Analyzing the active time and dissolved oxygen levels of Melnesium tardigradum in its anoxybiotic state in both acidic and basic conditions.
By: Christopher A. Acevedo
Biology 402 Senior Seminar
Research project and thesis
Due: May 7, 2008
Table of contents
SectionsPage number
Abstract2
Introduction3-13
Hypothesis 13
Material and Methods13-17
Active Test14-16
Dissolved Oxygen Test16-17
Result17-22
Discussion22-28
Acknowledgments29
Literature Cited30
Abstract
Tardigrades, or water bears, are invertebrates that survive extreme environmental conditions by entering a state of suspended animation. The study examined the tardigrade, Milnesium tardigradum’s ability, to withstand extreme changes in pH. The study examined how the change in pH affected the activity of M. tarigradum and the dissolved oxygen in the water. Each sample was placed in 2 mL of water and 3 mL of pH buffer. The dissolved oxygen doubled the samples size. Tardigrade movement was used to indicate health.M. tardigradum survived pHs of 1.54 and 12.5 for 1 minute, and increased in time the closer the pH was to 7. The dissolved oxygen showed similar increases the closer the pH was to 7. Overall M. tardigradum are more active at higher pHs, whiletheactive time and dissolved oxygen showed similar changes with the change in pH. Because many tardigrades need specific environmental and physical conditions to remain active, the understanding of the change in activity to the change in environment may allow humans to use tardigrades as an indicator species for any changes in the surrounding environment.
Introduction
The phylum Tardigrada includes more than 700 species of microscopic metazoans (Garey et al., 1996). Known commonly as water bears, the name Tardigrade comes from Spallanzini in the 18th century, with “tardi” meaning slow and “grade” meaning walker (Romano, 2003). Tardigrades range from 0.1 to 1.5 millimeters in length. Their body consists of five segments: a cephalic or head segment, and four trunks or body segments. The tardigrade has four pairs of legs that have either claws or suction discs. The suction discs are found on marine and some fresh water tardigrades, while the claws are associated with all terrestrial tardigrades (Garey et al., 1996). The first three pairs of legs are directed ventrally, while the fourth pair is directed posteriorly (Romano, 2003). Tardigrades inhabit marine and freshwater sediments and terrestrial habitats with a water film. Such terrestrial habitats include soil, mosses, lichen, and liverworts (Garey et al., 1996). Tardigrades feed on the cells of bacteria, algae, mosses, liverworts, lichen, protozoans, rotifers, nematodes, larvae, and other micro-invertebrates using their hardened stylets. These are the parts of the mouth used to grab and break down food (Romano, 2003).
The phylum Tardigrada is currently composed of only two classes, Heterotardigrada and Eutardigrada. These classes were established by Marcus in 1929. “Eutardigrada,” means true tardigrades, and “Heterotardigrada” means the other tardigrades (Romano, 2003). Heterotardigrada consists of marine and armored terrestrial species that have a thick cuticle. Eutardigrada consists of mainly unarmored freshwater and terrestrial species (Garey et al., 1996). A third class was reported in a hot spring in Nagasaki, Japan, the Mesotardigrada, (meso- meaning middle). However, the only sample was lost during an earthquake in 1937, so Mesotardigrada was removed as a class of Tardigrada by the Eighth International Symposium on tardigrades in 2000 (Romano, 2003). Even though the sample was lost, the discovery showed just how little scientists knew about tardigrades. With more frequent discoveries, scientists will be better able to define what tardigrades are.
