Radiation Tolerance in Tardigrades
Samuel Emmanuel Soentoro
Program Studi Biologi, Universitas Pelita Harapan
Jl. M.H Thamrin Boulevard 1100, Lippo Karawaci Tangerang, Banten 15811
*Corresponding e-mail: 01113190014@student.uph.edu
ABSTRACT
Tardigrades or commonly known as water bears, are small aquatic animals.
Some tardigrade species tolerate almost complete dehydration and are able to
exhibit tolerance to various physical extremes in the dehydrated state. Their
extremophilic behavior has long been a topic of interest towards researchers who
seek to understand its evolution as well as its physiology. Fortunately, recent
advances in genomics and sequencing technology have given researchers new
insights towards these organisms. By obtaining whole-genome sequences,
researchers are now able to obtain information regarding the unique genes being
expressed in tardigrades which are responsible for its unique properties. Hopefully
in the near future, information regarding the molecular protection system of
tardigrades cells can be applied into the field of medicine. This paper aims to review
the current information on the unique genes in tardigrades – with specific focus
towards its radiation resistance as well as its potential applications in the field of
medicine.
Introduction
Tardigrades, also known as water bears, are microscopic eight-legged
aquatic animals – with their sizes ranging from 0.05mm - 1.2mm in body length.
Its physical features are comprised of a head segment as well as four body segments
that each contains two legs with claws (Møbjerg et al., 2011). Currently, it has been
reported that there are over 1,000 species of tardigrades from a plethora of various
habitats including marine, fresh water or even terrestrial environments (Guidetti &
Bertolani, 2005). Regardless of their habitats, tardigrades generally require water
around their bodies in order to stay active. In the case of terrestrial species, this is
provided by the fact that these species usually maintain a thin film of water on the
surface of their bodies (Møbjerg & Neves, 2021).
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Although there exists a large number of tardigrades species with a multitude
of morphological and physiological characteristics, these organisms are most wellknown for the having the unique evolutionary adaptations to survive extreme
environmental conditions. In fact, they are one the most resilient living creatures
that have been studied with individual species that are able to withstand the harshest
conditions that would usually be fatal for other forms of life including exposure to
extreme temperatures, extreme pressures, air deprivation, dehydration, and
starvation (Horikawa et al., 2008). As a result of this, they are classified as
extremophiles – which are usually single-celled organisms such as archaea and
bacteria.
Their extremophilic nature has been shown to be related to their ability to
tolerate almost complete dehydration. In general, tardigrades that inhabit
terrestrial environments such as lichens, mosses and soil are constantly under the
threat of drying. As a result, these species have also developed a unique trait that
functions to protect them against the effects of dehydration. When a tardigrade
encounters desiccation, they are able to lose water and enter a dehydrated state
known as anhydrobiosis (Wełnicz et al., 2011). When this occurs, the dehydrated
tardigrades are able to withstand a broad range of physical extremes that would
normally be fatal to most organisms such as being able to survive despite
exposure to high doses of radiation as well as even direct exposure to the vacuum
of open space (Jönsson et al., 2008). Although the tolerance of some tardigrades
has long been a topic of interest in the field of biology, the molecular mechanisms
for such unusual tolerance have not been extensively studied yet.
Radiation Tolerance
Proper maintenance of genomic DNA is important for preserving correct
genetic information and normal cellular functions (Wilson & Hunt, 2002). It is well
known that ionizing radiation can have adverse effects towards living organisms by
affecting DNA. Generally, there are 2 ways in which ionizing radiation can damage
DNA – known as direct and indirect effects (Figure 1). Through its direct effects,
ionizing radiation has the potential to damage the double helix structure of DNA
through inducing DNA single and double-strand breaks – resulting in cellular
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responses such as apoptosis, necrosis and abnormal mitosis. On the other hand,
through its indirect effects, ionizing radiation damages DNA through intermediates
such as reactive oxygen species (ROS) and free radicals generated in cells by
radiation (Desouky et al., 2015).
