Austin Byron and Austin Morales Macomb Mathematics Science Technology Center




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Genetic Variation in Tardigrada


Austin Byron and Austin Morales
Macomb Mathematics Science Technology Center
Biology
Mr. Estapa, Mr. Supal, Mr. Acre
5 December 2014

Genetic Variation in Tardigrada

On earth, humankind stares above at the stars and the whole of the universe, curious, desiring to expand into this great unknown. Now, researchers have found an organism that possesses the ability to take us there with proper experimentation. This creature has been classified and named as the Tardigrade, also commonly known as the water bear.

The purpose of this experiment was to test and prove that this life form’s durability is based on its own genetic pattern. To conduct research on the Tardigrade, two analysts observed, compared and contrasted survival rates between samples of water bears. The researchers placed each sample in a freezer set to an average of -24° Celsius for 35-45 seconds. The rate of surviving water bears was calculated, and the same sample would be placed in the freezer again for the same amount of time. The second survival rate was recorded and it was concluded that this rate was, on average, 21% higher than the first; leading to substantial evidence that the survivability of the Tardigrade is governed by its particular genetic code.

Table of Contents
Introduction……………………………………………………………………....….........1
Review of Literature……………………………………………………………....………4
Problem Statement………………………………………………………………...……...8
Experimental Design…………………………………………………………….…......…9
Data and Observations…………………………………………………………….….....11
Data Analysis and Interpretation…………………………………………………..........13
Conclusion…………………………………………………………………………...…..20
Appendix A: Exposure to Cold Time………………………………………………........23
Works Cited……………………………………………………………………………...24

Introduction

During the beginning of the human race, our ancestors looked to the stars in wonder and amazement for a number of things: calendars, navigational aids, even dreams of traveling them. Today, humans still look to the stars for answers, and ever more prevalent is a desire to travel the outer reaches of space. Doing so is not possible. Perhaps if it were possible to be in a state of suspended animation from cryobiosis, the stars could finally be explored. In fact, cryobiosis, a branch of cryptobiosis, may be a key factor in solving a number of issues, rather than just a desire to travel long distances in space. Specifically, these issues include the preservation of food, seeds, and even blood (Balser and Lindahl). The study of such fields in cryptobiosis are widely thought of as a part of a science-fiction movie, however the research is more prevalent than ever due to the amazing Tardigrada.

Tardigrades possess the uncanny ability to enter into a crytobiotic state, and more specifically a cryobiotic state. If scientists would want to break down how they are able to do this at a genetic level, using a sample of water bears known to be more resistant than others eliminates a portion of Tardigrades useless to their research. In other words, it gives the scientists a better sample to work from for their research. One particular company named Biomatrica has made incredible advancements in the preservation of cells, tissue, and biopsies. The bewildering Tardigrades are the key inspiration for their research. Biomatrica designed a thin film that mimics the chemistry of the Tardigrade; the film wraps around the sample, and preserves its contents until needed, no refrigeration required. When the sample is needed, water is added and it is rehydrated without tissue or DNA damage (McKeag). Although this is not cryobiosis, the method is still from branch of cryptobiotics associated with the Tardigrada known as anhydrobiosis (McKeag).

The researchers believed finding a solution to preservation issues begins with finding if certain Tardigrades survive in certain environmental conditions better than other Tardigrades. In the case of the experiment, the researchers wanted to find if some Tardigrades survive the extreme cold better than other Tardigrades, or if after exposed to the cold, a certain percentage die off each time. To test this, the researchers placed drops of water from a Tardigrade sample onto a depression slide, and then the number of Tardigrades was counted under a light microscope. Afterwards, the slide was placed into an industrial freezer and the Tardigrades were exposed to the cold for 30 to 45 seconds, depending on the temperature of the freezer, which ranged from -35° C to -11° C. The survivors were counted under a light microscope one minute after removal from the freezer. The number was recorded, and the exposure process was repeated to measure if a similar number died off, or if the survivors from the first exposure survived the second exposure as well. If the initial survivors once again survived the second exposure to the cold, it would suggest genetic variation in the species and that some Tardigrades survive in cold conditions better than other Tardigrades, which in further research would ultimately produce a better initial sample population for a relevant experiment. The data and observations collected from this experiment may aid future research regarding how the Tardigrada survive in such drastic climates, for both preservation of organic materials and suspended animation purposes, by giving future researchers a more environmentally resilient population to begin their research. Whether the future research involves temperature extremes or preservation strategies such as Biomatrica’s thin film, preparing populations of Tardigrada for studies is important. No matter what, Tardigrades may possess the key to unlocking the mysteries of suspended animation and the preservation of organic material for the future.