Defined in Kinchin (1994), one of the most common species, and earliest species to be named was Milnesium tardigradum. M. tardigradum are a terrestrial Eutardigrade, and they are one of the largest species of tardigrades with some growing to lengths of 1 mm in length. However, most of the species are around 0.5 mm in length. Another defining characteristic in the species M. tardigradum was that they are one of the few actual carnivorous species in the phylum, feeding on rotifers and small tardigrades. For physical characteristics, they have a lateral and oral papillae, a pear-shaped pharyngeal bulb, and a mouth region surrounded by six triangular lamellae. Another external characteristic was that the species had a smooth exterior compared to other species with hook like extensions on their bodies. However, researchers have found some subspecies of M. tardigradum that do not have a smooth exterior. The body of M. tardigradum is wider around the region with its three pairs of appendages that point anteriorly. The coloration for the species is translucent pink to a brownish color. The eggs are smooth and spherical to oval in shape, with a brown coloration. Usually there are six to nine eggs per caste, and the eggs range in sizes from 70 µm to 110 µm. The species are found in moist terrestrial habitats, such as soil and slightly dry moss and lichen.
Meiofauna are organism that are classified as too small to be collected by 500 µm sieves, but large enough to be collected in 62 µm sieves (Palmer, 1990). This is in between the classification macrofauna, which can be seen by the naked eye, and microfauna which requires a microscope to observe. The phyla Tardigrada are classified as meiofauna, because their size range is within the classified range for meiofauna. One of the characteristics of meiofauna that may limit them to certain habitats is the absence of any oxygen regulatory system. The organisms are too small to regulate or store oxygen, so meiofauna take in oxygen by diffusion across the outer tissue of their body wall. Because they need water for the diffusion of the dissolved oxygen, meiofauna are found in aquatic or moist environments. These environments are fresh water, seawater, and moist terrestrial area. The terrestrial areas are places that have water within them, such as soil, moss, lichen, and other plants (Kinchin, 1994). Because meiofauna need specific conditions to survive, such as the correct amount of light, heat, water, pH, and nutrients depending on the phyla to the specific species of the organisms, meiofauna can be found in different areas of a specific habitat. For example, different species of tardigrades could be found on different levels of one piece of moss, depending on their specific needs. These different levels are the second characteristic of meiofauna, which is the formation of zones or layers (called zonation) in the habitat. These zones form recognizable bands in the specific habitat, in which the species can survive. These zones might delineate a range of water depth or a range of height (NOAA’s Coral Reef Information System, 2005). For terrestrial habitats like moss, the zones are formed by the amount of water and chemicals that are exposed to the moss. For example, higher up in the moss is where more water can be obtained year round, however it is also were it is exposed to the sun making it hotter in temperature than the base of the moss. Organisms found toward the top need to be adapted to withstand constant exposure to the sun. A study by F. Mihelĉiĉ (1954/55 as cited in Kinchin, 1994) classified different moss habitats by their exposure to water. With the study he created five classes of moss. The first group was mosses that were permanently exposed or soaked in water. The second group, are mosses that were frequently soaked in water or floated in water. Like group one, group two mosses never experienced periods of dry out and were usually located near waterways or waterfalls. Group three was mosses that were found in shady or humid areas away from direct sunlight. Group four was mosses that were exposed to direct sunlight and experienced frequent dry outs. Group five was mosses that were exposed to direct sunlight for prolonged periods, in which the moss could be without moisture for long periods of time. These zonations can be formed by the need for moisture or the prevention of drying out. Another type of zonation is pH. Some species of tardigrades have been found to thrive in alkaline conditions, in which case these species can be found in urban areas with population. These preferences can determine where and how easy a species can live in an area. The species M. tardigradum has no preference in the difference pH surfaces (Kinchin, 1994).
Terrestrial tardigrades are some of the few invertebrates that have the ability to slow their metabolic rates to improve their survival under harsh environmental conditions. This ability, known as cryptobiosis, is also found in nematodes, rotifers, and some protozoans. Cryptobiosis was discovered in 1702, when Dutch microscopist Anton van Leeuwenhoek described the revival of rotifers from re-wetted gutter sediment. However, this observation did not prompt scientific inquiry until 1743 when Needham observed that blighted wheat grains “took to life” upon wetting. The animals being observed were nematodes (Wright et al., 1992).
