Understanding Tardigrades to Discover Life Beyond Earth One of the prominent goals of astrobiology is to discover life or signs of life on planets beyond Earth. While most organisms require oxygen and water to survive, some organisms on Earth can survive without either of those. Tardigrades, for example, can remain unharmed in extreme environments, but only when they are in a dehydrated state. Tardigrades have become one of the most well-known test subjects for astrobiology research because of their ability to cope with a wide range of environments.
To better understand why tardigrades are suitable for space esearch, this paper will introduce the characteristics of the tardigrade, the range of environments it can withstand (explained by the numerous coping mechanisms while mainly focusing on anhydrobiosis) and how several astrobiologists consider tardigrades as the key to finding life outside of our planet. Characteristics and the Extreme Tolerance of the Tardigrade Tardigrades, or water bears, can range from 0. 1 to 1. 0 mm in size (Fig. 1).
These aquatic invertebrates can thrive in terrestrial habitats such as moss cushions, ocean depths, mountain peaks and even in the Antarctic (Ramazzotti and Maucci, 1983; Nelson, 2002). Based on the various habitats, tardigrades are considered to be one of the most tolerant extremophiles because they are tolerant to a wide variety of extreme environments rather than one specific environment (Horikawa et al. , 2008). For example, thermophiles thrive in high temperatures but could die in temperatures below 1040F (Madigan and Martino, 2006) while adult tardigrades can withstand temperatures of -460 to 3040F (Horikawa et al. 2008). They also have a radiation tolerance of 3000 to 6200 Gray (adsorbed dose of radiation, Gy), about a 1000 times higher than the tolerance of humans (Anno et. l. , 2003), and can withstand pressures as great as 6000 atm (Seki and Toyoshima, 1998). FIG. 1 Comparison of size between average tardigrade and cubic grain of table salt (Armstrong, 2011). These remarkable protections against damages to cellular components further strengthen the potential for tardigrades to become space research models. In 2007, tardigrades became the first multi-cellular organism to survive the lethal exposure of outer space.
The Tardigrade Resistance to Space Effects (TARSE) project, conducted by the European Space Agency, sent tardigrades on mission FOTON-M3 to orbit 260 kilometers above Earth. Dehydrated tardigrades were exposed to direct solar radiation, heat and the vacuum of space for 12 days (Miller, 2011; Rebecchi et al. , 2009). The survival rates of four groups of adult tardigrades in four different states were recorded (Fig. 2) (Rebecchi et al. , 2009). The highest survival rate was obtained by the desiccated tardigrades collected from leaf litter (Fl) with 94. % survival rate, dried on paper (F2) had the second best survival rate of 78. 9%, starved and active tardigrades (F4) with 60. 6% survival rate, and fed and active tardigrades (F3) did the worst with 6. 8% survival rate (Rebecchi et al. 2009). The collected data suggests that there must be a supporting mechanism behind the high survival rate of the least active group of tardigrades. FIG. 2 Survival of tardigrade specimens involved in the flight experiments: Fl (dry within leaf litter), F2 (dry on paper), F3 (fed and active), and F4 (starved and active, Rebecchi et al. , 2009). It was noted that “during the flight mission… emales laid eggs” with several hatched eggs “exhibit[ing] normal morphology and behavior” (Rebecchi et al. , 2009). After examining this data set, one obvious question stands: what methods do tardigrades tilize to withstand such hostile environments? Regardless of their developmental stage, most tardigrade species can survive extremities by entering a phase known as cryptobiosis (Clegg, 2001). The cryptobiotic stage can be induced by low temperatures (cryobiosis), osmotic pressure (osmobiosis), the lack of oxygen (anoxybiosis) or, most commonly, by the lack of water (anhydrobiosis.
Keilin, 1959). Coping Mechanisms and Experimental Data Cryptobiosis As mentioned previously, tardigrades have the ability to undergo cryptobiosis, a state in which an organism show ‘no visible signs of life… when its metabolic activity ecomes hardly measurable, or comes reversibly to a standstill’ (Keilin, 1959). An organism undergoing cryptobiosis enters a death like stage in order to endure external stresses. Tardigrades then become immobile and shrink up into the tun’ state (Fig. ) for the purpose of reducing areas of their bodies with high permeability in response to low humidity levels (Crowe, 1972; Seki and Toyoshima, 1998). Cryptobiosis, as a result, will reduce the organism’s metabolic rate to endure environmental challenges. Tardigrades in their cryptobiotic stage can be stored for up to nine years at normal atmospheric pressure and room temperature O?¶nsson, 005). Furthermore, cryptobiosis is a reversible ametabolic (the suspension of metabolism) state. FIG. 3 Tardigrade in a tun’ compared to a normal tardigrade (Science Photo Library).
Osmobiosis The least common type of cryptobiosis preformed is osmotiosis, the metabolic inactivity in response to extreme salinity (Miller, 2011). Upon immersion in saline solutions, tardigrades rapidly contract into the tun stage due to osmosis, suggesting that the primary adaptation is dehydration (Kinchin, 1994). In one study done by Wright et al. in 1992, tardigrades were immersed in solutions with different concentrations of NaCl(aq). Tardigrades show a survival rate of over 11 hours in concentrations with 2% NaCl(aq) and falling to 30 minutes in 10% NaCl(aq).
When osmobiotic tardigrades are returned to distilled water, the revival time from the tun state to their active state increased exponentially as the concentration of NaCl(aq) increased (Wright, 1987). Thus Mability decreases with prolonged immersion”. Anoxybiosis The metabolic suspension in response to oxygen deficient environments is called anoxybiosis (Rebecchi et al. , 2006). Tardigrades are very sensitive to changes in oxygen levels and can undergo osmoregulatory failure due to the lack of oxygen. As a result, the organism cannot maintain the homeostasis of its water content.