Review of Literature

           Just try to imagine it, the vastness of space. It is unfathomably large, especially in comparison to the Earth’s own size. As unfortunate as it may sound, humans could never really even attempt to explore any of it. With today’s technology, that is. Humans can only travel so far and the furthest so far being the moon. However, if humans were able to harness the power of cryogenic freezing, the ability to safely freeze something living and bring it back to life (“What is Cryogenic Freezing?”), the furthest reaches of space could then possibly be explored. Upon this, through the use of cryogenic freezing, a sick person could possibly be frozen until a cure is found for his or her disease.

           This all may sound like it came from a science-fiction movie or novel of some sort, but it is fact extremely prevalent to research being conducted today – mostly via the mighty water bear. The Tardigrada, also known as water bears, are a species that are very hearty in nature. They have the incredulous ability to survive in the most extreme conditions, that of which a human could never dream of surviving in. They have the ability to survive in extreme heat and cold temperatures, exposure to radiation, extreme dryness, and even in the harsh vacuum of space (Encyclopedia of Life).

           The word Tardigrada means “slow moving animal” (Mach). It is nicknamed the water bear because when it walks it resembles a lumbering bear. Tardigrada is a phylum of invertebrates that was discovered by Goetz in 1773 (Balser and Lindahl). As seen on the following page in figure 1, the water bear has four pairs of limbs, with each limb containing between four and eight claws, as well as a short and stubby. Although mighty, these creatures are only about a single millimeter in length, making them easily seen under a light microscope (Balser and Lindahl).

Figure 1. Picture of Tardigrada (Lam)

           Since its discovery in 1773, over 400 different species of the Tardigrada have been discovered. In fact, they have actually been discovered all across the globe. This ranges from even the highest points of the Himalayas, to the deep-freezing parts of Antarctica, and then all the way to a suburban backyard in North America. In short, they are any and everywhere. Even though they are found just about anywhere, they are actually considered aquatic animals because they are mainly found at locations on or near water – mostly in a moss of some sort. Due to this, they have rightfully earned the nickname of “moss piglets” (Bensinger). These “moss piglets” can be found in an array of different colors; they have been found to be blue, gray, brown, yellowish-brown, or even red. Similarly to crustaceans and insects, a water bear will shed its exoskeleton as it grows and develops. A water bear’s diet typically consists of plants, nematodes, and algae, however there are a few species that are carnivorous and consume other Tardigrades (“Water Bear”). This fascinating species can actually reproduce both sexually and asexually. They reproduce sexually like most other animals (Bordenstein). However, a water bear can reproduce asexually through Parthenogenesis, a process through which embryos grow without fertilization (“Parthenogenesis”).

As said by widely acclaimed scientist, Neil deGrasse Tyson, “They’ve (Water Bears) been in business for half a billion years.” He also says, “The Tardigrades have survived all five mass extinctions (McDermott). This does not happen by mistake. Rather, the timely process called evolution. The idea of evolution was first researched by none other than Charles Darwin. He basically said that evolution occurred due to natural selection; the process by which nature selects traits most suited to the environment a species lives in (O’Neil). The hearty Tardigrada have evolved to possess many interesting attributes. These include its ability to go into a cryptobiotic state when exposed to an extreme stress such as lack of water, lack of oxygen, and even extreme hot and cold temperatures.



Tardigrada have the uncanny ability to resist almost all types of extreme environments. This is made possible by the use of its cryptobiosis. When a water bear enters cryptobiosis, its metabolism is slowed to nearly zero (Meyers). The adaptive biochemical processes that allow this cryptobiotic state to occur involve large amounts of polyhydroxy compounds, most notably trehalose or sucrose, which are disaccharides (Clegg). To organize the forms of cryptobiosis, it has been divided into sub-classes, those of which being: Anoxybiosis, a response to lack of oxygen by which an animal takes in water and becomes turgid and immobile. Chemobiosis, a response to enhanced levels of toxins or radiation. Osmobiosis, the least studied type of cryptobiotic states, occurs in response to elevated levels of solute concentration. Anhydrobiosis, the most studied form of cryptobiotic types, which occurs when animals are subject to extreme desiccation. And finally, Cryobiosis which takes place when an organism reacts to freezing temperatures.