The term “cryptobiosis” describes a stasis-like state in which the animal slows down its metabolic state to survive dangerous conditions in its environment. Stasis is when the organism ceases normal blood flow, body fluid, and metabolic rates for a period of time (McGraw-Hill, 2007). The major characteristic of cryptobiosis is the loss of more than 95% of the organism’s body water and the compression to a fraction of original body size. However, cryptobiosis is a general term that encompasses numerous stases, each with its specific environmental condition. The types of stases are anhydrobiosis, cryobiosis, osmobiosis, and anoxybiosis. Anhydrobiosis is induced by slow dehydration and is a stasis to protect the organism from low water levels. The major characteristic of anhydrobiosis is the production of the sugar trehalose to protect major cells from water loss, reducing the change of cell damage. Cryobiosis occurs in freezing conditions. Chemicals known as osmolytes, which include glycerol, inositol, and trehalose, are produced to prevent ice crystals from forming in the organism’s remaining water, causing damage. Osmobiosis is for osmotic extremes. This means the salinity of the environment has changed in which the outer water of the environment, and the water in the tardigrade may interact in a hypertonic or hypotonic way. Anoxybiosis is for conditions with low oxygen levels. Cryptobiosis is not an individual stasis, but a combination of each of stases. When an organism is said to be in a cryptobiotic state, it means that the organism is using one or more of stases for protection (Wright et al., 1992). Anoxybiosis is unlike the other types of cryptobiosis. The other types of cryptobiosis are characterized by the loss of water and for the organism to be inert until the condition improves. Anoxybiosis is an active state in which the organism continually takes in water to diffuse as much dissolved oxygen in the water. This causes the organism to increase in size and mass, which is different than the usual shrinking and shriveled body of the other states of cryptobiosis. Because of this some researchers do not consider anoxybiosis a form of cryptobiosis (Kinchin, 1994).
There have been many studies done on different organisms’ capacity to induce cryptobiosis; the two major experimental organisms are tardigrades and nematodes. Studies have found how long tardigrades can stay dehydrated, how much radiation they can endure, and the range of temperatures in which they can survive. However, their ability to survive in extreme pH has not been studied in detail. Most information that pertains to pH is either pH preference, which was pointed out in zonation, and the mentioning of the pH of the soil that some researchers discussed in their study. Most cryptobiotic studies that dealt with tardigrades have been focused on anhydrobiosis or cryobiosis. The studies of pH and cryptobiosis were on flagellated protozoans rather than tardigrades. So, the impact of pH on tardigrades is believed to be unknown. What we do know from the research on tardigrades and other organisms with similar cryptobiotic functions are the capacity and parameters of cryptobiosis, which have been found via experimental testing.
One of the parameters of tardigrade’s cryptobiotic state was tested by Crowe and Higgins (1967). The experiment was on the species Macrobiotus areolatus Murray, and examined the external factors that had an effect on the tardigrade’s ability to revive from cryptobiosis. The external factors consisted of different types of chemicals and temperatures to see if the changes would slow the revival time of M. areolatus M. External chemical changes had little effect on the rate of cryptobiosis. What was found to affect the rate of revival from cryptobiosis was the duration of the stasis, the age of the tardigrade, the number of times the tardigrade had entered cryptobiosis, and the duration of cryptobiosis. Crowe and Higgins (1967) found that cryptobiosis and revival from cryptobiosis costs high amounts of energy, so the longer a tardigrade is in cryptobiosis, the longer the tardigrade takes to come out of its stasis. Age of the tardigrade was shown to be a factor in revival; older tardigrades had a slower revival rate from cryptobiosis, while younger tardigrades could quickly come out of the stasis. Age was determined by measuring the stylet, which gets larger as the tardigrade grows older. The number of times a tardigrade has entered into cryptobiosis also affected revival time. The longer a tardigrade was in cryptobiosis, the more damaging it was to tissues and more energy storage was used. These two factors are important for the tardigrades’ survival. First, tardigrades cannot repair damaged tissue. Tardigrades are eutely, meaning that each entire body has a fixed number of cells that do not divide to replace lost or damaged ones (Moment, 2007). The study showed that tardigrades used their trehalose to maintain their cryptobiosis. Since the trehalose is being used by the body, the longer the tardigrade is in cryptobiosis, the less trehalose it has. If it runs out, then it can no longer maintain cryptobiosis and is now affected by the condition that it went into stasis for. If the tardigrade uses too much trehalose, it can starve even if the conditions are optimal to come out of cryptobiosis. Less tissue damage and the energy to regain the metabolic energy needed to replace the trehaloseis why younger tardigrades can survive better than older ones. These circumstances need to be considered when experimenting on tardigrades.
