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
Sections
Page number
Abstract
2
Introduction
Hypothesis
3-13
13
Material and Methods
Active Test
Dissolved Oxygen Test
13-17
14-16
16-17
Result
17-22
Discussion
22-28
Acknowledgments
29
Literature Cited
30
1
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, while the active 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.
2
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, (mesomeaning 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
3
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
4
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
5
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
6
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
7
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
8
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 trehalose is 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
9
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
10
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.
Another study of cryptobiotic organisms and acidity was conducted by Zuo and Woo
(1996). This study experimented on Cryptobia spp., which are flagellated parasitic protozoans,
that are pathogenic to fish. They compared acid phosphates in both pathogenic and
nonpathogenic Cryptobia spp, by testing the activity rate in the organism under different acidic
conditions, ranging from a pH of 5.5 to 3. The maximum activity of the Cryptobia spp. was
recorded at a pH of 5. The research showed that other organisms that go into cryptobiosis can
survive in acidic conditions. This research has been stated for Harada and Ito (2006) research;
however this study shows a cryptobiotic organism both surviving and active in strong acid pHs.
If these organisms can survive pH of 3, then how will other cryptobiotic organism fare? With a
comparison of the organism to tardigrades, assumptions can be made on how tardigrades might
react to equally strong pHs. First, like tardigrades, the protozoans have the ability to undergo
cryptobiosis. Second, it shows how an organism that can go into cryptobiosis reacts to an acidic
environment.
There have been experiments with higher pH’s than 4 and 5, for example the experiment by
Ardelli and Woo (2001). Like the study done by Zuo and Woo (1996), they examined pathogenic
and nonpathogenic Cryptobia spp. and studied how the pathogenic species were able to survive
in vitro conditions with pHs ranging from 6.0 to 7.3. The study showed that for Cryptobia spp. to
survive in the environmental conditions, they released hydrogen peroxide into the system to
increase the organism’s survivability to that pH range. The study showed that the parasites were
11
active for a week with the release of hydrogen peroxide in the system. However, in a saline
solution with no hydrogen peroxide, the Cryptobia spp. became inactive within twenty-four
hours and all were dead in about one week. This suggests that Cryptobia spp. are sensitive to
changing pH, and in order to survive these changes, they need to produce and release hydrogen
peroxide to stabilize the surrounding environment to conditions under which they can survive.
How do tardigrades survive changes in pH? They might release a chemical to maintain their
environment like the Cryptobia spp. Tardigrades produce compounds like trehalose to maintain
their internal conditions (Wright et al., 1992), so they might have a substance for surviving
changes in pH.
The two experiments with Cryptobia spp. both dealt with a changing pH environment.
Even though Cryptobia spp. are not tardigrades, their ability to survive in changing pH may
suggest how pH affects tardigrades’ cryptobiosis. Tardigrades produce many different chemicals
in their different stases forms. They might be able to produce a similar buffer to survive extreme
pH, where the results might be similar to the two studies with Cryptobia sp.
As mentioned earlier, research on the reaction of tardigrades to extreme pH conditions
has been limited. According to Harada and Ito (2006), it is known that some species can survive
acidic soil. However, very little has been studied on the effects of basic conditions. We do not
know what mechanism tardigrades might use to survive basic pH conditions or how high of a pH
they can survive.
The purpose of the study was to find the effects of the changing pH on the activity of M.
tardigradum. From the research into the change of tardigrades optimal state, it was believed that
M. tardigradum would show a progressive decrease in activity the further the pH was from
neutral (ph of 7). This was based on the assumption that the pH of 7 is the optimal pH, and that
12
the pHs of 6 and 8 would show similar activity. In addition, it was believed that M. tardigradum
would show longer activity times in lower pHs compared to higher pHs. This hypothesis was
based on studies that have found different tardigrade species in acidic soil and neutral soil from
around 8 to 4.5. Because the ion concentration was changing with the changing pH it was
believed that the dissolved oxygen in the samples would also be affected the further the pH was
from neutral. Because tardigrades need the dissolved oxygen in the water it was believed that the
change in pH would also show a similar progressive decrease in the amount of dissolved oxygen
in the sample. These changes in conditions were believed to reduce the activity levels of M.
tardigradum.