Figure 1 Direct and indirect DNA damage by ionizing radiation
However, as mentioned previously, there are some organisms which possess
special mechanisms to mitigate radiation-induced DNA damage. Tardigrades have
been shown to be able to tolerate radiation doses up to 1,000 times greater than
other animals are able to. To illustrate this, several species of tardigrades have been
recored to be able to withstand doses of 5,000 Gy (of x-rays) and 4,700 Gy (of
gamma rays) whereas 5 to 10 Gy would already prove to be fatal in human beings
(Horikawa et al., 2006). In tardigrades, the only explanation found in earlier studies
for this ability was that their lowered water state provides fewer reactants for
ionizing radiation – which is one of the reasons why this property is attributed to
the same mechanism that has evolved to cope with desiccation-induced stress
(Jönsson et al., 2008). However, more recent research has shown that even when
hydrated, tardigrades are somehow still able to remain resistant towards UV
radiation when compared to other animals (Horikawa et al., 2012). This finding
would mean that tardigrades are able to express proteins that are specifically able
to repair damage to their DNA resulting from radiation exposure.
Whole Genome Sequencing of R. varieornatus
In order to study radiation tolerance in tardigrades, The genome of
Ramazzotius varieornatus (R. varieornatus), one of the most stress-tolerant species
of tardigrades, was sequenced by Hashimoto et al. (2015) from the University of
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Tokyo. The main aim of their study was to look for expressed tardigrade-unique
genes and present evidence for the relevance of tardigrade-unique proteins to
tolerability. The genome sequence of R. varieornatus was determined by using a
combination of the Sanger and Illumina technologies. In addition to genome
sequencing, RNA was also extracted from 3 states to monitor gene expression
profiles during dehydration and rehydration as well as to obtain general
transcriptome data. Based on transcriptome data, it was discovered that in R.
varieornatus, there were only several minor changes in gene expression profiles
during dehydration and after rehydration. This would suggest that the proteins
involved in radiation tolerance are constitutively expressed and could therefore be
unrelated to the molecular system related to desiccation response.
All things considered, the R. varieornatus genome was estimated at around
55 Mb. Along with Hypsibius exemplaris (previously named Hypsibius dujardini)
from the Hypsibidae family, these also became the first tardigrades to have their
genomes fully sequenced. By obtaining a full genome sequence, this finding
allowed for the production of the comprehensive gene set for R. varieornatus which
can be seen in Figure 2.
Figure 2 Summary of the final gene model of R. varieornatus
When compared to other metazoans, the R. varieornatus genome contains
several unique characteristics that give it an edge in terms of its desiccationtolerance and subsequent resistance towards radiation such as:
1. Expansion of superoxide dismutases (SODs)
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The R. varieornatus genome contains sixteen different genes coding for the
enzyme superoxide dismutase (SOD) whereas less than ten SODs are found in
most animals. SOD is a detoxifying enzyme that plays a role in removing
superoxide radicals which are a type of reactive oxygen species (ROS) (França
et al., 2007). As desiccation induces oxidative stress, an expanded SOD gene
set could potentially contribute to better tolerance against desiccation
(Hashimoto et al., 2015).
2. Expansion of MRE11 genes
MRE11 is another gene family that plays important roles in the process of
repairing DNA double-strand breaks (DSBs) (Lamarche et al., 2010). Four
MRE11 genes are found in the R. varieornatus genome, whereas less than one
copy is found in most animals. Because DNA in tardigrade cells experience
DSBs during a dehydrated state, a greater number of MRE11 genes could
potentially be beneficial as it would allow for the efficient repairing of damaged
DNA (Hashimoto et al., 2015).
3. Loss of peroxisomal oxidative pathway
The R. varieornatus genome reveals that it lacks the peroxisomal boxidation pathway found in other animals. This could be advantageous to
tardigrades as it would most likely lead to decreased hydrogen peroxide
production during fatty acid metabolism - preserving the ability to respond to
oxidative stress when exposed to environmental stress (Hashimoto et al., 2015).
4. Loss of 8 genes from stress-responsive signalling pathways
The tardigrade genome is also missing in several key signalling pathways
that mediate stress stimuli to inactivate mTORC1. When this happens, it can in
turn can cause autophagy and subsequent degradation of damaged components
within the tardigrade cells. As a result of missing key pathways in this process,
the tardigrade could potentially avoid excessive degradation of cellular
components after severe stress by suppressing the induction of autophagy –
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allowing it to resume cellular activity by using partially damaged biomolecules
after rehydration. (Hashimoto et al., 2015).
5. Constitutive abundant expression of tardigrade-unique genes
It is revealed that tardigrades are able to constitutively express proteins such
as the tardigrade-unique heat-soluble proteins, CAHS and SAHS. Both of these
proteins maintain solubility even after heat treatment and are thought to play an
important role in the protection of biomolecules during desiccation (Yamaguchi
et al., 2012).