Another experimental study that examined how the environment affects a tardigrade’s ability to enter and survive in cryptobiosis was conducted by Rebecchi et al. (2006). Their study questioned the common belief that tardigrades can survive for one hundred years in anhydrobiosis. The concept came from Franeshi’s (1948, as cited Rebecchi et al., 2006) observation of a single tardigrade that was revived from an Egyptian exhibit, which had been sealed. This tardigrade had not been hydrated for hundreds of years. There had been evidence that rotifers and nematodes could survive at least 39 years while dehydrated. However, there had been no study that gave any evidence that a tardigrade could survive for that long. Rebecchi et al. (2006) studied 10,370 tardigrades: 1,586 belonging to Heterotardigrada and 8,728 belonging to Eutardigrada. The study dehydrated the tardigrades and periodically checked on them sixty times within the 1,604 day experiment. The researcher would rehydrate the tardigrades to observe and compare the revival from cryptobiosis. There were twenty checks done within the 1,604 days of experiment, which was approximately four years and five months. The results showed that Heterotardigrada had a better survival rate than Eutardigrada in long-term dehydration, but only one Heterotardigrada was able to be revived at the end of 1,604 days. Their results questioned the belief in tardigrades long-term anhydrobiosis and revealed time limitations of cryptobiosis in tardigrades.
These two studies showed the consequences of continuous cryptobiosis on survival of tardigrades. The longer the tardigrades were in cryptobiosis, the fewer tardigrades that were revived. This means that the duration of any organism in a cryptobiotic state must be taken into consideration in any study that is observing them in that state. If the longer an organism is in cryptobiosis, the harder it is to revive, the timing of the study must be monitored and controlled.
Harada and Ito(2006) examined tardigrades in acidic conditions, looking for a correlation between communal soil-inhabiting tardigrades and the forest they lived in. The experiment studied nine forests in Japan, each with its own environmental conditions. Two types of tardigrades were tested, predatory and herbivorous. The herbivorous species assumed the role of nematodes, which the predatory variety would eat in that habitat. The experiment studied the soil’s pH, hardness, and moisture, as well as porosity and leaf litter. The soil pH was at a range of 4.83 to 6.15, with the most acidic soil being in the forest at Futago, which had a pH of 4.83 ± 0.56. The experiment showed a correlation between tardigrades and the forest environment. Specifically, the study showed that tardigrades were active in the acidic environment, even the soil with a pH of 4.83 had active tardigrades living in it. Not only were tardigrades alive, but they were hunting and moving around. The limited numbers in the most acidic environment was related to the limited number of rotifers and nematodes, which the tardigrades feed on. The study showed that tardigrades can handle acidic environments and are even active in them. The reduction in numbers could be directly because of pH or because of lack of food. However, an active state in an acidic surrounding still leaves the inactive cryptobiosis state in question on how long they can survive in an extreme acidic environment. The research shows that tardigrades will react to acidic conditions. In addition, the research reports a range of activity for tardigrades in acidic soil. The research is one of the few so far that shows a reaction between tardigrades and low pH, which will add in the creation of parameters in the study of cryptobiosis reaction to strong pH.