Materials and Methods
The tardigrade that was used for the study was Milnesium tardigradum, which are
terrestrial Eutardigrades around 0.5 to 1.0 mm long (Kinchin, 1994). They were obtained from
the Carolina® Biological Supply Company in form specimen cultures used for a class of thirty.
Three cultures were ordered and received in January 2008, and used one week after they were
received for the pH test. This is different than studies by Rebecchi et al. (2006) and Crowe and
Higgins (1967), who observed, identified, and tested tardigrades caught on moss either around
their study site or a nearby location. Because the samples were ordered, they did not need to be
identified, and unlike the studies that tried to remove the samples, the M. tardigradum were kept
in the same water in which they were sent. The study took place in the preparation room of Saint
Martin’s University Biology Laboratory. When the samples were not being tested they were
stored in a store room at room temperature, which ranged from 20oC to 26oC. This is similar to
Rebecchi et al. (2006), in which they stored their samples at room temperature in a paper bag.
The pH of the cultures was recorded at 7 to 8.3 using a Fisher Scientific pH thermometer.
13
Organisms in the cultures were different sizes, indicating that some of the samples may
have been procreating. This resulted in a random sampling of both size and age for the pH test.
Even though there were differences in the sizes of the samples, the variables of age, recent
molting, and body length were not calculated due to the lack of time and equipment. No M.
tardigradum were tested more than once and all ages were tested, which was different than
Rebecchi et al. (2006), who tested only adults and kept testing the same samples over and over
again until the last sample died after 1,604 days.
Activity test
The purpose of this test was to induce an anoxybiotic state in the samples of M.
tardigradum. Inducing anoxybiosis has been done in many tests. However, the procedure for
inducing tardigrades into anoxybiosis was mostly used for collecting samples for study not as a
study on its own. The anoxybiotic state caused the tardigrade to release from the moss and to
become more visible with their increased size (Romano, 2003). To find the limit of anoxybiosis,
a buffer set at a specific pH was added until there was an absence of movement in the test
sample. An absence of movement, defined as no movement of any of the eight appendages, body
segments, body compression, or in a stationary form for more than five seconds, was used for all
tested samples. The stationary forms were a ball form, in which the sample rolled its segments
into a ball, or a hollowed form, in which the sample’s outer tissue remained while its remaining
cells collected together. The hollow form resembled how tardigrades molt leaving eggs behind in
the dead outer tissue, although only one mass of cells remain instead of multiple eggs (Kinchin,
1994).
Samples were divided into three groups, which were the three cultures ordered. There
were two test samples taken from each group, giving a total of six test samples for each pH. The
14
pHs tested ranged from 1.5 to 12.53, which came from buffer solutions, either in liquid or
powder form. The buffers that were used were from 2 to 12 pH. Any of the pH buffers that were
in powder form were dissolved in deionized (DI) water prior to usage. The difference in buffer
pH that was on the label and actual pH resulted from preparing the buffers of 2 and 12, which
when made from the powder read a pH less than 2 and greater than 12. This was most likely
caused by either the age of the chemical in the buffer or the temperature in which each buffer
was made, which could have affected the actual pH of both buffers. The pHs were divided into
two groups, diluted and non-diluted buffer. The diluted buffer was made from the same buffer
solution as the non-diluted buffer, a 1:10 concentration using DI water in the preparation. Before
the tests the pH of the buffer and the samples were taken using the pH meter to make sure it was
set at the correct pH. For the experiment only one buffer concentration type was tested per day.
All tested samples were observed using a compound microscope at 400X magnification. The
microscope was kept at its lowest light intensity to prevent overheating the samples.