Cryobiology is signified as the science of life at freezing temperatures. The branch is mainly interested in the study of living material at any temperature below their stable range. Some applications include preservation of organic substances, cryosurgery for the destruction of unhealthy tissue, Lyophilization (the freezing of pharmaceutical medicines) and the education of evolutionary aspects due to cold in animals (“What is Cryobiology?”).

The cryobiotic state occurs when an organism’s temperature reaches its lowest threshold. For the Echiniscus Testudo and the Hypsibius Dujardini, this point happens around freezing temperatures or zero degrees Celsius. Certain species of water bears have been known to survive in near absolute zero environments due to their specific cryobiosis. The study of water bears entering into a cryobiotic state applies to the study of Cryogenics and would be most helpful in the area of Cryonics.

Cryonics is the general preservation of animals and even humans using low temperature means. Often incorrectly specified as Cryogenics in science fiction or popular culture, the term Cryonics is used to describe the freezing of living organisms that may have incurable diseases, in hopes that one day, future medicine can revive these organisms. This technology is being performed already, but it still is young in its scope. The preservation of humans depends on the actual death that they have gone through. In order to undergo this procedure, a person must be pronounced “legally dead” which means their heart has stopped beating. This differs from “total death”- the point at which all brain functions cease (Watson).

Problem Statement

Problem:

To determine if certain Tardigrada have a genetic advantage over other Tardigrada in the same culture.



Hypothesis:

The same sample of Tardigrades that survive a first “deep-freeze” will remain alive with a significant increase in survival rate during subsequent freezes.



Data Measured:

The independent variable of the experiment is the exposure of the Tardigrades to a cold environment. The dependent variable of the experiment is the percentage of Tardigrades that survived in a given sample. The data was analyzed with descriptive statistics. A total of 34 trials were run throughout the experiment.

Experimental Design

Materials:

Tardigrade culture (2)

Microscope

Depression microscope slide (5)

Deep-freeze freezer (-10°C – -40°C)

Temperature probe (0.1°C precision)



Procedure:

1. Extract Tardigrades from cultures and place in depression slides so that the depression is filled completely.

2. Observe and count the number of living Tardigrades under microscope.

3. Place depression slides in freezer.

4. Wait 35-45 seconds according to the temperature of the freezer. (See Appendix A)

5. Remove Tardigrade slides from freezer.

6. Wait 1 minute to allow surviving Tardigrades to exit cryptobiotic state.

7. Observe and count number of Tardigrades that survive the cold exposure.

8. Repeat steps 3-7 for second cold exposure.

9. Repeat steps 1-8 until trials are completed.



Diagram:


Deep-freeze freezer

Temperature probe

Depression slides

Figure 2. Materials for Experiment

Figure 2, above, pictures the deep-freeze freezer, and sitting on the paper towel that was used for contrast in the image are the depression slides, as well as the temperature probe.

Data and Observations


Table 1

Data of Trials



Trial

Initial Amount

Temp (°C)

First Survivors

Second Survivors

1

30

-18

17

17

2

45

-18

30

29

3

33

-20

23

21

4

28

-24

16

15

5

7

-19

5

5

6

37

-21

30

29

7

4

-22

4

4

8

24

-20

19

17

9

19

-28

11

11

10

31

-30

25

22

11

22

-31

21

20

12

15

-30

13

11

13

18

-29

10

10

14

39

-28

31

29

15

23

-11

18

18

16

27

-12

22

20

17

19

-14

19

18

18

32

-14

29

28

19

29

-18

28

28

20

21

-23

19

18

21

22

-23

19

19

22

16

-25

13

13

23

25

-26

20

20

24

27

-28

20

19

25

14

-26

9

8

26

31

-27

25

24

27

30

-31

26

24

28

18

-32

8

6

29

6

-30

5

5

30

9

-28

8

7

31

25

-27

21

21

32

36

-27

29

28

33

40

-26

20

18

34

12

-25

10

10

Average

23.94

-23.85

18.32

17.41

Table 1 shown on the previous page contains all of the data from the 34 trials conducted in the experiment. Each trial began with approximately 24 water bears at an average temperature of -23.85° Celsius. Also, on average, after the first exposure to the stress of extreme cold, about 18 water bears survived. On an interesting note, when the same water bears were exposed to the same extreme cold temperature as before, only approximately a single water bear died off. This is indicative of genetic variation within the Tardigrada, in that some do have a genetic advantage over the others.

