filaments (by ionizing the mineralized sheaths) for extraction and subsequent metagenomic sequencing using NGS platforms (Illumina MiSeq and Oxford Nanopore) to determine the communities responsible for filament formation. Confocal laser scanning microscopy (CLSM) will be used to localize nucleic acids and EPS. Single-cell sequencing will also be attempted if the DNA quantity is extremely low. Isolation of microbial strains from the organic matter inside the filaments will be attempted using suitable culture media. Detailed mineralogical and stable isotope analyses (δ13C, δ15N) will be performed using secondary-ion mass spectrometry (SIMS/Nano-SIMS) to determine the biogenicity and microbial involvement in filament formation. Finally, metagenomics and chemical composition studies of the filament substrate (i.e., bulk sample without the filaments) will also be performed to corroborate the findings. We hope this research workflow will finally solve the enigma of reticulated filaments and establish them as strong morphological biosignatures to search for life in Martian subsurface environments like lava tubes, where mineralized microbial fabrics may outlast cells and preserve evidence of past or present biological activity. This work is timely in the context of upcoming Mars subsurface exploration, including the ExoMars Rosalind Franklin mission, and may contribute to defining microtextural, mineralogical and molecular criteria for recognizing biosignatures in volcanic subsurface environments.

Keywords: Subsurface environments, reticulated filaments, lava tubes, biosignatures, FIB-SEM, Martian subsurface

Acknowledgements: This work was supported by the HERMES project (ref. PID2024-162087NB-C21) funded by the Spanish Ministry of Science, Innovation and Universities (MCIN/AEI/10.13039/501100011033) and by the ISSalus project (ref. 4000148673) funded by the European Space Agency and the Spanish Space Agency.

References: [1] Miller, A.Z. and Saiz-Jimenez, C. (2025), Intricate microbial structures: The enigma of reticulated filaments. Sedimentology, 72: 1025-1039. [2] Miller A.Z. et al. (2012a), Reticulated filaments in subsurface granite. Environmental Microbiology Reports, 4: 596-603. [3] Miller, A. Z. et al. (2012b). Biogenic Mn oxide minerals coating in a subsurface granite environment. Chemical Geology, 322-323, 181-191. [4] Melim, L.A., et al. (2008) Reticulated filaments in cave pool speleothems: microbe or mineral? J. Cave Karst Stud., 70, 135–141