For the test, the pHs of 6 to 8 were considered controls, which was based on the
experiment by Crowe and Higgins (1967) that showed no effect at a pH of 6 to 8. Before the
samples were retrieved the culture was stirred using a dropper. The purpose for stirring the
sample was to add some O2 to the culture, break apart the moss in the culture, separate the M.
tardigradum from each other, and separate the M. tardigradum from the moss to better observe
them. The samples were prepared using a 0.20mL to 0.25 mL drop of one of the three cultures in
a Petri dish with 2 mL of DI water. After M. tardigradum was observed, which ranged from one
to ten samples per test, 3 mL of a preselected pH was added. The M. tardigradum was then timed
from the moment the first drop of selected pH entered the water to when the M. tardigradum
stopped moving. After the tardigrade stopped moving, the water was tested for its pH. The pH
15
data was compiled to find the average for both concentrations. An ANOVA test was used to test
for significant differences between the treatments of the different pHs. Specifically, the test was
used to show any significant differences in the time collected for each pH in both concentrations.
The ANOVA test was set at a 95% confidence level, and was set to run a Tukey multiple
comparison test if p<0.05. The average time and standard deviation were calculated to visually
compare the movement time of each pH group.
Dissolved oxygen test
Townsend and Cheyne (1944) showed trends that increased hydrogen ions caused by
increased toxin and pH could affect the ability of salmon to resist areas of low dissolved oxygen.
Because the chemicals and concentration of chemicals dissolved in the buffer may have affected
the amount of O2 in the water, a dissolved oxygen (DO) test was taken at each pH and at both
concentrations. For each pH there were six samples taken giving a total of 132 test samples for
pH and six samples for normal DI water, which acted as a control for the DO test. The DO test
was conducted using the Micro-Winker Procedures for small amounts of water samples. The
procedure used equal volumes of MnSO4, KOH-KI, H2SO4, and starch. These chemicals were
used to react with sodium thiosulfate solution (Na2S2O3). When starch was added it turned the
solution blue, and eventually Na2S2O3 was titrated until the blue color disappeared. The amount
of Na2S2O3 added to turn the solution clear gave the amount of dissolved O2 that was in each
sample of water when used with the Winker’s formula (Equation 1).
Equation 1. 11 X amount of Na2S2O3 = DO of the sample (ppm or mg/L)
11mL of sample
Because the amount of water in the pH test was small, and observing a color change would have
been difficult at 5.25 mL, the samples for the DO test were doubled to 11 mL to get a more
accurate O2 reading. Each DO sample was made from 5 mL of DI water and 6 mL of a specific
16
pH and concentration. This gave a total of 11 mL, because the calculation was similar to the
titration procedures followed if the samples were 10 mL from Sumich and Dudley (2005). The
control used 11mL of DI water with no buffers added. The amount of DO in each sample was
analyzed using the Minitab©2007 program. The pH concentrations were tested separately using
ANOVA test. The test was used to show any significant difference in the amount of DO in each
sample taken at a specific pH concentration. The ANOVA test was set at a 95% confidence level,
and set to run a Tukey multiple comparisons with a p value of < 0.05. This compared the
difference between diluted and non-diluted buffers in the amount of dissolved O2 that was
present in the water sample for the pH tests without using any M. tardigradium.
Results
The active test observed the time it took the M. tardigradium to go from an active state to
an inactive state after the pH buffer was added, with a maximum time of 90 minutes set for each
sample. This test showed four general stages of M. tardigradum. Samples that were near the
control (pH 7) were active for the entire 90 minutes of observation (Figure 1).
Figure 1. M. tardigradum actively moving. All samples resembled this before
the buffers were added.
17
Samples tested in pH levels further from pH 7 had three types of inactive forms. The stiff, ball,
and hollowed forms (Figure 2).
A)
B)
C)
D)
Figure 2. The three non active forms M. tardigradum observed in the active test. (A) shows the
stiff form, with a straight body and translucent legs. (B) Shows the ball form with the sample’s
body rolled into a ball near moss. (C) Is the hollow form in which the external body cells
remained while the remaining internal body cells collected in one area. (D) Is a picture from the
Carolina catalog of a M. tardigradum with eggs, as a comparison to the hollow form.