Table 2

Observations



Day

Observations

28-Oct

Trials 1-8 conducted. Temperature ranges between -18 and -24. Initial data looks very promising. Trial 7 began with a low sample size of 4, resulting in a 100% survival rate through two stages of freezing the water bears, whereas trial 2 began with a high number of 45, resulting in an initial survival rate of 66.67% after the first freezing.

29-Oct

Trials 9-14 conducted. Much colder temperatures today. Ranges between -28 and -31. The freezer is proving to be troublesome; it is very hard to keep it close to a constant temperature. Exposure time to the temperature was decreased in order to compensate for the colder temperatures. Other researchers needed to use the freezer and were incapable of conducting more trials today.

30-Oct

Trials 15-21 conducted. Slightly warmer temperatures than previous days, however a much larger range in temperatures is prevalent. The temperature ranges from -11 to -23. Range of temperatures is once again a challenge, but data is holding true with previous days. Trials cut short because of other researchers using the freezer.

31-Oct

Trials 22-34 conducted. Temperature ranges between -25 and -32. Good trials and good data now that everything is going smooth. Unfortunately no more trials can be conducted because of a high demand for use of the freezer from other researchers and their own experiments.

Table 2 shown above contains observations collected for the experiment. Unfortunately, only 34 trials were conducted; however, this is enough for data to be considered normal because of the Central Limit Theorem, as more than 30 trials were conducted. Due to an overwhelming demand for the freezer, trials were cut short.

Data Analysis and Interpretation


Initial Number of Water Bears

4

45

24.5

18

31

Figure 3. Initial Number of Water Bears at Start of Each Trial

The purpose of this boxplot is to show the normality of the extraction of water bears from the cultures. The mean and median of this data set is very close, with the mean being 23.94 and the median being 24.5. With more trials, the mean and median would eventually become equal due to the Law of Large Numbers. The lower and upper quartiles are equidistant from the median, each being 6.5 units away at 18 and 31 respectively. The initial number of water bears ranges from 4 to 45. The boxplot looks very symmetric, with virtually no skewness, further supporting statistical regularity with the initial number of water bears for each trial.


Initial Survival Rate

0.444

1

0.867

0.806

0.697

Figure 4. Initial Survival Rate



Above in figure 4 is a boxplot for the survival rates of Tardigrada after the first freeze. The data in the graph is calculated by dividing the number of surviving water bears by the number of initial water bears. The percentages range anywhere from 43 to 100 percent; however, the box is skewed to the right, indicating that most (77.7291% of) water bears have a natural resistance to cold temperature. The median of the lower half of the data, or Q1, is 69.7% while the median of the upper half of the data, or Q3, is 86.7%. The median of all the data is 80.6%. This is the first of two graphs necessary to answer the problem statement.


Second Survival Rate

0.75

0.846

0.913

0.963

1

Figure 5. Second Survival Rate



This is the second of two graphs necessary to answer the problem statement. The boxplot above describes the data for the survival rates of Tardigrada after the second freeze. With a range from 84.6% to 100%, the graph is skewed extremely right, which supports the estimated hypothesis greatly and can help the researchers come to a conclusion. This second survival rate data is noticeably higher than the first survival rate data. The boxplot contains a median of 96.3%, a mean of 94.9%, a lower quartile of 91.3% and an upper quartile equal to the positive end of the range. The boxplot contains only one outlier, which can be omitted.


0.75

Initial Survival Rate and Second Survival Rate

Initial

Survival

Rate

Second

Survival

Rate

0.846

0.913

0.963

1

0.444

0.697

0.806

0.867

1

Figure 6. Comparison of Survival Rate Boxplots


Figure 6, above, shows the comparison of the initial survival rate of water bears against the second survival rate of water bears side by side. Each boxplot’s individual statistics can be observed closer in figures 4 and 5 on pages 14 and 15, respectively. Some important notes to take into consideration include that the majority of the data in the second survival rates (Q1 to Maximum) is above even the third quartile of the initial rate. Furthermore, the lowest rate of survival for the second set of data, which is an outlier, is still higher than the first quartile of the initial survival rates.

Figure 7. Amount Die off Each Time

The mean amount of water bears that die off during the first freeze is 5.61765, or around six water bears. The mean amount of water bears that die off during the second freeze is 0.911765, approximately one water bear. The standard deviation for each sample is 4.53949 and 0.865768 respectively. These statistics show there are no patterns or lurking variables that skew the data.