Dsup – a novel tardigrade-unique DNA-associating protein
Considering the fact that DNA is a major target of radiation damage,
Hashimoto et al. (2015) suggested the existence of tardigrade-unique proteins that
are able to associate with DNA in order to shield and/or effectively repair DNA
within the tardigrade cells. This was achieved through the isolation of the chromatin
fraction from the tardigrade using tandem mass spectrometry to identify the
different proteins found in the bands belonging to those from the chromatin fraction.
This revealed the presence of a tardigrade-specific protein, termed Dsup for damage suppressor. At the time, the Dsup protein showed no sequence similarity
to any proteins or motifs found in databases such as BLASTP and InterProScan.
Due to its novel nature, it was also necessary to study the function behind this
tardigrade-unique DNA-associating protein. When the Dsup gene is expressed in a
human cell line, there was a decrease in DNA fragmentation induced by X-ray
radiation and even treatment with hydrogen peroxide. In addition to this, the Dsupexpressing human cells showed higher viability than cells in the control group
(Hashimoto et al., 2015) (Figure 3). These studies suggest that Dsup is a unique
protein that can protect DNA either through a direct or indirect mechanism.
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Figure 3 Schematic model of DNA protection by Dsup
Because ionizing radiation and hydrogen peroxide both generate hydroxyl
radicals as a major reactive oxygen species in cells (Riley, 1994), Chavez et al.
(2019) investigated whether purified Dsup affects hydroxyl radical-mediated DNA
cleavage. This study revealed an important piece of information regarding the
molecular mechanism behind the Dsup protein and its role in the tardigrade genome
which is that the Dsup protein from R. varieornatus not only protects DNA from
damage by binding to chromatin but also that it is able to protect the tardigrade
genome from hydroxyl radical-mediated damage in general (Figure 4). Due to this
fact, this protective mechanism could be the reason why tardigrades are able to
survive even after extended periods of being in its anhydrobiotic state.
Figure 4 Model for Dsup binding to nucleosomes and protecting chromatin from hydroxyl radicals.
In addition to this, this study also revealed that the Dsup protein is able to
protect chromosomal DNA from hydroxyl radical-mediated damage in a wide
variety of environmental conditions conditions. This observation is consistent with
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the previous studies that have proven that tardigrades are able to survive high doses
of ionizing radiation in both the active hydrated state as well as anhydrobiotic state.
Relevance of Tardigrade Stress Mechanisms for Research
Due to the fact that the protection and repair of DNA is an important
component of all cells as well as the main aspect of many human diseases, research
looking into radiation tolerance in tardigrades show great potential especially in
terns if applying the molecular protection system of tardigrades cells into the field
of medicine.
One of the areas where studies on the tolerance of tardigrades have a huge
potential to contribute to medical research is in diseases related to oxidative stress.
Oxidative stress is a central aspect in the development of many diseases, including
cancer, aging, diabetes, inflammation, and Parkinson’s disease (Liguori, 2018). One
reason this is relevant is due to the fact that the oxidative stress with respect to the
production of ROS is an important consequence of both desiccation and ionizing
radiation. As mentioned previously, tardigrades are tolerant towards desiccation
and ionizing radiation and therefore studying the proteins involved in its antioxidant
system as well as experimental studies where tardigrade genes are tested in human
cells can be beneficial.
In addition to its potential in studies directly involving diseases related to
oxidative stress, studies more directed towards the response of the tardigrade
protective system as well as those related to radiotherapy and the development of
cancer would also be an important step towards making this research more relevant
to cancer research. For example, experiments related to the exposures of tardigrades
to radiation may be used to study, for example, the effects of dose rate,
fractionation, hypoxia, and bystander effects, as well as cell biology responses to
radiation such as cell cycle arrest, apoptosis, senescence, and autophagy (Jönsson,
2019). In addition to this, there is also the further possibility of applying the
protective mechanisms which are responsible for the tardigrades’ stress tolerance
capabilities and expressing them in human cells to further improve the stress
resistance of human cells.
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Hopefully in the near future, this could be applied in the field of medicine
and be beneficial to people undergoing radiation therapies for specific diseases. At
the time of writing this, these are areas where any research or study has yet been
reported in tardigrades, but which are undoubtedly important in current cancer
research.
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