The time it took to go from an active state to an inactive state depended on how basic or acidic
the buffer was from the control.
Once the physical comparison between the active and the three inactive states was done
the amount of time it took each sample to remain active or become inactive was analyzed using
an ANOVA test. For both the 1:10 concentration (Figure 3) and the non-diluted buffer (Figure
4) the pH for each buffer and the time each sample remained active were averaged (n=6) and
each concentration was compared to the pHs of that specific concentration.
18
Figure 3. Comparison of activity time across different pHs from the use of 1:10 diluted
buffer. It shows the type of pH and how long the tardigrades remained active. Error bars
represent one standard deviation about the mean (n=6). Any data without an error bar had
a standard deviation of zero.
Figure 4. Comparison of activity time across different pHs using non-diluted buffer for
the source of the pH. It shows the type of pH and how long the sample remained active.
Error bars represent one standard deviation about the mean (n=6). Any data without an
error bar had a standard deviation of zero.
Figures 3 and 4 showed a decrease in time of activity the further the pH was from neutral and the
active time of the sample for the non-diluted pH buffer was lower than the 1:10 concentration.
19
Both 1:10 concentration and non-diluted pH buffer show a trend of progressively decreased
active time of M. tardigradum in the pH further from 7. Whereas, the tardigrades in the pHs
closer to 7 remained active like the samples tested at the pH of 7. For both 1:10 concentration
and non-diluted buffers, the pH buffer set at 7 was used as both a control and the basis of
comparison for any significant difference within the data. For both concentrations, the
tardigrades that were introduced to a pH of 7 reached the maximum active time, which was 90
minutes. For the 1:10 concentration there were significant differences in the active time of M.
tardigradum (F= 264.61, DF= 10, P= 0.0001). The Tukey analysis set at 99.86% confidence
indicated that the activity time at buffers 2, 3, 4, 11, and 12 was statistically lower than the pH of
7. The samples at those pH levels went into an inactive state. The other buffers at 1:10
concentration remained active for the entire 90 minutes, showing no significance in activity
compared to the M. tardigradum at a pH of 7. The data for the non-diluted pH buffers had more
significant differences (F= 2177.72, DF= 10, P= 0.0001) in its pHs. For the non-diluted pH
buffer the Tukey analyses had an individual confidence level of 99.86%. The analysis showed
the pH buffers 2, 3, 4, 5, 10, 11, and 12 had times significantly lower active times compared to 7.
Similar to the 1:10 concentrated buffer the pH buffers 2, 3, 4, 5, 10, 11, and 12 for the nondiluted buffer had all of their samples go inactive. The other buffers for non-diluted had similar
active times, so their data was not significant.
The amount of dissolved oxygen in each sample (n=6) for both 1:10 concentration and
non-diluted buffers were run together in the ANOVA test. The samples were compared to the
amount of dissolved oxygen from samples of DI water set at room temperature (23.5oC) (Figure
5).
20
Figure 5. Comparison of the dissolved oxygen test for both 1:10 dilution (dark bars) and
non-diluted (light bars). Error bars represent one standard deviation above the mean
(n=6). Any data without an error bar had a standard deviation of zero.
Figure 5 has both concentrations together for a comparison of the amount of DO in that specific
buffer. The 1:10 concentration (light bar) showed higher DO concentrations compared to the
non-diluted (dark bar) samples, when compared side by sided. The comparison was used to find
any significance in the data (F= 68.11, DF= 20, P= 0.0001) after the ANOVA test. With an
individual confidence level of 99.96% from the Tukey analysis, the test shows a significant
difference in the data for pHs 4, 5, 6, 7, 8, and 9 for the 1:10 concentration, and a significant
difference in the pHs 2, 3, 4, 5, 7, 8, 9, and 10 for the non-diluted buffers when compared to the
control pH. For the pHs 4, 5, 7, 8, and 9 for the 1:10 concentration and pH 7 for the non-diluted
21
buffer the data were significantly higher in the amount of DO compared to the amount in DI
water samples. Whereas, the non-diluted pHs 2, 3, 5, 8, 9, and 10 were significantly lower in the
amount of DO from the samples taken. The analysis showed no significant difference in activity
in pHs 2, 3, 10, 11, 12 for the 1:10 concentration, and no significance with the buffer 6 for the
non-diluted buffers in the amount of DO compared to the amount in the DI samples.
