Tardigrades as Environmental Bio-Indicators

Part of the Young Naturalist Awards Curriculum Collection.

by Amber, Grade 12, Iowa - YNA WINNER

The time seemed to slip away so suddenly, almost as if it was sand sifting through an hourglass, yet at the same time the days were filled with experiences and encounters that one could never forget. For me, the Arctic Workshop on tardigrades in Greenland was an experience of a lifetime -- one I will always remember and to which I will always refer. I would like to start from the beginning and share some of my experiences and learning moments with you.

My love for tardigrades began as a sixth grade science project when my friend and mentor, Ernie Schiller, first introduced me to them. I was hooked right away by their bear-like appearance! I am interested in tardigrades asbio-indicators and have studied their behavior when exposed to several environmental contaminants including ultra-violetlight, sulfuric and nitric acid, and sulfur dioxide. But many people are not familiar with tardigrades, and wonder what exactly they are.

A microscopic image of a Eutardigrade clinging to a moss habitat, shown as an irregular-shaped form with several small appendages.
Eutardigrade clinging to moss habitat

Tardigrades, tiny microscopic organisms that inhabit mosses and lichens, are found in almost every country in the world. They were discovered over 200 years ago, and more than 400 different species have been classified (Kinchin, 1994). Lichens are the primary habitat of these tardigrades and are very reliable indicators of air quality.

More than 20,000 species of lichens have been identified worldwide (Kiester, 1991). Lichens absorb all of their nutrients from the air, including any contaminants that are a part of the air. They sponge up everything the air brings their way -- carbon dioxide, sulfur dioxide, heavy metals, radiation, and dust -- tucking it away whether they need it or not.Sulfur dioxide emissions are currently a major air pollution problem in the United States. It is estimated that 30-40million tons of sulfur dioxide are emitted into the atmosphere of the U.S. each year (EPA, 1997). This material can be very harmful to plant and animal life.

After exhaustive research, I concluded that there have not been any studies done comparing tardigrades on lichen samples from pristine environments and those exposed to various air pollutants in the state of Missouri. With this knowledge I decided to pursue a field study in the area of a coal burning power plant. But the plot thickens and twists. For what was beginning as a simple study grew more complex by the minute. The results left numerous possibilities to be tended to in the near future. What unfolded was a true experience that seems almost impossible and unbelievable.

A photo of the James River Power Plant in Springfield, Missouri. Cylindrical emissions stacks release smoke; trees and a body of water are behind it.
The James River Power Plant emission stacks in Springfield, MO

Tardigrades inhabit lichens worldwide. This field study examines the diversity, distribution, density, and abundance of tardigrades, rotifers, nematodes, and lichens that inhabit oak trees upwind and downwind from a coal-fired power plant in Springfield, Missouri. The significant differences found in the density and diversity of tardigrades suggests that a model for their use as a bio-indicatorcould be developed as an environmental check-and-balance system.

The James River Power Plant is a coal burning facility located just south of the town of Springfield, Missouri. The plant is publicly owned and is currently governed by a Board of Public Utilities. It went into operation in 1957 and serves the electrical needs of communities in a 320 square mile area. The plant has four emission stacks that are some of the shortest in the Midwest. In 1990, the power plant began working toward compliance with the Clean Air Act. In1996, the plant began burning 75% Wyoming coal (a much lower sulfur content) and only 25% Illinois coal. By 1998, sulfur dioxide emissions were reduced to compliance levels (DNR, 1998).

The area around the plant is primarily a native oak and maple forest broken by development and agricultural fields. Because the James River Power Plant lies in a river valley, the wind around the plant has an anomalous circulation,blowing primarily from the northwest to the southeast (Airport, 1998).

This field study compares the diversity, distribution, and abundance of tardigrades, nematodes, rotifers, and lichens inhabiting trees both upwind and downwind from the James River Power Plant. The purpose of my research was to analyze relationships between microfauna and microflora. I hypothesized that there would be no significant difference in the diversity, distribution, or abundance of lichens, tardigrades, rotifers, or nematodes living on trees both upwind and downwind from the power plant.

A map titled, "20 mile radii from James River Power Plant where lichen samples were extracted."
20 mile radii from James River Power Plant where lichen samples were extracted (Click to enlarge.)
A person extracting lichen samples from the bark of the trunk of a tree.
Extraction technique used to remove lichen samples from bark of Quercus macrocarpa

Resource materials -- including topography maps, tree and lichen identification guides, climatological wind readings, and SO2 monitoring reports -- were gathered for this study. I contacted the Iowa State Forestry Department to make sure that the sampling technique was safe for the trees. Sampling sites were identified around the power plant area using a circular radius of 3, 4, 5, and 20 miles upwind and downwind from the power plant. Three oak trees were identified at each site and a leaf sample was gathered from each tree. After measuring the circumference, a compass was used to determine cardinal north, east, south, and west side of each tree. I used a gasket bore to sample a one-inch bark circle (with lichen) between 2-4 feet above ground level from each tree. The area was then scraped with a scalpel and placed in a labeled paper sack. The same procedure was repeated for all trees at each sample site.