Figure 8. Survival Rates

The one variable statistics above in figure 8 shows the data behind the boxplots in Figures 4 and 5 on pages 14 and 15, respectively. The mean of the initial survival rate is 77.7291%. In short, this means that after exposure to the cold just one time, on average, 77.7291% of the water bears in the sample survived. This also means on average, 22.2709% of the water bears in a given sample died after the first exposure to the cold. For the second survival rate, the mean is 94.9249%; a significant increase. This means that after the second exposure to the cold, on average, 94.9249% of the remaining water bears survived in a sample. Likewise, after the second exposure to the cold, on average, only 5.0751% of the water bears died off in a given sample.

Interpretation:

The reason to show the descriptive analysis of all the data found in this experiment is to answer the problem statement, “Do certain water bears have a genetic advantage over others?” The two major data sets that influence the support of the hypothesis include figure 4 on page 14, the initial survival rate of water bears, along with figure 5 located on page 15, the second survival rate of water bears. The main concept this supports is whether the second survival rate was higher than the initial survival rate. The data indicates this to be so, because the mean of the boxplot in figure 5 is, in fact, higher than the mean of the boxplot in figure 4. The difference in the boxplots is clearly evident in figure 6 on page 16.

The second rate of surviving water bears is approximately 1.5 standard deviations from the initial rate. This was calculated using the initial rate’s standard deviation, since the mean of the second rate is 22% higher than the initial rate, and the standard deviation of the initial rate was 14%. Any number of standard deviations above two would be logically impossible, because this would imply that survival rate percentages have the capacity to go over 100%. Using this knowledge, it can be said that 1.5 standard deviations is statistically significant in this specific case. The rest of the data shown on previous pages displays normality, regularity, and consistency during experimentation. Overall, the experiment shows minimal signs of patterns and/or lurking variables. With all the knowledge gathered in the Data Analysis section, the researchers can come to a conclusion.

Conclusion

The purpose of this experiment was to test and observe the genetic variation of different Tardigrada and observe if some had a genetic advantage over others. The hypothesis that the same sample of water bears that survive a first “deep-freeze” will remain alive with a significant increase in survival rate during subsequent freezes was accepted. A total of 34 trials were run between two Tardigrade culture samples. The water bears were exposed to frigid temperatures in a freezer measured to be about -24°C on average, and it was measured as to how many entered a cryptobiotic state, and then revived afterwards. Subsequently they were exposed to the frigid cold a second time to see if a similar percentage would die off each trial, or if the survivors had the genes to survive such temperatures again. The collected data of the survival rates of the water bears was analyzed using descriptive statistics. In comparison, the first survival rate of the water bears was 77.7291%, while the survival rate of the Tardigrades after the second “deep-freeze” was 94.9249%; an increase of 17.1958% in the survival rate of the water bears. This data meant that the ability to go into a cryobiotic state and survive cold, harsh temperatures was governed by the Tardigrades’ own genetic code. Certain water bears were “programmed” to survive in colder climates than others did. It is Darwinism at its finest - survival of the fittest.

These results concur with other researchers in the field of cryobiology. Scientists in this field have discovered that Cryobiosis in animals is in fact genetic. Not only does the experimentation of survival rates in Tardigrada confirm their discoveries, it also paves the way for further research on the topic, leading to more complex ways to test the boundaries of the water bears’ resistance.

When doing pre-trials and regular trials, the researchers ran into some design flaws and errors. In pre-trials, the researchers initially found it difficult to count and to keep track of the Tardigrades if one large drop was placed onto the depression slide. To overcome this obstacle, the researchers developed a better plan for counting the water bears on the slides; instead of a random pattern, the researchers counted the water bears in a grid-like fashion. With that problem overcome, another arose. It was incredibly difficult to gain an understanding of how long the water bears be exposed to the cold. At first, they were left for around 72 hours, in which none survived. Time exposed was decreased repeatedly, as all the Tardigrades kept dying. It was then realized that when the water froze it was crushing the water bears, and likely impaling them on a microscopic level.


Figure 9. Dead Tardigrade after being frozen

Figure 9 shown above is a picture taken by the researchers of a dead water bear that was part of a sample in which the water froze during pre-trials under a light microscope. To combat the freezing, the researchers took a break from freezing water bears and tested how long it took to freeze 1 cubic centimeter of water at about -24°C; the answer: between 35 and 45 seconds, depending on the definite temperature of the freezer at the time. With that knowledge, the rest of pre-trials were successful and smooth as well.