Discussion
The data from the active test showed a decrease in M. tardigradum active time the further
the tardigrades were from the pH of 7. For both the 1:10 concentration buffer and non-diluted
buffer, tardigrades in the pHs of 6, 7, 8, and 9 remained active for the entire test. The data also
showed that M. tardigradum remained more active at higher pH than lower pH. This was shown
in both concentrations of pH buffers where both pHs of 8 and 9 had M. tardigradum remaining
active while only pH of 6 had M. tardigradum remain active for both concentrations. Other pHs
such as 5 and 10 had different results between the two concentrations. In comparing the two
concentrations of pH buffers, both showed progressively decreasing activity in M. tardigradum
the further the pH was from 7. However, there was a difference in the reactions with the buffers
other than a decrease in time. There was a difference in the active time between the buffers of 11
and 12 when the two concentrations are compared. Both showed similar pHs with the buffer of
11 having a pH greater than 10.5 and the buffer of 12 having a pH of around 12.5. Both
concentrations show a pH difference of 2, which has 100 times more ion concentrations between
the two basic pH buffers. The non-diluted buffer had a tardigrade active time difference between
the two pHs of 14.12 minutes (subtraction between the averages of the two n=6). In the 1:10
concentration, the tardigrade had an active time difference between the two pHs of 54.91
minutes. Both concentrations had similar pHs, but the 1:10 concentration had a larger drop in
tardigrade activity, even when compared with the pHs of the same concentration.
22
The DO test showed similar results compared to the activity test in the context that both
tests showed similar decreases the further the pH was from 7. The differences in the results are
that the activity test showed a change in tardigrade’s active time, while the DO test showed a
decrease in dissolved oxygen. Both concentrations of buffers had decreases in dissolved oxygen
the further the pH was from 7. For deionized water, the dissolved oxygen was measured at 6.5
ppm. The non-diluted had dissolved oxygen ranges of 1.72-8.88 ppm, while the 1:10 concentrate
had dissolved oxygen ranges of 6.25-10.17 ppm. From the data there is a difference between the
amount of dissolved oxygen and the concentration of the pH buffers. The 1:10 concentration had
most of its dissolved oxygen higher than DI water, while the non-diluted buffers had dissolved
oxygen levels lower than DI water. The dissolved oxygen level of the buffers 11 and 12, for the
non-diluted concentration, could not be retrieved due to the reaction between the chemical in the
buffers and the DO kit. The significance in the change in dissolved oxygen and the tardigrade’s
activity is unknown due to the few studies of oxygen consumption by tardigrades. Compared to
the salmon study the dissolved oxygen for the entire test was low. The data for the dissolved
oxygen level of salmon comes from Maun (2008). The sheet from the Nisqually River
Education Project defines the optimal DO levels of salmon in rivers as 9 ppm, with 7-8 ppm as
acceptable, 3.5-6 being poor, and lower than 3.5 as fatal to salmon. Even though salmon and
tardigrades cannot fully be compared due to the difference in size and modes of oxygen
consumption, the data can be used to show the importance of decreased oxygen to the survival of
the organisms. Also the DO test did not have any M. tardigradum, so the correlation between
activity and DO cannot be adequately compared.