Once I returned home, the lichen samples were inverted in culture dishes with 6 ml of spring water. After 48 hours, each sample was observed for 15 minutes. I recorded the number of tardigrades, nematodes, and rotifers within a sample,identified the lichen growth form, and took the lichen coverage on the one-inch bark circle using a R-40 circle template.

Three photos: one showing hands placing something in a test tube with tweezers, one placing an object under a microscope, and one holding another test tube.
Left to right: Sample preparation for extracting tardigrades; tardigrade observation using a stereo dissecting microscope; sample preparation for preserving tardigrades

Next came the preserving and identifying process. 2 ml of 70% ethyl alcohol was added to each sample. After 30 minutes, the solution was removed, and once again I added 2 ml of 70% ethyl alcohol. The debris and alcohol were then transferred to a vial using a pipette. I topped the sample with 0.2 ml of ethyl alcohol to keep a set concentration, and placed them in a two-inch petri dish. The tardigrade was teased to the surface and lifted out with an Irwin loop. I placed the tardigrade on the slide with one small drop of Hoyer's preserving medium and a cover slip was placed on top. Slides were labeled and placed in a drying oven at 150 degrees for three hours. Once the slides had cooled, the edges were coated with clear fingernail polish. Tardigrades and lichens were then identified to genus.

A bar chart titled, "Number of Animals versus Tree Species and Direction."
A bar chart titled, "Total Number of Animals versus Direction from Power Plant."
A chart titled, "Average Density of Animals versus Distance from Power Plant."
A bar chart titled, "Tardigrade Genera versus Direction from Power Plant."

Statistical analyses show that within a site, the three oak trees were not significant to the animals (63%<95%), as well as lichen growth form (44%<95%), and sample position on the tree (44%<95%). However, the species of the tree (99%>95%) and the sample distance from the power plant was significant to the animals (99.5%>95%).

This research has shown that there were twice as many tardigrades upwind of the power plant than downwind. Rotifer populations were greater upwind and lower downwind. Nematode populations were lower upwind and greater downwind. The density of the tardigrades (number of tardigrades percm2 of lichen) gradually increased as the distance from the power plant increased.

Lichen coverage did not affect the number of tardigrades found in a sample. Five lichen genera were found upwind of the power plant while only three were found downwind (Candelaria and Pertusaria were found upwind but not downwind). Four tardigrade genera (Macrobiotus, Milnesium, Minibiotus, Ramazzottius) were found upwind of the power plant while four tardigrade genera (Echiniscus, Macrobiotus, Milnesium, Minibiotus) were found downwind. Echiniscus was only found at the downwind sites while Ramazzottius was only found at the upwind sites.

The proposal to use the diversity, distribution, and abundance of tardigrades as bio-indicators of environmental conditions seems to be supported by the significant differences I found between the upwind and downwind populations of tardigrades relative to the power plant in Springfield, Missouri. My results suggest that airborne conditions, both natural and man-made, may influence the tardigrade diversity, distribution, and abundance found at the time of sampling.

This study also establishes a model for future baseline research. It can be used as a "model" that other researchers may use when conducting field studies. Because there was no significant difference between samples taken from direction on tree, or tree within a site, selecting sites randomly will produce meaningful data. Once a potential center is decided,and wind direction determined, one must develop concentric rings for sampling. It would be best to limit the study to one species of tree to eliminate variables. Because collections do not exist from 10 or 20 years ago, change cannot be measured for tree, lichen, or animal occurrence, density, or diversity. But now a baseline has been established to aid in future work. This allows for the possibility of measuring change in the future.

This research would not be complete without acknowledging those who assisted me. Mr. Ernest Schiller has been my day-to-day advisor as my project progressed and questions arose. He has provided the high school's stereo dissecting microscope for my use. Acknowledgements must go to Dr. Chris Dionigi, U.S. Department of Agriculture, who aided in the statistical treatments, and to Dr. William R. Miller, who assisted me with the preserving/mounting techniques, and also the identification of the tardigrades to genus. I would also like to recognize Thomas Moninger of the Central Microscopy Research Facility at the University of Iowa, who provided access to the training in the use of the microscopes necessary to identify specimens and proper photographic techniques. Access to data and information about the James River Power Plant's sulfur dioxide emission monitoring procedures and equipment was provided by Dr. David Fraley, Director of Environmental Compliance at City Utilities in Springfield, Missouri. Dr. A.J. Mumm, Hazard Mitigation Planner, helped in the running of the Geographic Information Systems program, while Dr. Lois Tiffany, Professor of Botany at Iowa State University (ISU), assisted in the identification of lichens to genus. I would also like to thank Paul Way, Extension Forrester at ISU, who confirmed leaf and tree identifications.

