Abstract
Antony van Leeuwenhoek, “the Father of Microbiology”, discovered in the year 1702 under his microscope unique microscopic creatures that he resurrected when adding water to dry leaf litter. One of these micro-organisms were bdelloid rotifers which are some of the smallest animals to be found, being less than 1mm in size and comprising approximately 1,000 cells. These smallcreatures astonished van Leeuwenhoek because they were still alive after rehydrating leaves kept dry for months. Bdelloid rotifers are remarkable animals for their ability to survive desiccation at any stage of their life cycle, but also to resist other abiotic stresses including freezing and high doses of ionizing radiation. During these stresses, bdelloid rotifers reversibly halt their metabolism and enter a suspended animation state called ”tun”. Upon experiencing favorable conditions, these microscopic animals exit the tun state and enter their active, hydrated lifestyle where they continue feeding and reproducing asexually.
Besides their extreme stress tolerance, another remarkable feature of bdelloid rotifers became apparent, even more unusual among animals, which is the absence of males, vestigial male structures, or hermaphrodites in any of the populations studied within the 460 described morphospecies. Bdelloid rotifers are females, apparently cloning themselves for millions of years. In 1986 the
evolutionary biologist John Maynard Smith referred to this group as an ≪ evolutionary scandal≫ because they defy all theories claiming that sexual reproduction is essential to persist and diversify. Bdelloid rotifers are therefore a unique model system in evolutionary biology to study their long-term persistence in the absence of canonical sexual reproduction. Moreover, they are one of the toughest animal clades on earth together with nematodes and tardigrades that can resist complete desiccation and high doses of ionizing radiation.
The KVD group sequenced the first bdelloid genome and recently published the first chromosomelevel genome assembly of A. vaga, showing a diploid genome composed of 6 pairs of homologous chromosomes with signatures of ancient tetraploidy (paleotetraploidy). Both in the assembled genomes and the transcriptomes of distinct bdelloid rotifer species, researchers highlighted
the high content of horizontally transferred genes (HTGs) from non-metazoan origin. Within this catalog of foreign genes identified, a substantial amount included antioxidants. Interestingly, while homologous chromosomes were identified in bdelloid genomes, recent research in the KVD group also detected recombination signatures in A. vaga (Simion et al) and modified meiosis with the first division being non-reductional. In addition, a drop in linkage disequilibrium (LD) was detected with increasing distances across A. vaga populations [19], also confirming ongoing recombination. Finally, reports of allele sharing between bdelloid individuals have been given by several research groups. [19, 20, 21, 22, 23]. These recent studies highlight a parthenogenetic mode of reproduction in A. vaga through a modified meiosis, including homologous recombination, and potential genetic exchanges. How these exchanges take place among bdelloid rotifers and whether they contribute to their evolution remains a mystery.
The research of also showed in A. vaga a highly invariant development scheme with approximately 1k cells, where somatic cells are post-mitotic (present in the G1 phase of the cell cycle) and primary oocytes (germline) arrested in the G2 phase until they resume oogenesis and go through non-reductional meiosis. Post-mitotic somatic cells have been observed to withstand increased genome fragmentation following exposure to high doses of ionizing radiation, as compared to germline cells. This difference is the consequence of a distinct spatio-temporal DNA repair dynamic in A. vaga with an onset of DNA repair commencing following oogenesis, while somatic cells undergo rapid repair likely through the non-homologous end-joining mechanism (NHEJ). This
study therefore highlighted a difference in radiation tolerance between somatic and germline cells. Given the exceptional tolerance of somatic cells to extreme genotoxic stresses, the current PhD dissertation aimed to understand the biochemical mechanisms involved with protein protection under extreme abiotic stresses, primarily focusing on the antioxidant response of A. vaga. Krisko et
al. have previously observed an increased resistance to IR-induced protein carbonylation among the bdelloid species A. vaga as compared to C. elegans. In a recent transcriptomic study by Moris et al., the authors found constitutive expression of canonical enzymatic antioxidants during desiccation and IR stresses. However, the contribution of non-enzymatic antioxidants has never been explored in bdelloid rotifers and rarely in understanding extreme stress tolerance across eukaryotes. Therefore, this dissertation aims to characterize antioxidants that are shared across various eukaryotic extremotolerant species with a particular emphasis on manganese-based antioxidants, similar to those reported within the extremotolerant prokaryote Deinococcus radiodurans.
To explore oxidative stress resistance in extremotolerant eukaryotes, with a main focus on the bdelloid species A. vaga, this thesis comprises 4 major chapters, briefly detailed below.
Chapter 2: Extreme stress tolerance across eukaryotes: Reduce, Rescue, Recycle, Repair, and Remove
This review introduces the concept of oxidative stress, with a specific focus on oxidative stress induced by radiation and desiccation. It also provides an overview of the responses observed in various extremotolerant species. These responses are categorized as follows: i) REDUCE: Primarily referring to a reduction in cell divisions, also known as eutely. ii) RESCUE: Involves rescuing active cellular macromolecules through the accumulation of bioprotectants and enzymatic antioxidants. iii) RECYCLE: Entails recycling protein-free low molecular weight antioxidants, with a focus on manganese-based antioxidants, to maintain redox equilibrium. iv) REPAIR: Involves repair pathways that actively participate in DNA and protein repair. v) REMOVE: Includes processes
necessary to remove harmful and damaged cellular macromolecules that cannot be repaired.
Chapter 3: Manganese-Based Antioxidants: Unraveling the Mechanisms of Radiation Resistance across Prokaryotic and Eukaryotic species
This chapter is the main publication of this dissertation, compiling most experimental results obtained during this PhD research. We report for the first time, a correlation between extreme radiation tolerance and and a high Mn/Fe ratio within eukaryotes, as has been demonstrated across prokaryotes. An in-gel superoxide scavenging assay demonstrated the same unique superoxide
scavenging profiles across the three radio-resistant organisms in the presence of Mn, which were not detected in radio-sensitive organisms. Upon Mn starvation, these profiles changed, the sensitivity to radiation increased and the levels of protein oxidation also increased. However, in both A. vaga and D. radiodurans, when Mn is absent and MnSOD is not functional, an alleviation in radiation-induced mortality seems to occur through an unknown mechanism, suggesting a manganese-independent rescue. Lastly, we identified two horizontally acquired MnSOD genes of non-metazoan origin in A. vaga. These genes may account for the unique Mn-dependent superoxide scavenging zones observed in A. vaga, similar to those seen in D. radiodurans.
Chapter 4: Protection of extremotolerant lysates against oxidative damages performed ex-vivo and in-vitro
This chapter includes preliminary results, not included in any publication. It covers experiments investigating the antioxidant potential of A. vaga and D. radiodurans lysates through in-vitro superoxide scavenging assays, enzymatic protection of irradiated restriction enzyme BamH1, and protection of oxidatively damaged human cells (both primary and immortalized).
Chapter 5: General Discussion
Includes the general discussion, perspectives and the conclusion of this entire PhD thesis.
| Date of Award | 4 Jul 2024 |
|---|---|
| Original language | English |
| Awarding Institution |
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| Sponsors | FNRS-FRIA |
| Supervisor | Karine Van Doninck (Supervisor), Emilien NICOLAS (Co-Supervisor), Xavier De Deken (President), Vinciane Debaille (Jury), Jean-Yves Matroule (Jury), Anjana Badrinarayanan (Jury) & Leandro Tabaras (Jury) |
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