It was believed that a change in pH would decrease the amount of dissolved oxygen. I
hypothesized that M. tardigradum activity would decrease the further the pH was from 7, and
23
this was supported by the data in both buffer concentrations. The pHs that were tested and had
decreases in activity showed significant differences in activity when compared to the control of 7
in both 1:10 concentration (F= 264.61, DF= 10, P= 0.0001) and the non-diluted buffer (F=
2177.72, DF= 10, P= 0.0001). For the hypothesis that M. tardigradum would remain active
longer in lower pH compared to higher, the results showed data that would reject the hypothesis.
From the activity test M. tardigradum remained active in more samples that had higher pH
compared to samples with lower pHs. For the hypothesis that there would be a decrease in
dissolved oxygen similar to the decrease in activity the results showed data that would support
the hypothesis. The DO decrease with the change in pH and the change from the ANOVA was
significant (F= 68.11, DF= 20, P= 0.0001) in the change in DO levels in the samples. However, a
full comparison between activity and DO cannot be made due to the fact that the active test did
not have DO taken and there were no tardigrades tested for the DO test. The comparison that
supports the hypothesis comes from the visual similarity between Figure 3 and 4 to Figure 5.
There are few studies that focus on the need for oxygen by tardigrades. One study that
does focus on oxygen and tardigrades is a study by Klekowski and Opalimski (1988). The
research studied the oxygen consumption of seven different tardigrade species in changing
temperatures. The research studies how the change in temperature might affect the tardigrades
weight, metabolic rate, and oxygen consumption. The study focused on temperatures of 2oC to
25oC in some areas. The test showed that with an increase in temperature the tardigrades showed
a change either an increase or decrease in wet body weight, metabolic rate, and oxygen
consumption in all samples. One species showed fluctuations in the data, Doryphorybius
smreczynkii. Species Doryphorybius smreczynkii showed an increase in wet body weight,
metabolic rate, and oxygen consumption between the temperatures of 2oC and 6oC. However,
24
between 6oC and 10oC the wet body weight and oxygen consumption decrease, while the
metabolic rate increased. Although none of the species were M. tardigradum, the data from the
study gives information on the change of metabolic rates and oxygen consumption of tarigrades
and specifically some of the species that are in the class Eutardigrada, which is the same class as
M. tardigradum.
Like the study on the affects of oxygen on tardigrades, the affects of pH and the changes
of pH on tardigrades have few studies on it. Most of the studies that focus on pH mostly study
zonation and not the affect on the organisms itself. A study by Meininger and Spatt (1988) study
the movement and zonation of arctic species of tardigrades on how dust and pollution affect their
habitat. They focused on the Dalton Highway, which is the road that goes to the trans-Alaska
Pipeline. The traffic on the road generates calcium-rich dust that settles on moss inhabited by
tardigrades. The dust affects the habitat by increasing the pH of the moss. The study examined
distances of 5 to 500 meters from the road. The pH range for the distances was 4.00 to 4.55 for
distances of 500 meters, while distances of five meters had pHs of 5.25 to 6.05. Even though the
pHs were still acidic, a change in pH from 4 to 6 is a 100 time the change in hydrogen ions in the
moss. The study focused on how the change in pH would affect frequency and density of
tardigrade species. The test showed some species reduced in both frequency and density the
closure to the road the moss was, while other species actually increase. The study showed how
humans created zones within the habitat, by creating areas of different pH levels. The test also
showed that different species of tardigrades have different preferences in the pH of its habitat.
One of the topics that was not limited in research is the zonation of tardigrades and other
meiofauna. There have been many studies that focused on the impact of physical conditions,
such as salinity and water on the formations of these zones. With these studies researchers have
25
gotten a better understanding the environmental needs of tardigrades and other meiofauna. In the
Antarctic by Treonis et al. (1999) focused on the zonation of invertebrate species in dry valley
soil and sediments. The study observed nematodes, rotifers, tardigrades, and other invertebrates.