Further research of measuring the survival rates of Tardigrada can be performed by subjecting the Tardigrades to different factors. Some factors that could potentially be applied to the experiment include a bath in toxic chemicals, exposure to radiation, heat to high temperatures, placement in a centrifuge, and placement in a zero-oxygen vacuum. Further research could also be done by increasing the intensity of the cold temperatures used in the experiment; possibly bringing the temperature down to -50°C or even -60°C. Also, for further research, after the water bears were exposed twice so that the survivors had the genetic advantage over the others, one could possibly control the water bears’ reproduction and then measure if the offspring show the same genetic advantage the parents had. Factors could also be combined for trials: the waters bears bathe in toxic chemicals while also in a freezer, for instance, and then repeating that to measure survival rates and having the offspring reproduce to measure their genetic variation as well. By testing these different factors, researchers can begin to understand the process of cryptobiosis in water bears to an even higher degree.

Appendix A: Determination of Cold Exposure Time

Table 3

Point of Freezing Times



Temperature (°C)

Estimated Time that Water freezes (seconds)

-11

45

-12

44

-13

44

-14

43

-15

43

-16

43

-17

42

-18

41

-19

41

-20

40

-21

40

-22

39

-23

39

-24

39

-25

39

-26

38

-27

38

-28

37

-29

36

-30

36

-31

35

-32

35

Above is the table used to determine how long water bears should be left in the industrial freezer, given the temperature in degrees Celsius. The temperature in the table ranges from -11° C to -32° C to agree with the range of the accepted time exposure (35-45 seconds). The times above are the estimated points at which the water nearly freezes on the depression slides. Since the experiment depends on the cold climate and not the additional variable of crushing ice, it is important not to reach these times; rather, to approach a time two to five seconds below them.

Works Cited

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Bensinger, Lou. Tiny Invaders in the Water. New York: PowerKids, 2010. Print.

Bordenstein, Sarah. "Tardigrades (Water Bears)." Tardigrades. Carleton College, 17 Dec. 2008. Web. 20 Sept. 2014. .

Clegg, James. “Cryptobiosis- a peculiar state of biological organization”. ScienceDirect. N.p., June 2001. Web. 6 Oct 2014. .

Encyclopedia of Life. "Water Bears (Tardigrada) - Information on Water Bears - Encyclopedia of Life." Encyclopedia of Life. Eol.org, n.d. Web. 01 Oct. 2014. .



Lam, Mickey. "Nature's Tough Guys Turned Cosmonaut." Epoch Times. N.p., n.d. Web. 1 Oct. 2014. .

Mach, Martin. "Microscopy-UK Micscape Microscopy and Microscopes Magazine." Microscopy-UK Micscape Microscopy and Microscopes Magazine. N.p., n.d. Web. 15 Sept. 2014. .

McDermott, Maeve. "5 Reasons Why The Tardigrade Is Nature’s Toughest Animal." Nat Geo TV Blogs. National Geographic, 19 Mar. 2014. Web. 05 Oct. 2014. .

McGill, Lorraine. “A Molecular and Phylogenetic Analysis of Cryobiosis in Nematodes of the Genus Panagrolaimus”. Maynooth University. [Online], 3917 (2012). Web. 6 October 2014. .



McKeag, Tom. "The Biomimicry Column." GreenBiz.com. N.p., 24 Feb. 2011. Web. 26 Nov. 2014. .

Meyers, Ashley. “Cryptobiosis Protects from Extremes: Waterbear”. AskNature.org. N.p., n.d. Web. 6 Oct 2014. .

O'Neil, Dennis. "Early Theories of Evolution: Darwin and Natural Selection." Early Theories of Evolution: Darwin and Natural Selection. Palomar College, 2013. Web. 02 Oct. 2014. .

"Parthenogenesis." Fact Monster. N.p., n.d. Web. 3 Oct. 2012. .

"Water Bear." Water Bear. N.p., 31 July 2006. Web. 18 Sept. 2014. .

Watson, Stephanie. “How Cryonics Works.” Howstuffworks.com. N.p., 5 Jan 2005. Web. 6 Oct 2014. .

“What is Cryobiology?”. SocietyForCryobiology.org. N.p., 2013. Web. 6 Oct 2014.



.

"What Is Cryogenic Freezing?" What Is Cryogenic Freezing? Innovateus.net, 12 Mar. 2011. Web. 01 Oct. 2014. .





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