Several people standing on a rocky formation gathering moss and lichen samples for tardigrade research.
Tardigradologists gathering moss and lichen samples for tardigrade research

The story now unfolds. When I started this research I never dreamed that it would have led me to where I am today. My knowledge of tardigrades has grown, and my curiosity has blossomed. When this 17-year-old girl was asked how she spent her summer, many would not believe me when I told them. If asked a year ago, I never would have dreamed that I would spend 10 days in Greenland with 15 prestigious tardigradologists from around the world. Greenland has such a unique environment that it is home to many different groups of tardigrades. Approximately 80 species are known in the neighborhood of the Arctic Station, where we conducted our work. They are found in the mosses, lichens, corals,barnacles, cryconite holes, freshwater, and many other places. We learned from one another and conducted research during our stay.

Disko Island, Greenland. Seven motorboats in the foreground. On the far shore is a community of about ten houses, with low mountains in the background.
Disko Island, Greenland

We left Copenhagen late Monday night, August 7, 2000, and arrived in a small town in Greenland where we switched flights. Then off to Illussat where we took a six-hour boat ride to the Arctic Station. Godhaven, where we stayed, is a very small village on the south coast of Disko Island. Disko Island is the largest island in Greenland and the interior part of it is dominated by a local ice cap. Situated right on the coast, it had everything I needed to survive.

The Arctic Station was founded in 1906 by botanist Morten P. Porsild and has served as an arctic scientific station. In 1953 the station served as a biological station under the Botanical Central Institute. Today it is placed directly under the Faculty of Science and a board of scientists of botany, geology, geography, and zoology at the University of Copenhagen. In addition there is a modern lab building with a library, darkroom, herbarium, and a scientific research vessel. 

The Arctic Station on Disko Island, with a two-story building and a smaller adjacent building. A barren hillside is in the background.
The Arctic Station, Disko Island

Outside the door was a rippling body of water that led into a vast ocean that gleamed in the sunshine. Some days we would have glaciers passing through for only a few hours,while others would stay for the day. With luck, one would hear a vibrant crack, and see pieces of ice fall gracefully into the salty heterogeneous solution, starting their own journey through the Arctic. Whales skimmed the surface gasping for air, just enough for the human eye to catch a glimpse of the beauty of nature. A few seals bobbed here and there in the deep, vast ocean searching for food, but were shortly gone upon human arrival. An arctic hare set against the landscape, tried desperately to nibble its supper in peace. 

Iceberg in a bay
A typical iceberg floating in or out of the bay

The group had a daily ritual of hiking to a different location and gathering samples. Each day we saw something different. We would gather samples, take turns analyzing them in the lab building or classroom, ask other people's opinions, and stay up late discussing tardigrades. Each day I found myself trying things I had never done before, and pushing myself to new limits. One afternoon I was scouting around at the base of the ice cap. The group decided to take a short jaunt down Englishman's harbor. It was a stream that went down a cliff to the ocean. Very narrow, steep, and full of vegetation, I found it fascinating to be climbing right above glaciers in the sea. A radioactive spring was nestled right below the ridge and it was a "hot zone" for tardigrades. I am very lucky that I went down the cliff for otherwise I would not have found what I did that day.I carefully extracted several moss samples and placed them in my bag. Not too many, however, because I knew there was along climb ahead of us. We climbed on the edge of the cliff with only a foot's width on which to balance. Always using our hands, we climbed and climbed for what seemed an eternity before we returned to the Arctic Station.

The next day I soaked my samples, and while wading through grams and grams of sand and soil I found a rare occurrence intardigrade behavior. There was a Milnesium eurystonum tardigrade feeding on a Macrobiotus hufelandii sp. The Macrobiotus was still in Milnesium's mouth. Milnesium is a carnivorous tardigrade; it had pierced Macrobiotus with its stylets in order to use its sucking pharynx to bring Macrobiotus through its buccal tube. Everyone in the lab was thrilled! I decided to take the chance and try to mount the specimens. To my dismay, I lost the tardigrades in the pipette and thought for sure my find was ruined. But, after a second try, the specimens were successfully mounted on a slide and ready for DIC (differential-interference-contrast) pictures back in the States.

Upon return to the States, I took photographs of this rare occurrence in tardigrade behavior. I am now pursuing a different expedition into the world of scientific journal publishing. I hope that this discovery will be met with excitement in the scientific world and that I can make an important contribution to the discovery of scientific truth.



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Coombes, Allen. Trees. New York: DK Publishing, Inc., 1997.

EPA Aerometric Information Retrieval System: Air Quality Subsystem, Raw Data Report (SO2), Springfield, Mo.: EPA, 1997.

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Miller, William R. Tardigrades -- Bears of Moss. Kansas: Emporia State University, Vol. 43, No.3, 1997.

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Missouri Department of Natural Resources. Environmental Service Program: St. Joseph Levee (Site 4), James River South, Springfield (Site 68). Retrieved from the World Wide Web on October 2000: http://www.state.mo.us/deq/esp/aqm/jamesr68.htm

Nelson, Diane R. Ecology and Classification of North American Freshwater Invertebrates. New York: Academic Press, 1991.

Ramazzotti, G. and W. Maucii. The Phylum Tardigrada. Texas: McMurry University, 1994.