The research studied the horizontal zones between the dry valley soil and soil near streams. The
study showed that the abundance of smaller invertebrates and moisture of the soil did not affect
the abundance in the population of rotifers, nematodes, or tardigrades. However, the study did
show data that sagest a difference in community in the zones. The data samples showed most of
the invertebrates in wet low-salinity sediments in the center of the stream. Adjacent to the
stream, in the hyporheic zone, was dominated by three species of nematodes. The hyportheic
zone was influenced by salt deposit from the stream. Further from the stream in dry soil, was
only one species of nematodes. The data from the study suggest that niches for the invertebrate
community are based on the distance of the source water and the amount of salinity.
There are other types of zonations other than spatial. Zones can also be created by time,
which is usually created by the change in seasons. For meiofauna both spatial and temporal
affect its habitat. A study by Albuquerque et. Al (2007) studied the meiofauna communities in
the sands of Brazilian beaches. For the study samples were taken from July 1998 to June 1999
from four areas. The areas were the saturation zone, resurgence zone, retention zone, and drysand zone. These areas or zones are separated by the amount of water they receive. The study
found 12 taxa in the studied areas. For the test the study focused on Turbellaria, Nematoda,
Tardigrada, Copopoda, and other invertibrates. The study did show data presenting special zone
in the areas. The tardigrades in the area showed pick population in August and December, and
low populations in June. Turbellaria had higher populations in July and October, with low
populations in August and March. Nematodes had its highest population in June, and its lowest
26
in December and August. Copepoda had its highest population in July with its lowest in
December and January. For spatial the study found the highest density of organisms at 0-10 cm
in the retention zone. The study shows that both physical and temporal changes can affect a
population. Another study that focused on temporal and spatial zones of meiofauna was Palmer
(1990). The study was on Goose Creek in Virginia. The stream flows for year round and had a
sandy substrate. The samples were collected 180 cm deep in the stream. The studied identified 5
taxa in the area. Like Brazil both nematodes and tardigrades were among them. The data showed
the pick in tardigrade population in the month of February. Overall the data from the study
showed that the meiofauna were abundant mostly in the depths in the hyporheic zones most of
the year. The times with the lowest population were July and August, which have less water
flow. The reduction of water flow reduced the amount of oxygen that would penetrate the layers
of the soils preventing organism growth and survival. These studies show the affect that oxygen
and other factors can affect the habitat of tarigrades.
Humans still know very little about the phylum Tardigrada. Researchers are finding new
species and subspecies very year. Also the understanding of cryptobiosis is improving every year
with more research on the limitations on the organism and chemicals used by to induce
cryptobiosis. What is known is organism such as the tardigrade need specific environmental
conditions to survive. They can induce cryptobiosis if the environment changes, but they cannot
stay in that state forever. The physical conditions like pH, water, and salinity affect the
zonations and survival of tarigrades. Changes in the physical conditions could affect the
population dynamics of the different species in the area. This was seen in the study Meininger
and Spatt (1988) with species of tarigrades changing were they are found due to the change in
the pH caused by the Alaska road. The test showed that the change in pH can affect the dissolved
27
oxygen and activity/ survival of M. tardigradum. This study was one of the few actual studies
that showed the interaction between the changes in pH with the activity of a specific species of
tardigrades. It showed how receptive tardigrades are to the change in the pH in their habitat.
Tardigrades can survive from the ocean to dry moss. They can survive being dry for years and
many conditions that would be fatal to humans. Even though tardigrades can survive these
conditions, they can only survive them for so long. The observation of tardigrades can reveal the
conditions of the environment. If there are many dead tardigrades or if many of them are in
cryptobiosis then the physical conditions might be savvier to other organisms, such as humans.
For this and their ability of cryptobiosis more research must be done on these organisms.
28
Acknowledgments
I would like to thank the people at the Carolina Biological Supply Company for culturing
and shipping M. tardigradum. Also I would like to thank them for answering my questions that
helped in the creation of some of the parameters for the study. I would like to thank Cheryl
Guglielmo, the science lab technician for Saint Martin’s University, who order of supplies and
helped me create the work area for my study. I would like to thank my instructors Doctors Aaron
Coby, Mary Jo Hartman, and Margaret Olney who helped in focusing the study, data
interpretation, and evaluation of the study.
29
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