Unlike any other forms of cryptobiosis, water uptake rather than desiccation occurs, causing the animal to become plump (Kinchin, 1994). As of now, scientists still have a limited understanding of why this occurs. Anhydrobiosis The most well understood and most common type of cryptobiosis performed by tardigrades is anhydrobiosis. Anhydrobiosis is metabolic suspension caused by virtually complete desiccation (Miller, 2011). Every type of cryptobiosis is a slight playoff of anhydrobiosis, with anoxybiosis being the exception.
By depriving their cells of water, the tardigrade enters the tun state to reduce metabolic activity. With only 2% water content, the tun state also serves to lower the rate of evaporation by reducing the surface area of their bodies (Crowe, 1971). With absolutely no movement and close to zero biological activities, the tardigrade is close to dead and can remain in this state for over a decade. Yet upon rehydration, tardigrades can resume their activity within 1 5 minutes to two hours (Guidetti and J?¶nsson, 2002).
However, to survive extreme desiccation, tardigrades must be allowed to dry gradually. Crowe (1971) examined the rate of evaporative water loss under controlled relative humidities (RH). Specimens of tardigrades were starved for 24 hours to reduce metabolic activity, washed with distilled water, picked up individually and were blotted with filter paper to remove the remaining water before the experiment. The animals were then held in a chamber at 250C and the percent water content of tardigrades were recorded at each RH level.
The percentage water content of tardigrades after each experiment can be calculated according to the following equation: where Wt = weight at time t and Wd = dry weight. The average weight of fully hydrated tardigrades is around 98 pg, with 12 pg in dry matter and 86 pg in water. All calculations were carried out on groups of 25+3 animals (Crowe, 1971). Over the range of 70-95% relative humidity, the active animals with lower metabolic activities reduced from 80-90% to 50-60% water content within an hour (Fig. 4).
As RH decreases, the rate of evaporative water loss becomes greater. For instance, at 0% RH, the sample tardigrades reduced their water content to 2-3% in about 1 5 minutes. Fully active tardigrades were killed under direct exposure to RH. However, ardigrades that reduced their water content to 20% previously at 80% RH did survive exposure to dry air, reducing their water content to 2-3% without killing them (Crowe, 1971). FIG. 4 Percent water content over time (hr) kept at various relative humidities (Crowe, 1972).
The greatest survival rate was from tardigrades dried at relative humidities greater than 70%, suggesting a slow rate of water loss is necessary for survival from desiccation. Due to a higher RH, tardigrades are able to retain the water in their bodies for an extended period of time. This gradual change is easier for their cells to andle (Crowe, 1972). Developmental stages and its effects on desiccation tolerance were then examined. Three developmental stages of the tardigrade samples were observed: embryo, Juveniles, and adults (Fig 4).
After 10 days of desiccation, survival rates were found to be greater than 90% for all three developmental stages of the tardigrade (Hortkawa, 2008). Fig. 4 Percent recovery following 10 d anhydrobiosis in eggs, Juveniles, and adults tardigrades. No significant differences were shown in recovery rates among the three developmental stages. Error bars indicate standard deviations of 3 or 4 experiments From the data above, tardigrades seem to be inherently prepared for anhydrobiosis at birth. It is apparent that tardigrades have no trouble entering the anahydrobiotic stage.
Survival after desiccation is also very favorable if dehydration is gradual. The Effect of Anhydrobiosis on Extreme Tolerances Based on the results of the TARSE and anhydrobiotic experiments, desiccated tardigrades were in a state of anhydrobiosis for both cases (Rebecchi et al. , 2009). It is clear that being in the state of anhydrobiosis enabled the specimen to endure desiccation, radiation, heat and the vacuum of space but to what extent does nhydrobiosis aid tardigrades through other extremes?
When examining the tolerance of extreme environments, both active and anhydrobiotic tardigrades were observed under four environmental factors; high and low temperatures, high irradiation, and chemical surroundings (Fig. 5) (Horikawa, 2008). FIG. 5 Survival rate of hydrated and anhydrobiotic tardigrades after exposure to extreme conditions. (A) 900C for 1 h, (B) -1960C for 15 min, (C) 4000 Gy of irradiation with 4He, and (D) 99. 8% acetonitrile for 1 h. Asterisks denote significant differences in survival between the two groups. Error bars indicate standard deviation for 3 experiments (Horikawa, 2008).
When exposed to gamma rays and heavy ions, the median lethal dose were 5000 Gray (gamma-rays) and 6200 Gray (heavy ions) in hydrated animals, and 4400 Gray (gamma-rays) and 5200 Gray (heavy ions) in anhydrobiotic ones (Horikawa, 2008). Short-term survival rates were recorded after 2, 24, and 48 hours post-irradiation (Fig. 6). FIG. 6 LD50 + standard error at 2, 24, and 48 h after irradiation of gamma-rays and 4He2+ in hydrated and anhydrobiotic tardigrades (Horikawa, 2008). Overall, there was no significant diffe ence in the survival rates under both radiation exposures between the two groups.
When seen without deviations, hydrated tardigrades have a higher survival rate than anhydrobiotic tardigrades when dealing with gamma-rays and 4He irradiation, suggesting that tardigrades can survive in environments without atmospheres and outer space (Horikawa, 2008). CAHS and SAHS proteins When comparing tardigrades to other extremophiles with anhydrobiotic abilities such as the nematodes and rotifers, the accumulation of tregalose, a sugar that has expressed an important role in aiding desiccation, was much less (